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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
ISSN 1064-3745
E-ISBN 1-59259-957-5
Library of Congress Cataloging-in-Publication Data
Pharmacogenomics : methods and applications / edited by Federico Innocenti.
p. ; cm. -- (Methods in molecular biology, ISSN 1064-3745 ;311)
Includes bibliographical references and index.ISBN 1-58829-440-4 (alk. paper)
1. Pharmacogenomics. I. Innocenti, Federico.II. Series: Methods in molecular biology (Clifton, N.J.);
v. 311.[DNLM: 1. Pharmacogenetics--methods.
2. Genomics--methods.
3. Variation (Genetics) QV 38 P53193 2005] RM301.3.G45P427 2005 615'.7--dc22
2005010328](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-10-320.jpg)
![6 Kalow
led to the recognition of a pharmacogenetic difference between human popula-
tions were first considered to be odd cases. In 1921, Paskind (23) injected atro-
pine sulfate into 20 Caucasian and 20 African-American men in Chicago. He
found that the drug caused an initial slowing of the heart rate in the white but
not the African-American subjects. In 1929, Chen and Poth (24) measured the
pupillary size after applying various mydriatic eye drops into the eyes of a
number of people. The increase in size was largest in Caucasians, intermediate
in Asians, and smallest in African-Americans; the authors thought that the color
of the iris affected its movability.
During World War II, American soldiers stationed in tropical countries
received primaquine (25) as antimalarial prophylaxis; it turned out that only
soldiers of African descent developed hemolysis from the administration of
primaquine. The explanation came later (26): The affected soldiers had a genetic
deficiency of glucose-6-phosphate dehydrogenase; this deficiency was frequent
in Africans because it protected the carrier from malaria, but it was rare in
countries without malaria. After discovery of the genetic deficiency of iso-
niazid acetylation (5,27), other investigators found substantial interethnic dif-
ferences in the frequency of this deficiency (28). The deficiency was rare in
Eskimos (Inuit), relatively frequent in Europeans and Africans, and intermedi-
ate in East Asians.
In the early 1970s, my laboratory studied the metabolism of amobarbital (an
at that time widely used drug) in a class of students (29). When we did not
observe its normal metabolite in 7 of the 140 students, we assumed a labora-
tory error. When calling the students back for reinvestigation, it turned out that
all 7 were of Asian origin and that our first measurements had been correct. At
the same time, our laboratory ran tests with debrisoquine (30) because a genetic
variation of its metabolism had just been discovered (14). Again, we saw a sub-
stantial difference of its metabolic destruction between students of Asian and
non-Asian origin (the difference was later defined in terms of DNA variation
by Swedish investigators [31]). These unexpected observations with amobar-
bital and debrisoquine caused us to search the literature and to publish in 1982
the first article on interethnic differences in drug metabolism (32).
In the mean time, studies of interethnic differences in drug response or metabo-
lism have become frequent research projects. Computerized Pubmed lists more
than 2000 articles dealing with the combined entries “drug” and “race.” It is
now quite clear that the interethnic differences may be divided into two kinds:
first, a given mutation of a particular gene may occur with different frequen-
cies in different populations. Second, there are mutations that appear to be
specific for a particular population. Some of the differences may be there
because they provide a population with a biological advantage; however,](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-22-320.jpg)
![8 Kalow
consisted of twin studies (35): that is, the magnitude of differences between the
two members of a pair of identical and a pair of fraternal was measured. Repeat-
ing such studies in many twins and averaging and comparing the differences
allowed a calculation of heritability. Unfortunately, the recruiting of a suffi-
cient number of twins often is difficult.
However, a simpler method is now available (36) that is based on the fact
that one can give a drug repeatedly to a person and measure each response and
the difference between the responses. When giving a drug at an appropriate
interval two or more times to a group of people, one can measure two magni-
tudes: first, one can calculate the average and standard deviation of the response
differences between the first and subsequent applications in the same people;
second, one can equally calculate and record the difference between subjects.
Let us designate the standard deviations of the within-subject variations as SDw,
of the between-subjects as SDb, and the genetic component of the between-
subject variation as rGC. Squaring the standard deviations, the genetic compo-
nent is then calculated by the following equation:
rGC = (SDb
2 – SDw
2)/SDb
2 [37]).
A value close to 1.0 indicates overwhelming heredity, close to 0 indicates
mostly environmental influence. A recent example of the use of this method
has been the assessment of genetic and environmental determinants of cyto-
chrome CYP3A4 activity (38).
Often forgotten is the fact that there may be a clinically significant drug
response difference between two populations even if the average response dif-
ferences are small, perhaps not even statistically significant (39). If population
data are represented by a normal distribution (Gauss) curve, the persons with
abnormal responses may be represented by one of the edges of the curve.
For example, let us consider a normally distributed metabolic destruction
rates of drug X. Assume that 2% of the persons have a destruction rate low
enough to suffer toxicity from the drug. In another group of people, destruction
of the same drug may have a somewhat lower average rate, a fact that is imma-
terial for most subjects; however, if the distribution curves have equal spread
in the two populations, many more people in the second population will have
the critically low drug destruction rate and will be intoxicated than in the first
population (Fig. 1). In short, the difference between the edges of the distribu-
tion curves may be of clinical and statistical significance even if the averages
are similar.
3. Pharmacogenomics
As described previously, pharmacogenetics began with the study of single
gene differences between individuals but developed into a broad science. Meth-
odological advances expanded the science further into pharmacogenomics; one](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-24-320.jpg)
![Pharmacogenomics: History and Overview 9
may say that pharmacologists followed the geneticist’s adoption of genomic
techniques. The consequence should be a better understanding of the multi-
plicities and complexities of drug–gene interactions; not only may genes affect
drug action, but drugs may affect gene function. Pharmacogenomics repre-
sents attempts by researchers to medically use these new understandings
together with the old ones. The hope is to optimize the efficacy of drugs, to
minimize adverse drug reactions, and to facilitate drug discovery, develop-
ment, and approval.
4. The Aims of Pharmacogenomics
4.1. Aim 1: Creation of a Basis for Personalized Medicine
Current medicine is based on statistical likelihood and often fails the indi-
vidual. The incidence of serious or fatal drug reactions depends in many or
most cases on genetic variation. Studies from U.S. hospitals (40) suggested
that 6.7% of patients had serious adverse and 0.32% had fatal, drug reactions.
The latter caused approx 100,000 deaths per year in the United States. Many
researchers hope that adverse reactions or therapeutic failures will be elimi-
nated by the introduction of personalized medicine (41), meaning that the drug
to be given to a patient will be determined by the patient’s genes.
To reach this aim, we must learn much more about the genetic variants that
may affect drug action. The most frequently occurring genetic variants are
single-nucleotide polymorphisms (SNPs [42]). The human genome contains
approx 3 billion basepairs, and SNPs occur on the average in approx 1 per
1000 bases; thus, they cause genetic variation of many human proteins. These
variants are important objects of study because human individuals usually dif-
fer from each other by less than 1% of their genes; thus, SNPs are important.
Fig. 1. Variation of drug clearance in two populations. The figure illustrates a hypo-
thetical case showing a 20-fold variation of drug clearance between the individuals
within each population, and a twofold difference between the population means. Let
us assume that a slow clearance will tend to produce overdose toxicity in affected
subjects. Obviously, the proportion of such subjects would be much larger in popula-
tion B than in A.](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-25-320.jpg)
![Wasting the public money! Wasting the public money! Whigs damned
rogues! Forster damned fool!"
DUBLIN,[1]
November 4, 1880.
MY DEAR MRS. O'SHEA,—I take advantage of almost the first
moment I have had to myself since leaving you to write a few hasty
lines. And first I must again thank you for all your kindness, which made
my stay at Eltham so happy and pleasant.
The thunderbolt, as you will have seen, has at last fallen, and we are
in the midst of loyal preparations of a most appalling character.
I do not suppose I shall have an opportunity of being in London again
before next Thursday, but trust to be more fortunate in seeing Captain
O'Shea then than the last time.—Yours very truly, CHAS. S. PARNELL.
DUBLIN,[1]
Saturday.
MY DEAR MRS. O'SHEA,—I hope to arrive in London on Tuesday
morning, and trust to have the pleasure of seeing you before I leave. Do
you think you shall be in town on Tuesday?
Kindly address 16, Keppel Street.—Yours very truly, CHAS. S.
PARNELL.
On November 5th that year the village was great on the subject of
"gunpowder, treason, and plot," and during dinner that evening there was
such a noise and shouting outside my house that I asked the maid who was
waiting what all the excitement was about.
She answered breathlessly that "the procession, ma'am, have got Miss
Anna Parnell in a effigy 'longside of the Pope, and was waiting outside for
us to see before they burnt 'em in the village."](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-43-320.jpg)
![This electrifying intelligence was received with grave indifference by
Mr. Parnell till the disappointed maid left the room; then with a sudden
bubble of laughter—"Poor Anna! Her pride in being burnt, as a menace to
England, would be so drowned in horror at her company that it would put
the fire out!"
The cheering and hooting went on for some time outside the house, but,
finding we were not to be drawn, the crowd at last escorted the effigies
down to the village and burnt them, though with less amusement than they
had anticipated.
DUBLIN,[2]
November 6, 1880.
MY DEAR MRS. O'SHEA,—You can have very little idea how
dreadfully disappointed I felt on arriving here this evening not to find a
letter from either you or Captain O'Shea. I send this in hope that it may
induce you to write in reply to my last letter and telegram, which would
appear not to have reached you.—Yours very sincerely, CHAS. S.
PARNELL.
AVONDALE,
Monday.
MY DEAR MRS. O'SHEA,—I enclose keys, which I took away by
mistake. Will you kindly hand enclosed letter to the proper person[3] and
oblige,—Yours very truly, CHAS. S. PARNELL.
DUBLIN,
Wednesday night, November 11, 1880.
MY DEAREST LOVE,—I have made all arrangements to be in
London on Saturday morning, and shall call at Keppel Street for a letter
from you. It is quite impossible for me to tell you just how very much
you have changed my life, what a small interest I take in what is going
on about me, and how I detest everything which has happened during the
last few days to keep me away from you—I think of you always, and
you must never believe there is to be any "fading." By the way, you must](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-44-320.jpg)
![not send me any more artificial letters. I want as much of your own self
as you can transfer into written words, or else none at all.—Your always,
C. S. P.
A telegram goes to you, and one to W.,[4] to-morrow, which are by no
means strictly accurate.
DUBLIN,
December 2, 1880.
MY DEAR MRS. O'SHEA,—I succeeded in getting the train at
Euston with just ten minutes to spare, and, arriving here this morning,
found that my presence to-day was indispensable.
I need not tell you how much I regretted leaving Eltham so suddenly;
but we cannot always do as we wish in this world.
My stay with you has been so pleasant and charming that I was
almost beginning to forget my other duties; but Ireland seems to have
gotten on very well without me in the interval.
Trusting to see you again next week on my way to Paris.—Yours very
sincerely, CHAS. S. PARNELL.
I have been exceedingly anxious all day at not receiving your
promised telegram to hear how you got home.
[1] These letters were really written from London.
[2] Sent to Dublin to be posted.
[3] Myself.
[4] Captain O'Shea.](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-45-320.jpg)
![his horror, for, although used to such homage in Ireland, he disliked it
extremely, and he told me with some reproach that he had expected to be
quite free from that sort of thing in my house.
At Christmas he tipped my servants generously, and indeed Ellen and the
parlourmaid Mary vied with each other in their attention to his comfort. The
enthusiasm of the cook was so great that she bought an enormous gold
locket, and, having inserted a portrait of Mr. Parnell in it, wore it constantly.
Mary, not to be outdone, thereupon bought a locket of identically the same
design, and wore it with an air of defiance, when bringing in tea, on New
Year's Day.
This was against all regulations, and I said laughingly to Mr. Parnell that
he was introducing lawlessness into my household. He answered, "Leave it
to me," and when Mary appeared again he said gently to her, "Mary, that is
a magnificent locket, and I see you are kind enough to wear my portrait in
it. Mrs. O'Shea tells me that Ellen has bought one also, but I just want you
and Ellen not to wear them outside like that, for Mrs. O'Shea lets me come
down here for a rest, and if people know I'm here I shall be worried to death
with politics and people calling." So Mary promised faithfully, and Ellen
came running in to promise too, and to threaten vengeance on "the others" if
absolute silence was not observed. The lockets went "inside," and only a
tiny bit of chain was allowed to show at the throat in evidence of homage
continued, though hidden.
Meanwhile, events were fusing in Ireland. Parnell had gone over there
immediately after Christmas. From Dublin he wrote:—
DUBLIN,
Monday evening, December 27, 1880.
MY DEAR MRS. O'SHEA,—I have been exceedingly anxious all
day at not receiving your promised telegram to hear how you got home;
trust I may have something to-morrow morning that it is all right.[1]—
Yours in haste, C. S. P.](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-53-320.jpg)
![MORRISON'S HOTEL,
Tuesday, December 28, 1880.
MY DEAREST WIFE,—You will be delighted to learn that
everything is proceeding first-rate so far.
The jury sworn to-day cannot possibly convict us, and there is a very
fair chance of an acquittal. I do not think the Government will attempt to
prevent me from being present at the opening of Parliament, though I am
not quite sure yet whether it will be prudent for me to leave until
Wednesday evening. So far as I can see there is no necessity for the
presence of any of the Traversers; one of them, Gordon, who has broken
his leg, has not appeared at all, and his absence has not been even
mentioned or noticed.
I was immensely relieved by your letter this morning. You must take
great care of yourself for my sake and your and my future.—Yours
always, C. S. P.
I have wired and written to Madrid[2] explaining situation lest my
observations at yesterday's meeting as to doubt of my being in
Parliament, intended to throw dust in eyes of Government, might be
literally interpreted.
DUBLIN,
Thursday, December 30, 1880.
MY DEAREST LOVE,—Your letters have reached me quite safely,
and you cannot tell how much pleasure they give me. I fear I was very
foolish to allow you to come with me the day of my departure; I felt sure
it would do much harm, and until your first letter arrived I was in a
continual panic lest some dreadful disaster had happened.
That my poor love should have suffered so much makes my heart
very sore, and she must take great care of herself for the sake of our
future....
I enclose letter from W.S.[3]—Yours always affectionately, C. S. P.
Will send you photo to-morrow.](https://image.slidesharecdn.com/81919-250721212402-1d19cf5b/85/Pharmacogenomics-Methods-and-Protocols-1st-Edition-Werner-Kalow-Auth-54-320.jpg)
Pharmacogenomics Methods and Protocols 1st Edition Werner Kalow (Auth.)
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- 5. Pharmacogenomics Methods and Protocols 1st Edition Werner Kalow (Auth.) Digital Instant Download Author(s): Werner Kalow(auth.), Federico Innocenti MD, PhD (eds.) ISBN(s): 9781588294401, 1588294404 Edition: 1 File Details: PDF, 2.50 MB Year: 2005 Language: english
- 6. Edited by Federico Innocenti, MD, PhD Pharmacogenomics METHODS IN MOLECULAR BIOLOGY™ 311 Methods and Protocols Edited by Federico Innocenti, MD, PhD Pharmacogenomics Methods and Protocols
- 8. M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, SERIES EDITOR 323. Arabidopsis Protocols, Second Edition, edited by Julio Salinas and Jose J. Sanchez-Serrano, 2006 322. Xenopus Protocols: Cell Biology and Signal Transduction, edited by X. Johné Liu, 2006 321. Microfluidic Techniques: Reviews and Protocols, edited by Shelley D. Minteer, 2006 320. Cytochrome P450 Protocols, Second Edition, edited by Ian R. Phillips and Elizabeth A. Shephard, 2006 319. Cell Imaging Techniques, Methods and Protocols, edited by Douglas J. Taatjes and Brooke T. Mossman, 2006 318. Plant Cell Culture Protocols, Second Edition, edited by Victor M. Loyola-Vargas and Felipe Vázquez-Flota, 2005 317. Differential Display Methods and Protocols, Second Edition, edited by Peng Liang, Jonathan Meade, and Arthur B. Pardee, 2005 316. Bioinformatics and Drug Discovery, edited by Richard S. Larson, 2005 315. Mast Cells: Methods and Protocols, edited by Guha Krishnaswamy and David S. Chi, 2005 314. DNA Repair Protocols: Mammalian Systems, Second Edition, edited by Daryl S. Henderson, 2005 313. Yeast Protocols: Second Edition, edited by Wei Xiao, 2005 312. Calcium Signaling Protocols: Second Edition, edited by David G. Lambert, 2005 311. Pharmacogenomics: Methods and Protocols, edited by Federico Innocenti, 2005 310. Chemical Genomics: Reviews and Protocols, edited by Edward D. Zanders, 2005 309. RNA Silencing: Methods and Protocols, edited by Gordon Carmichael, 2005 308. Therapeutic Proteins: Methods and Protocols, edited by C. Mark Smales and David C. James, 2005 307. Phosphodiesterase Methods and Protocols, edited by Claire Lugnier, 2005 306. Receptor Binding Techniques: Second Edition, edited by Anthony P. Davenport, 2005 305. Protein–Ligand Interactions: Methods and Protocols, edited by G. Ulrich Nienhaus, 2005 304. Human Retrovirus Protocols: Virology and Molecular Biology, edited by Tuofu Zhu, 2005 303. NanoBiotechnology Protocols, edited by Sandra J. Rosenthal and David W. Wright, 2005 302. Handbook of ELISPOT: Methods and Protocols, edited by Alexander E. Kalyuzhny, 2005 301. Ubiquitin–Proteasome Protocols, edited by Cam Patterson and Douglas M. Cyr, 2005 300. Protein Nanotechnology: Protocols, Instrumentation, and Applications, edited by Tuan Vo-Dinh, 2005 299. Amyloid Proteins: Methods and Protocols, edited by Einar M. Sigurdsson, 2005 298. Peptide Synthesis and Application, edited by John Howl, 2005 297. Forensic DNA Typing Protocols, edited by Angel Carracedo, 2005 296. Cell Cycle Protocols, edited by Tim Humphrey and Gavin Brooks, 2005 295. Immunochemical Protocols, Third Edition, edited by Robert Burns, 2005 294. Cell Migration: Developmental Methods and Protocols, edited by Jun-Lin Guan, 2005 293. Laser Capture Microdissection: Methods and Protocols, edited by Graeme I. Murray and Stephanie Curran, 2005 292. DNA Viruses: Methods and Protocols, edited by Paul M. Lieberman, 2005 291. Molecular Toxicology Protocols, edited by Phouthone Keohavong and Stephen G. Grant, 2005 290. Basic Cell Culture, Third Edition, edited by Cheryl D. Helgason and Cindy Miller, 2005 289. Epidermal Cells, Methods and Applications, edited by Kursad Turksen, 2005 288. Oligonucleotide Synthesis, Methods and Applications, edited by Piet Herdewijn, 2005 287. Epigenetics Protocols, edited by Trygve O. Tollefsbol, 2004 286. Transgenic Plants: Methods and Protocols, edited by Leandro Peña, 2005 285. Cell Cycle Control and Dysregulation Protocols: Cyclins, Cyclin-Dependent Kinases, and Other Factors, edited by Antonio Giordano and Gaetano Romano, 2004 284. Signal Transduction Protocols, Second Edition, edited by Robert C. Dickson and Michael D. Mendenhall, 2004 283. Bioconjugation Protocols, edited by Christof M. Niemeyer, 2004 282. Apoptosis Methods and Protocols, edited by Hugh J. M. Brady, 2004 281. Checkpoint Controls and Cancer, Volume 2: Activation and Regulation Protocols, edited by Axel H. Schönthal, 2004 280. Checkpoint Controls and Cancer, Volume 1: Reviews and Model Systems, edited by Axel H. Schönthal, 2004 279. Nitric Oxide Protocols, Second Edition, edited by Aviv Hassid, 2004 278. Protein NMR Techniques, Second Edition, edited by A. Kristina Downing, 2004 277. Trinucleotide Repeat Protocols, edited by Yoshinori Kohwi, 2004
- 9. M E T H O D S I N M O L E C U L A R B I O L O G Y™ Pharmacogenomics Methods and Protocols Edited by Federico Innocenti, MD, PhD Committee on Clinical Pharmacology and Pharmacogenomics Section of Hematology/Oncology, Department of Medicine University of Chicago, Chicago, IL
- 10. © 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular BiologyTM is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Jennifer Hackworth Cover design by Patricia F. Cleary Cover illustration: Figure 2 from Chapter 4, “Genome-Wide Analysis of Allele-Specific Gene Expression Using Oligo Microarrays," by Maxwell P. Lee. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: orders@humanapr.com; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829-440-4/05 $30.00 ]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 ISSN 1064-3745 E-ISBN 1-59259-957-5 Library of Congress Cataloging-in-Publication Data Pharmacogenomics : methods and applications / edited by Federico Innocenti. p. ; cm. -- (Methods in molecular biology, ISSN 1064-3745 ;311) Includes bibliographical references and index.ISBN 1-58829-440-4 (alk. paper) 1. Pharmacogenomics. I. Innocenti, Federico.II. Series: Methods in molecular biology (Clifton, N.J.); v. 311.[DNLM: 1. Pharmacogenetics--methods. 2. Genomics--methods. 3. Variation (Genetics) QV 38 P53193 2005] RM301.3.G45P427 2005 615'.7--dc22 2005010328
- 11. v Preface For the first time in the published literature, Pharmacogenomics: Methods and Protocols describes the newest and most commonly adopted technologies in the field of pharmacogenomics, providing guidance for investigators in the selection and experimental application of such technologies. Many of the contributors to this book are leading experts in the field. Using the extensive information provided on materials and methods, investigators will be able to easily reproduce each technique in their laboratories. Moreover, this book high- lights problems that might be encountered in performing specific techniques and describes how to identify and overcome them. Pharmacologists, geneti- cists, molecular biologists, and physicians in academic institutions and the bio- technology and pharmaceutical industries will find Pharmacogenomics: Methods and Protocols an essential reference. Pharmacogenomics exists at the intersection of pharmacology and genomics. It aims to study the genetic basis of interpatient variability in response to drug therapy. Pharmacogenomics holds the promise that drugs may eventually be tailor-made for individuals and adapted to each person’s genetic makeup. En- vironment, diet, age, lifestyle, and disease state can all influence a patient's response to medicines, but understanding an individual’s genetic makeup is thought to be the key to creating personalized drugs with greater efficacy and safety. Pharmacogenomics combines traditional pharmaceutical sciences with annotated knowledge of genes, proteins, and single nucleotide polymorphisms. Various technologies are currently available and researchers must be capable of choosing the technology suitable for their purposes. After an introductory chapter about the history of pharmacogenomics and its current status, Pharmacogenomics: Methods and Protocols is divided in three parts. Part I comprises the methodologies for assessing the functional consequences of a certain polymorphism. Part II describes the variety of genotyping platforms currently available. Part III ends the book with two chap- ters devoted to the management of pharmacogenomic information. A large amount of data about the pattern of human genomic variation has been provided by the Human Genome Project and is now publicly available. However, the functional consequences of SNPs and haplotypes are, for the most part, unknown, and current research efforts are oriented toward the elucidation of the genetic basis of changes in function of expression of the coded protein. Chap- ter 2 reports the classical method of transient expression combined with site- directed mutagenesis to study the functional effect of naturally occurring
- 12. vi Preface variants in the UDP-glucuronosyltransferase 1A1 gene. Chapters 3–5 describe newer methodologies recently introduced to evaluate differences in gene expression between two genotypes/haplotypes. The allele-specific differential expression method described in Chapter 3 circumvents the analytic problems of confounding variation arising from environmental or physiologic factors during the analysis of subtle differences in expression between two different alleles. Chapter 4 provides a method for performing both genotyping and allele-specific gene expression for hundreds of genes using a chip system. Finally, it is crucial to take into account the haplotype structure of multiple variants when functional assays of single variants are performed. The HaploChIP is an in vivo cell-based assay that allows screening of haplotypes for differences in relative gene expression and is described in Chapter 5. Part III deals with genotyping techniques. Chapters 6–13 present a wide variety of methodologies, platforms, and chemistries for genotyping. This sec- tion provides an understanding of the factors influencing the efficiency of dif- ferent genotyping methods and the priorities required of different study designs. Readers will find technical information on several different types of assays, includ- ing denaturing high-performance liquid chromatography, pyrosequencing, kinetic-fluorescence detection assay, mass spectrometry, and TaqMan assay for insertion/deletions. Moreover, Part III describes the recent application in which genetic variation is surveyed in DNA pools from individuals enrolled in large studies. The integration of genome-information management systems with patient clinical data sets is the key needed to achieve personalized medicine. Disparate data sources, including public or proprietary biology databases and laboratory and clinical information management systems, pose significant challenges in converting this information into clinically applicable knowledge. In Part IV, Chapter 14 describes PharmGKB, a registration-free interactive tool display- ing genotype, molecular, and clinical primary data integrated with literature information and links to external sources. Finally, Chapter 15 gives an overview of the main technologies needed for the management of pharmacogenomic infor- mation. I am extremely grateful to all the authors for their excellent contributions making this book a comprehensive and up-to-date resource for investigators in pharmacogenomics. Federico Innocenti, MD, PhD
- 13. vii Contents Preface ..............................................................................................................v Contributors .....................................................................................................ix PART I. HISTORY AND OVERVIEW 1 Pharmacogenomics: Historical Perspective and Current Status Werner Kalow ....................................................................................... 3 PART II. FUNCTIONAL ANALYSIS OF GENE VARIATION 2 Transfection Assays With Allele-Specific Constructs: Functional Analysis of UDP-Glucuronosyltransferase Variants Hideto Jinno, Nobumitsu Hanioka, Toshiko Tanaka-Kagawa, Yoshiro Saito, Shogo Ozawa, and Jun-ichi Sawada ....................... 19 3 Snapshot of the Allele-Specific Variation in Human Gene Expression Hai Yan ............................................................................................... 31 4 Genome-Wide Analysis of Allele-Specific Gene Expression Using Oligo Microarrays Maxwell P. Lee .................................................................................... 39 5 HaploChIP: An In Vivo Assay Julian Charles Knight .......................................................................... 49 PART III. GENOTYPING TECHNIQUES 6 Aspects Influencing Genotyping Method Selection Peter Imle............................................................................................ 63 7 Denaturing High-Performance Liquid Chromatography for Mutation Detection and Genotyping Donna Lee Fackenthal, Pei Xian Chen, and Soma Das....................... 73 8 Pyrosequencing of Clinically Relevant Polymorphisms Sharon Marsh, Cristi R. King, Adam A. Garsa, and Howard L. McLeod .................................................................. 97 9 Kinetic Fluorescence-Quenching Detection Assay for Allele Frequency Estimation Ming Xiao and Pui-Yan Kwok ........................................................... 115 10 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Matthias Wjst and Dirk van den Boom ............................................ 125
- 14. viii Contents 11 Fluorescence-Based Fragment Size Analysis Peter Imle.......................................................................................... 139 12 Single-Nucleotide Polymorphism Genotyping in DNA Pools Ian Craig, Emma Meaburn, Lee Butcher, Linzy Hill, and Robert Plomin ........................................................................ 147 13 TaqMan Genotyping of Insertion/Deletion Polymorphisms Renato Robledo, William R. Beggs, and Patrick K. Bender .............. 165 PART IV. MANAGEMENT OF PHARMACOGENOMIC INFORMATION 14 PharmGKB: The Pharmacogenetics and Pharmacogenomics Knowledge Base Caroline F. Thorn, Teri E. Klein, and Russ B. Altman ....................... 179 15 Systems for the Management of Pharmacogenomic Information Alexander Sturn, Michael Maurer, Robert Molidor, and Zlatko Trajanoski ................................................................... 193 Index ............................................................................................................ 209
- 15. ix Contributors RUSS B. ALTMAN • Department of Genetics, Stanford University School of Medicine, Stanford, CA WILLIAM R. BEGGS • Division of Molecular Biology, Coriell Institute for Medical Research, Camden, NJ PATRICK K. BENDER • Division of Molecular Biology, Coriell Institute for Medical Research, Camden, NJ LEE BUTCHER • SGDP Centre, Institute of Psychiatry, London, UK PEI XIAN CHEN • Department of Human Genetics, The University of Chicago, Chicago, IL IAN CRAIG • SGDP Centre, Institute of Psychiatry, London, UK SOMA DAS • Department of Human Genetics, The University of Chicago, Chicago, IL DONNA LEE FACKENTHAL • Department of Human Genetics, The University of Chicago, Chicago, IL ADAM A. GARSA • Division of Molecular Oncology, Washington University School of Medicine, St. Louis, MO NOBUMITSU HANIOKA • Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, Tokyo, Japan LINZY HILL • SGDP Centre, Institute of Psychiatry, London, UK PETER IMLE • Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN FEDERICO INNOCENTI • Committee on Clinical Pharmacology and Pharmacogenomics, Section of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, IL HIDETO JINNO • Division of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan WERNER KALOW • Department of Pharmacology, University of Toronto, Toronto, Canada CRISTI R. KING • Division of Molecular Oncology, Washington University School of Medicine, St. Louis, MO TERI E. KLEIN • Department of Genetics, Stanford University School of Medicine, Stanford, CA JULIAN CHARLES KNIGHT • Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK PUI-YAN KWOK • Cardiovascular Research Institute, Department of Dermatology, University of California, San Francisco, CA
- 16. x Contributors MAXWELL P. LEE • Laboratory of Population Genetics, National Cancer Institute, Bethesda, MD SHARON MARSH • Division of Molecular Oncology, Washington University School of Medicine, St. Louis, MO MICHAEL MAURER • Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria HOWARD L. MCLEOD • Division of Molecular Oncology, Washington University School of Medicine, St. Louis, MO EMMA MEABURN • SGDP Centre, Institute of Psychiatry, London, UK ROBERT MOLIDOR • Christian Doppler Laboratory for Genomics and Bioinformatics, Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria SHOGO OZAWA • Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan ROBERT PLOMIN • SGDP Centre, Institute of Psychiatry, London, UK RENATO ROBLEDO • Department of Biology, University of Cagliari, Italy YOSHIRO SAITO • Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan JUN-ICHI SAWADA • Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan ALEXANDER STURN • Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria TOSHIKO TANAKA-KAGAWA • Division of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan CAROLINE F. THORN • Department of Genetics, Stanford University School of Medicine, Stanford, CA ZLATKO TRAJANOSKI • Christian Doppler Laboratory for Genomics and Bioinformatics, Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria DIRK VAN DEN BOOM • Director of the Molecular Applications Department, Sequenom Inc, San Diego CA MATTHIAS WJST • Molecular Epidemiology, GSF - Forschungszentrum für Umwelt und Gesundheit, Neuherberg/Munich, Germany MING XIAO • Cardiovascular Research Institute, University of California, San Francisco, CA HAI YAN • Department of Pathology, Duke University Medical Center, Durham, NC
- 17. Pharmacogenomics: History and Overview 1 I HISTORY AND OVERVIEW
- 18. 2 Kalow
- 19. Pharmacogenomics: History and Overview 3 3 From: Methods in Molecular Biology, vol. 311: Pharmacogenomics: Methods and Protocols Edited by: F. Innocenti © Humana Press Inc., Totowa, NJ 1 Pharmacogenomics Historical Perspective and Current Status Werner Kalow Summary Pharmacogenomics is an extension of pharmacogenetics, a science described here in terms of five stages of development: 1) some clinical observations predicted genetic alterations of drug response; 2) additional case discoveries led to the term “pharmaco- genetics,” a concept broadened by 3) many systemic case studies, and the realization of its wide applicability; 4) came the recognition of systematic pharmacogenetic differ- ences between human populations. Then it became clear that 5) most human drug– repsonse differences were multifactorial, caused by many genetic alterations plus environmental factors. The recognition of these complexities, and the advance of genet- ics into genomics led to the broader science of pharmacogenomics. This led to plans to create “personalized medicine,” that is, making drug use more effective and safer by giving drugs that fit a person’s genes. Much of the science of genetics, dealing with gene structure, was changed by the realization that gene expression and thereby gene function was variable; this leads to systematic studies of drug action on genes, reversing the tradi- tional studies of genes affecting drug action. Finally, the realization that gene–protein variations contribute to most common dis- eases leads to efforts of creating new drugs that act on these variants. Key Words: Pharmacogenetics; pharmacogenomics; personalized medicine; popu- lations; gene expression; drug targets. 1. Introduction Pharmacogenomics is a recent offspring of pharmacogenetics. Both sciences deal with hereditary impacts upon the action of drugs, and their goals are over- lapping. Any proper account of the history of pharmacogenomic must include a look at the development of pharmacogenetics. Thus, in this chapter pharma- cogenetic history will be outlined first, concentrating on its development in terms of succeeding stages.
- 20. 4 Kalow 2. Pharmacogenetics: Its Stages of Development 2.1. Stage 1: Visions and Some Predictive Observations Some visionaries and some keen observers predated pharmacogenetics as a science. Garrod’s 1902 studies (1) of alcaptonuria and of phenylketonuria indi- cated to him that there was such a thing as human biochemical individuality. Haldane (2) summarized his views in 1949 by stating, “It is an advantage to a species to be biochemically diverse. For the biochemically diverse species will contain at least some members capable of resisting any particular pestilence.” There were also some observational forerunners. In 1932, Snyder (3) described a heritable disability of some people to taste phenylthiocarbamide. In 1943, Savin and Glick (4) noticed a genetic lack of atropine esterase in some rabbits; these animals died while eating belladonna leaves, whereas most rab- bits were not affected. These cases were perceived as isolated observations; they preceded the definition of pharmacogenetics but they helped later investi- gators to establish pharmacogenetics as a science. 2.2. Stage 2: Pharmacogenetics Lives: Systematic Case Studies Several separate observations in the 1950s indicated clearly the dependence of drug effects on the genetic constitution of the recipient. The data were con- vincing because they were based on combinations of biochemical, clinical, and genetic observations. The cases included genetic variation of isoniazid acety- lation (5), failing cholinesterase activity affecting succinylcholine action (6), and primaquine-caused hemolysis owing to deficiency of glucoses-6-phosphate dehydrogenase (7). All these cases became subjects of subsequent studies that showed that each of the enzymes could vary in many ways because of different mutations. These clear-cut cases raised in several people the opinion that pharmaco- logical heritability was a clinically important subject. Thus, the American Medical Association (AMA) invited the geneticist Arno Motulsky (8) to con- sider the problem; he summarized it in a 1957 paper entitled “Drug Reactions, Enzymes, and Biochemical Genetics.” Vogel (9) in Germany was aware of the same problem and coined the term “Pharmacogenetics.” Kalow began to sum- marize all available knowledge in a book that appeared in 1962 (10), indicating that the concept of pharmacogenetics had been fundamentally accepted. 2.3. Stage 3: Broadening of Pharmacogenetic Knowledge In the following years, many centers contributed new data, but all the data represented monogenic variations, i.e., differences between individuals caused by mutations of single genes. Weber’s 1997 book (11) listed 15 variable drug- metabolizing enzymes, 11 variable drug receptors, and 14 other variable pro- teins in humans that affected drug actions. In 2001, Kalow (12) counted 42
- 21. Pharmacogenomics: History and Overview 5 variable drug-metabolizing enzymes. In short, the knowledge of different kinds of protein variants that may affect drug responses was growing. Drug-metabolizing enzymes represent the category with the largest number of known variants, which probably has historical and methodological reasons. To measure a change of drug metabolism, all one needs are chemical methods, many of which are dated. To find a drug receptor variation, one has to identify the receptor protein and its gene (13), processes that require sophisticated pro- cedures of more recent date. Thus, measurements of drug metabolism are older than some other procedures. In the history of drug metabolism, prominent were the discoveries of genetic variability of the metabolism of debrisoquine (14) and of sparteine (15). Subse- quent studies indicated that both drugs are metabolized by the same enzyme (16), which turned out to be the P450 cytochrome CYP2D6 (17). The enzyme’s variations were found to be complex (18): enzyme activity could be absent because of frameshift mutations, splicing defects, gene deletion, or the pres- ence of a stop codon. The enzyme may function slowly because of various kinds of mutation, whereby some mutations affected only the interaction with specific substrates. Enzyme duplication or multiplication could lead to very fast action. Many clinical case studies and observations could be mentioned. For example, as summarized by Meyer (19), patients with deficient CYP2D6 activity experi- enced exaggerated or prolonged responses to metoprolol, encainide, perhexiline, or thioridazine; however, codeine had no analgesic effect in such cases because it must be activated to morphine by CYP2D6. More than 60 alleles of CYP2D6 are known today, characterized by differ- ent combinations of some 45 mutations (20). Approximately 60 different drugs are metabolized by CYP2D6 (21). A Pubmed search indicated that there are more than 2300 publications dealing with CYP2D6. The studies of CYP2D6 helped to give pharmacogenetics the deserved clinical attention. Genetic failure of drug-metabolizing enzymes can lead to a patient’s death. For instance (22), mercaptopurine or thioguanine have been fatal in cases of failing activity of thiopurine methyltransferase. Regulatory agencies are con- sidering recommendations and official acceptance of pharmacogenetic testing before the administration of dangerous drugs. 2.4. Stage 4: Pharmacogenetic Differences Between Populations Pharmacogenetics began with the observation of interindividual differences of some drug responses or of drug metabolism that started pharmacogenetics. This recognition, that there are pharmacogenetic differences between popula- tions, truly widened and altered the science. Various older observations that
- 22. 6 Kalow led to the recognition of a pharmacogenetic difference between human popula- tions were first considered to be odd cases. In 1921, Paskind (23) injected atro- pine sulfate into 20 Caucasian and 20 African-American men in Chicago. He found that the drug caused an initial slowing of the heart rate in the white but not the African-American subjects. In 1929, Chen and Poth (24) measured the pupillary size after applying various mydriatic eye drops into the eyes of a number of people. The increase in size was largest in Caucasians, intermediate in Asians, and smallest in African-Americans; the authors thought that the color of the iris affected its movability. During World War II, American soldiers stationed in tropical countries received primaquine (25) as antimalarial prophylaxis; it turned out that only soldiers of African descent developed hemolysis from the administration of primaquine. The explanation came later (26): The affected soldiers had a genetic deficiency of glucose-6-phosphate dehydrogenase; this deficiency was frequent in Africans because it protected the carrier from malaria, but it was rare in countries without malaria. After discovery of the genetic deficiency of iso- niazid acetylation (5,27), other investigators found substantial interethnic dif- ferences in the frequency of this deficiency (28). The deficiency was rare in Eskimos (Inuit), relatively frequent in Europeans and Africans, and intermedi- ate in East Asians. In the early 1970s, my laboratory studied the metabolism of amobarbital (an at that time widely used drug) in a class of students (29). When we did not observe its normal metabolite in 7 of the 140 students, we assumed a labora- tory error. When calling the students back for reinvestigation, it turned out that all 7 were of Asian origin and that our first measurements had been correct. At the same time, our laboratory ran tests with debrisoquine (30) because a genetic variation of its metabolism had just been discovered (14). Again, we saw a sub- stantial difference of its metabolic destruction between students of Asian and non-Asian origin (the difference was later defined in terms of DNA variation by Swedish investigators [31]). These unexpected observations with amobar- bital and debrisoquine caused us to search the literature and to publish in 1982 the first article on interethnic differences in drug metabolism (32). In the mean time, studies of interethnic differences in drug response or metabo- lism have become frequent research projects. Computerized Pubmed lists more than 2000 articles dealing with the combined entries “drug” and “race.” It is now quite clear that the interethnic differences may be divided into two kinds: first, a given mutation of a particular gene may occur with different frequen- cies in different populations. Second, there are mutations that appear to be specific for a particular population. Some of the differences may be there because they provide a population with a biological advantage; however,
- 23. Pharmacogenomics: History and Overview 7 some mutations may simply differ because they have arisen in a population after it separated from others. This difference is suggested by some overview data (12). The occurrence of 11 mutations that affect the function of the P450 cytochrome CYP2D6 was tested by various investigators in populations from Europe, China, Japan, and Africa. Of these 11 mutations, Europeans carried 7, Chinese 4, Japanese 3, and Africans 2. Only one mutation (G4268C) was found in all countries, suggest- ing that it arose before humanity separated into different ethnicities. Inter-ethnic differences occur frequently. As stated previously (12), 42 drug- metabolizing enzymes have shown pharmacogenetic variability within one or other population. When checking the literature for the occurrence of intereth- nic differences between these variants, researchers discovered that 28 (66%) showed such differences. This percentage is considered high, particularly when noting the fact that interethnic comparisons had never been made for many drugs. In short, if we view a pharmacogenetic variation between people, it is likely absent in other populations or occurring with a different frequency. Is this a rule that holds for all mutations in any gene? 2.5. Stage 5: The Rise of Multifactorial Pharmacogenetics Differences between people in their response to drugs are regular occur- rences. This observation was formalized in 1927 by introduction of the term and the concept of ED50 (33); it indicates the dose of a drug sufficient to pro- duce a given effect in 50% of the members of a population. In other words, all drug effects are variable. This result is not surprising because there are numer- ous factors that can affect a drug response. As a simple example, let us consider the rate of metabolism of any particular drug. The metabolism may fail because of a genetic change of the enzyme structure. Perhaps the metabolism failed because not enough enzyme was formed, perhaps because of low gene expression or because of a failure of transcription or translation. Was there the absence of an inducing or regulating hormone, or was the enzyme degraded too quickly? Perhaps a genetic abnor- mality of the promoting region prevented the normal response to the inducer. Perhaps the drug could not reach the enzyme because it was bound somewhere else or a transporter was missing. Thus, even a single step in the drug’s fate may be complex and affected by many genes; the genes may interact, and envi- ronmental factors also may contribute to the variation. The causes of most differences generally remain uninvestigated, but the presence of both genetic and environmental causes is common (34). It is of considerable interest to know the relative contribution of the two causes. The classical method of investigation, used prominently, for example, by Vesell,
- 24. 8 Kalow consisted of twin studies (35): that is, the magnitude of differences between the two members of a pair of identical and a pair of fraternal was measured. Repeat- ing such studies in many twins and averaging and comparing the differences allowed a calculation of heritability. Unfortunately, the recruiting of a suffi- cient number of twins often is difficult. However, a simpler method is now available (36) that is based on the fact that one can give a drug repeatedly to a person and measure each response and the difference between the responses. When giving a drug at an appropriate interval two or more times to a group of people, one can measure two magni- tudes: first, one can calculate the average and standard deviation of the response differences between the first and subsequent applications in the same people; second, one can equally calculate and record the difference between subjects. Let us designate the standard deviations of the within-subject variations as SDw, of the between-subjects as SDb, and the genetic component of the between- subject variation as rGC. Squaring the standard deviations, the genetic compo- nent is then calculated by the following equation: rGC = (SDb 2 – SDw 2)/SDb 2 [37]). A value close to 1.0 indicates overwhelming heredity, close to 0 indicates mostly environmental influence. A recent example of the use of this method has been the assessment of genetic and environmental determinants of cyto- chrome CYP3A4 activity (38). Often forgotten is the fact that there may be a clinically significant drug response difference between two populations even if the average response dif- ferences are small, perhaps not even statistically significant (39). If population data are represented by a normal distribution (Gauss) curve, the persons with abnormal responses may be represented by one of the edges of the curve. For example, let us consider a normally distributed metabolic destruction rates of drug X. Assume that 2% of the persons have a destruction rate low enough to suffer toxicity from the drug. In another group of people, destruction of the same drug may have a somewhat lower average rate, a fact that is imma- terial for most subjects; however, if the distribution curves have equal spread in the two populations, many more people in the second population will have the critically low drug destruction rate and will be intoxicated than in the first population (Fig. 1). In short, the difference between the edges of the distribu- tion curves may be of clinical and statistical significance even if the averages are similar. 3. Pharmacogenomics As described previously, pharmacogenetics began with the study of single gene differences between individuals but developed into a broad science. Meth- odological advances expanded the science further into pharmacogenomics; one
- 25. Pharmacogenomics: History and Overview 9 may say that pharmacologists followed the geneticist’s adoption of genomic techniques. The consequence should be a better understanding of the multi- plicities and complexities of drug–gene interactions; not only may genes affect drug action, but drugs may affect gene function. Pharmacogenomics repre- sents attempts by researchers to medically use these new understandings together with the old ones. The hope is to optimize the efficacy of drugs, to minimize adverse drug reactions, and to facilitate drug discovery, develop- ment, and approval. 4. The Aims of Pharmacogenomics 4.1. Aim 1: Creation of a Basis for Personalized Medicine Current medicine is based on statistical likelihood and often fails the indi- vidual. The incidence of serious or fatal drug reactions depends in many or most cases on genetic variation. Studies from U.S. hospitals (40) suggested that 6.7% of patients had serious adverse and 0.32% had fatal, drug reactions. The latter caused approx 100,000 deaths per year in the United States. Many researchers hope that adverse reactions or therapeutic failures will be elimi- nated by the introduction of personalized medicine (41), meaning that the drug to be given to a patient will be determined by the patient’s genes. To reach this aim, we must learn much more about the genetic variants that may affect drug action. The most frequently occurring genetic variants are single-nucleotide polymorphisms (SNPs [42]). The human genome contains approx 3 billion basepairs, and SNPs occur on the average in approx 1 per 1000 bases; thus, they cause genetic variation of many human proteins. These variants are important objects of study because human individuals usually dif- fer from each other by less than 1% of their genes; thus, SNPs are important. Fig. 1. Variation of drug clearance in two populations. The figure illustrates a hypo- thetical case showing a 20-fold variation of drug clearance between the individuals within each population, and a twofold difference between the population means. Let us assume that a slow clearance will tend to produce overdose toxicity in affected subjects. Obviously, the proportion of such subjects would be much larger in popula- tion B than in A.
- 26. Other documents randomly have different content
- 27. Willie broke in to say that he and I were going to give some political dinners in London and would ask Parnell, though he was sure he would not come. The O'Gorman Mahon paid some idle compliment, but I was not interested particularly in their stories of Parnell, though I mentally decided that if I gave any dinners to the Irish Party for Willie I would make a point of getting Parnell. On the 26th of April the members of the Irish Party met in Dublin to elect a chairman, and the meeting was adjourned without coming to a decision, but in May Mr. Parnell was chosen as leader. Willie voted for him, with twenty-two others, and telegraphed to me to say that he had done so, but feared that Mr. Parnell might be too "advanced." The fact was that many people admired steady-going William Shaw, the then chairman, as being very "safe," and doubted whither their allegiance to Mr. Parnell would lead them. Years after, when their politics had diverged, Mr. Parnell said: "I was right when I said in '80, as Willie got up on that platform at Ennis, dressed to kill, that he was just the man we did not want in the Party." After the meeting of Parliament Willie was insistent that I should give some dinner parties in London, and, as his rooms were too small for this purpose, we arranged to have a couple of private rooms at Thomas's Hotel —my old haunt in Berkeley Square. There were no ladies' clubs in those days, but this hotel served me for many years as well as such a club could have done. We gave several dinners, and to each of them I asked Mr. Parnell. Among the first to come were Mr. Justin McCarthy (the elder), Colonel Colthurst, Richard Power, Colonel Nolan, and several others; but—in spite of his acceptance of the invitation—Mr. Parnell did not come. Someone alluded to the "vacant chair," and laughingly defied me to fill it; the rest of our guests took up the tale and vied with each other in tales of the inaccessibility of Parnell, of how he ignored even the invitations of the most important political hostesses in London, and of his dislike of all social intercourse—though he had mixed freely in society in America and Paris before he became a politician for the sake of the Irish poor. I then became determined that I would get Parnell to come, and said, amid laughter and
- 28. applause: "The uncrowned King of Ireland shall sit in that chair at the next dinner I give!" One bright sunny day when the House was sitting I drove, accompanied by my sister, Mrs. Steele (who had a house in Buckingham Gate), to the House of Commons and sent in a card asking Mr. Parnell to come out and speak to us in Palace Yard. He came out, a tall, gaunt figure, thin and deadly pale. He looked straight at me smiling, and his curiously burning eyes looked into mine with a wondering intentness that threw into my brain the sudden thought: "This man is wonderful—and different." I asked him why he had not answered my last invitation to dinner, and if nothing would induce him to come. He answered that he had not opened his letters for days, but if I would let him, he would come to dinner directly he returned from Paris, where he had to go for his sister's wedding. In leaning forward in the cab to say good-bye a rose I was wearing in my bodice fell out on to my skirt. He picked it up and, touching it lightly with his lips, placed it in his button-hole. This rose I found long years afterwards done up in an envelope, with my name and the date, among his most private papers, and when he died I laid it upon his heart. This is the first letter I had from Mr. Parnell:— LONDON, July 17, 1880. MY DEAR MRS. O'SHEA,—We have all been in such a "disturbed" condition lately that I have been quite unable to wander further from here than a radius of about one hundred paces allons. And this notwithstanding the powerful attractions which have been tending to seduce me from my duty towards my country in the direction of Thomas's Hotel.
- 29. I am going over to Paris on Monday evening or Tuesday morning to attend my sister's wedding, and on my return will write you again and ask for an opportunity of seeing you.—Yours very truly, CHAS. S. PARNELL. On his return from Paris Mr. Parnell wrote to me, and again we asked him to dinner, letting him name his own date. We thought he would like a quiet dinner, and invited only my sister, Mrs. Steele, my nephew, Sir Matthew Wood, Mr. Justin McCarthy, and a couple of others whose names I forget. On receiving his reply accepting the invitation for the following Friday, we engaged a box at the Gaiety Theatre—where Marion Hood was acting (for whom I had a great admiration)—as we thought it would be a relief to the "Leader" to get away from politics for once. On the day of the dinner I got this note:— HOUSE OF COMMONS, Friday. MY DEAR MRS. O'SHEA,—I dined with the Blakes on Wednesday, and by the time dinner was over it was too late to go to the meeting—the Post Office is all right here. I cannot imagine who originated the paragraph. I have certainly made no arrangements up to the present to go either to Ireland or America or announced any intention to anybody.—Yours, CHAS. S. PARNELL. He arrived late, but apologetic, and was looking painfully ill and white, the only life-light in his face being given by the fathomless eyes of rich brown, varying to the brilliance of flame. The depth of expression and sudden fire of his eyes held me to the day of his death. We had a pleasant dinner, talking of small nothings, and, avoiding the controversial subject of politics, Mr. Parnell directed most of his
- 30. conversation to my sister during dinner. She could talk brilliantly, and her quick, light handling of each subject as it came up kept him interested and amused. I was really anxious that he should have an agreeable evening, and my relief was great when he said that he was glad to go to the theatre with us, as the change of thought it gave was a good rest for him. On arrival at the theatre he and I seemed to fall naturally into our places in the dark corner of the box facing the stage and screened from the sight of the audience, while my sister and the others sat in front. After we had settled in our seats Mr. Parnell began to talk to me. I had a feeling of complete sympathy and companionship with him, as though I had always known this strange, unusual man with the thin face and pinched nostrils, who sat by my side staring with that curious intent gaze at the stage, and telling me in a low monotone of his American tour and of his broken health. Then, turning more to me, he paused; and, as the light from the stage caught his eyes, they seemed like sudden flames. I leaned a little towards him, still with that odd feeling of his having always been there by my side; and his eyes smiled into mine as he broke off his theme and began to tell me of how he had met once more in America a lady to whom he had been practically engaged some few years before. Her father would not dower her to go to Ireland, and Parnell would not think of giving up the Irish cause and settling in America. The engagement therefore hung fire; but on this last visit to America he had sought her out and found himself cold and disillusioned. She was a very pretty girl, he said, with golden hair, small features and blue eyes. One evening, on this last visit, he went to a ball with her, and, as she was going up the stairs, she pressed into his hand a paper on which was written the following verse: "Unless you can muse in a crowd all day On the absent face that fixed you, Unless you can dream that his faith is fast Through behoving and unbehoving,
- 31. Unless you can die when the dream is past, Oh, never call it loving." He asked me who had written the lines, and I answered that it sounded like one of the Brownings (it is E. B. Browning's), and he said simply: "Well, I could not do all that, so I went home." I suggested that perhaps the lady had suffered in his desertion, but he said that he had seen her, that same evening, suddenly much attracted by a young advocate named A——, who had just entered the room, and decided in his own mind that his vacillation had lost him the young lady. The strenuous work he had then put his whole heart into had driven out all traces of regret. After this dinner-party I met him frequently in the Ladies' Gallery of the House. I did not tell him when I was going; but, whenever I went, he came up for a few minutes; and, if the Wednesday sittings were not very important or required his presence, he would ask me to drive with him. We drove many miles this way in a hansom cab out into the country, to the river at Mortlake, or elsewhere. We chiefly discussed Willie's chances of being returned again for Clare, in case another election was sprung upon us. Both Willie and I were very anxious to secure Mr. Parnell's promise about this, as The O'Gorman Mahon was old, and we were desirous of making Willie's seat in Parliament secure. While he sat by my side in the meadows by the river he promised he would do his best to keep Willie in Parliament, and to secure County Clare for him should the occasion arise. Thus we would sit there through the summer afternoon, watching the gay traffic on the river, in talk, or in the silence of tried friendship, till the growing shadows warned us that it was time to drive back to London. Soon after my first meeting with Mr. Parnell, my sister, Mrs. Steele, invited Mr. Parnell, Mr. McCarthy and myself to luncheon. We had a very pleasant little party at her house. During lunch Mr. Parnell told us he was going to his place in Ireland for some shooting, and Mr. McCarthy and my sister chaffed him for leaving us for the lesser game of partridge shooting,
- 32. but he observed gravely, "I have the partridges there, and here I cannot always have your society." I had to leave early, as I was anxious to return to see my aunt; and Mr. Parnell said he would accompany me to the station. When we got to Charing Cross the train had already gone; and Mr. Parnell picked out a good horse from the cab rank, saying it would be much pleasanter to drive down on such a beautiful afternoon. We did so, but I would not let him stay, as I was not sure what state of confusion the house might be in, left in my absence in the possession of the children and governess. I told him I had to hurry over the park to my aunt, as really was the case, and he reluctantly returned to London. On the next Wednesday evening Mr. Parnell was to dine with me at Thomas's Hotel. He met me at Cannon Street Station as the train came in, and asked me to have some tea with him at the hotel there and go on to Thomas's together. We went to the Cannon Street Hotel dining-rooms, but on looking in he saw some of the Irish members there and said it would be more comfortable for us in his private sitting-room. I was under the impression that he lived at Keppel Street, but he told me he had just taken rooms in the Cannon Street Hotel. We had tea in his sitting-room, and he talked politics to me freely till I was interested and at ease, and then lapsed into one of those long silences of his that I was already beginning to know were dangerous in the complete sympathy they evoked between us. Presently I said, "Come! we shall be late!"; and he rose without a word and followed me downstairs. There were some members of his Party still standing about in the hall, but, as he always did afterwards when I was with him, he ignored them absolutely and handed me into a waiting cab. He and I dined at Thomas's Hotel that evening, and after dinner I returned home to Eltham. Mr. Parnell left for Ireland by the morning mail. From Dublin he wrote to me:— September 9, 1880. MY DEAR MRS. O'SHEA,—Just a line to say that I have arrived here, and go on to Avondale, Rathdrum, this evening, where I hope to
- 33. hear from you before very long. I may tell you also in confidence that I don't feel quite so content at the prospect of ten days' absence from London amongst the hills and valleys of Wicklow as I should have done some three months since. The cause is mysterious, but perhaps you will help me to find it, or her, on my return.—Yours always, CHAS. S. PARNELL. Then from his home:— AVONDALE, RATHDRUM, September 11, 1880. MY DEAR MRS. O'SHEA,—I take the opportunity which a few hours in Dublin gives me of letting you know that I am still in the land of the living, notwithstanding the real difficulty of either living or being, which every moment becomes more evident, in the absence of a certain kind and fair face. Probably you will not hear from me again for a few days, as I am going into the mountains for some shooting, removed from post offices and such like consolations for broken-hearted politicians, but if, as I hope, a letter from you should reach me even there, I shall try and send you an answer.—Yours very sincerely, CHAS. S. PARNELL. CHAPTER IX AT ELTHAM "But then—I supposed you to be but a fellow guest?" "Ah, no" he answered, he in that cold, unshaken voice, "I have but come home."—(THE BAGMAN) HONORA SHEE.
- 34. Whenever I went to town, or elsewhere, I always returned at night to see that my children were all right and to be ready to go to my aunt as usual every morning. One day, on my return from a drive with my aunt, I found that my old nurse Lucy, who still lived with me, was very ill, having had a stroke of paralysis while I was away. She lingered only a couple of days before she died and left a great void in my heart. My children missed their admiring old confidante sadly. She had always been devoted to me as the youngest of her "own babies," as she called my mother's children, and had shared in all my fortunes and misfortunes since I returned from Spain. She was always very proud, and so fearful of becoming a burden to anyone, that she rented a room in her sister's house so that she should feel independent. So often, when "times were bad" with us, she would press some of her savings into my hand and say that "The Captain must want a little change, Dearie, going about as he does!" In her earlier life she had had her romance, and had spent some years in saving up to marry her "sweetheart," as she called him; but shortly before the wedding her father's business failed, and she immediately gave him all her little nest-egg, with the result that her lover refused to marry her. So then, at the great age of ninety, after her blameless life had been passed since the age of sixteen in unselfish devotion to us all, we laid her to rest by the side of my father and mother at Cressing, Willie taking her down to Essex and attending the funeral. As she lay dying I got this note from Mr. Parnell:— DUBLIN, September 22, 1880. MY DEAR MRS. O'SHEA,—I cannot keep myself away from you any longer, so shall leave to-night for London. Please wire me to 16, Keppel Street, Russell Square, if I may hope to see you to-morrow and where, after 4 p.m.—Yours always, C. S. P. Owing to the piteous clinging to my fingers of my old Lucy I was unable to go to London even for an hour to meet Mr. Parnell, so I telegraphed to
- 35. that effect, and received the following letter:— EUSTON STATION, Friday evening, September 24, 1880. MY DEAR MRS. O'SHEA,—On arriving at Keppel Street yesterday I found that your wire had just arrived, and that the boy refused to leave it as I was not stopping there. Going at once to the district postal office I asked for and received the wire, and to-day went to London Bridge Station at 12.15. The train from Eltham had just left, so I came on to Charing Cross and sent a note by messenger to you at Thomas's, with directions to bring it back if you were not there, which turned out to be the case. I am very much troubled at not having seen you, especially as I must return to Ireland to-night—I came on purpose for you, and had no other business. I think it possible, on reflection, that the telegraph people may have wired you that they were unable to deliver your message, and, if so, must reproach myself for not having written you last night.—Your very disappointed C. S. P. From Dublin he wrote me: Saturday morning, September 25, 1880. MY DEAR MRS. O'SHEA,—In my hurried note to you last night I had not time to sympathize with you in this troublesome time you have been going through recently; how I wish it might have been possible for me to have seen you even for a few minutes to tell you how very much I feel any trouble which comes to you. I am just starting for New Ross, where there is a meeting to-morrow. If you can spare time to write me to Avondale, the letters will reach me in due course.—Yours always, C. S. P. September 29, 1880.
- 36. MY DEAR MRS. O'SHEA,—I have received your wire, but not the letter which you say you were writing me to Dublin for Monday. I suppose then you may have sent it to Rathdrum instead, whither I am going this evening, and that I may soon have the happiness of reading a few words written by you. I am due at Cork on Sunday, after which I propose to visit London again, and renew my attempt to gain a glimpse of you. Shall probably arrive there on Tuesday if I hear from you in the meanwhile that you will see me. On Friday evening I shall be at Morrison's on my way to Kilkenny for Saturday, and shall be intensely delighted to have a wire from you to meet me there.—Yours always, CHAS. S. PARNELL. Meanwhile Willie was in communication with Mr. Gladstone, Mr. Tintern (one of the Liberal agents) and others, in reference to a meeting held by him. Mr. Tintern wrote from Tenby commenting with satisfaction on the report of Willie's successful meeting, on Willie's kind mention of the Government, and on the good the meeting must do by promoting orderly progress and better feeling between one class and another. But he expressed surprise that Willie should think the Government had not treated him and West Clare well. He at least...! Mr. Gladstone wrote from Downing Street on the 21st September about the meeting in much the same terms. He expressed himself as gratified to think that the important local proceedings with regard to the land question showed the union of people and pastors against the extremists. Life at Eltham went on in the same routine. My aunt was well, and would sit for long hours at the south door of her house—looking away up "King John's Chase"—the ruins of King John's Palace were at Eltham, and my aunt's park and grounds were part of the ancient Royal demesne. In these summer evenings she loved to sit at the top of the broad flight of shallow steps with me, and tell my little girls stories of her life of long ago.
- 37. Sometimes her favourite Dr. Bader would bring his zither down from London and play to us; or my aunt and I would sit in the great tapestry room with all of the seven windows open, listening to the song of the æolian harp as the soft breeze touched its strings and died away in harmony through the evening stillness. Sometimes, too, she would sing in her soft, gentle old voice the songs of her youth, to the accompaniment of her guitar. "We met, 'twas in a crowd," was a favourite old song of hers, half forgotten since she used to sing it to the music of her spinet seventy years before, but Dr. Bader found the words in an old book, and the dear old lady crooned it sentimentally to me as we sat waiting for the hooting of the owls which signalled to her maid the time for shutting her lady's windows. And I was conscious of sudden gusts of unrest and revolt against these leisured, peaceful days where the chiming of the great clock in the hall was the only indication of the flight of time, and the outside world of another age called to me with the manifold interests into which I had been so suddenly plunged with the power to help in the making and marring of a destiny. In the autumn of 1880 Mr. Parnell came to stay with us at Eltham, only going to Dublin as occasion required. Willie had invited him to come, and I got in some flowers in pots and palms to make my drawing-room look pretty for him. Mr. Parnell, who was in very bad health at that time, a few days later complained of sore throat, and looked, as I thought, mournfully at my indoor garden, which I industriously watered every day. It then dawned upon me that he was accusing this of giving him sore throat, and I taxed him with it. He evidently feared to vex me, but admitted that he did think it was so, and "wouldn't it do if they were not watered so often?" He was childishly touched when I at once had them all removed, and he sank happily on to the sofa, saying that "plants were such damp things!" His throat became no better, and he looked so terribly ill when—as he often did now—he fell asleep from sheer weakness on the sofa before the fire, that I became very uneasy about him. Once, on awaking from one of
- 38. these sleeps of exhaustion, he told me abruptly that he believed it was the green in the carpet that gave him sore throat. There and then we cut a bit out, and sent it to London to be analysed, but without result. It was quite a harmless carpet. During this time I nursed him assiduously, making him take nourishment at regular intervals, seeing that these day-sleeps of his were not disturbed, and forcing him to take fresh air in long drives through the country around us. At length I had the satisfaction of seeing his strength gradually return sufficiently to enable him to take the exercise that finished the process of this building-up, and he became stronger than he had been for some years. I do not think anyone but we who saw him then at Eltham, without the mask of reserve he always presented to the outside world, had any idea of how near death's door his exertions on behalf of the famine-stricken peasants of Ireland had brought him. Once in that autumn, after he came to us, I took him for a long drive in an open carriage through the hop-growing district of Kent. I had not thought of the fact that hundreds of the poorest of the Irish came over for the hop- picking, and might recognize him. After driving over Chislehurst Common and round by the lovely Grays, we came right into a crowd of the Irish "hoppers"—men, women, and children. In a moment there was a wild surge towards the carriage, with cries of "The Chief! The Chief!" and "Parnell! Parnell! Parnell!" The coachman jerked the horses on to their haunches for fear of knocking down the enthusiastic men and women who were crowding up—trying to kiss Parnell's hand, and calling for "a few words." He lifted his cap with that grave, aloof smile of his, and said no, he was not well enough to make the smallest of speeches, but he was glad to see them, and would talk to them when they went home to Ireland. Then, bidding them to "mind the little ones," who were scrambling about the horses' legs, to the manifest anxiety of the coachman, he waved them away, and we drove off amid fervent "God keep your honours!" and cheers. These Irish hop-pickers were so inured to privation in their own country that they were very popular among the Kentish hop-farmers, as they did not
- 39. grumble so much as did the English pickers at the scandalously inefficient accommodation provided for them. Often before Parnell became really strong I used to watch for hours beside him as he slept before the drawing-room fire, till I had to rouse him in time to go to the House. Once, when he was moving restlessly, I heard him murmur in his sleep, as I pulled the light rug better over him: "Steer carefully out of the harbour—there are breakers ahead." He now had all the parcels and letters he received sent on to me, so that I might open them and give him only those it was necessary for him to deal with. There were hundreds of letters to go through every week, though, as he calmly explained, "If you get tired with them, leave them and they'll answer themselves." Often among the parcels there were comestibles, and among these every week came a box of eggs without the name and address of the sender. I was glad to see these eggs as the winter came on and with it the usual reluctance of our hens to provide us with sufficient eggs, but Mr. Parnell would not allow me to use them, for he said: "They might be eggs, but then again they might not," and I had to send them a good distance down the garden and have them broken to make sure of their genuineness, and then he would worry lest our dogs should find them and poison themselves. On his visits to Ireland he wrote to me continually:— DUBLIN, Tuesday. MY DEAR MRS. O'SHEA,—I have just a moment on my return from Ennis to catch the late post and reply to your wire. I received your two letters quite safely, and you may write me even nicer ones with perfect confidence. I blame myself very much for not having written you on my way through Dublin on Saturday, as you were evidently anxious about your notes, but I hope you will forgive me as there were only a few minutes to spare. I trust to see you in London on Tuesday next. Is it true that Captain O'Shea is in Paris, and, if so, when do you expect his return? ... I have
- 40. had no shooting, weather too wet, but shall try to-morrow, when you may expect some heather. DUBLIN, Friday evening, October 2, 1880. Have just received your wire; somehow or other something from you seems a necessary part of my daily existence, and if I have to go a day or two without even a telegram it seems dreadful. I want to know how you intend to excuse yourself for telling me not to come on purpose if I must return. (To Ireland.) Of course, I am going on purpose to see you; and it is also unhappily true that I cannot remain long. Shall cross Monday evening, and shall call at Morrison's for a message. Please write or wire me in London to 16 Keppel Street, Russell Square, where I shall call on Tuesday. DUBLIN, Monday night, October 4, 1880. Just arrived.... I write you on the only bit of paper to be found at this late hour (a scrap taken from one of your own notes), to say that I hope to reach London to-morrow (Tuesday) evening and to see you on Wednesday when and where you wish. Please write or wire me to Keppel Street. This envelope will present the appearance of having been tampered with, but it has not. DUBLIN, Tuesday evening, October 5, 1880. A frightful gale has been blowing all day in Channel and still continues.
- 41. Under these circumstances shall postpone crossing till to-morrow evening. Can meet you in London at 9 to-morrow evening anywhere you say. DUBLIN, Monday evening, October 17, 1880. MY OWN LOVE,—You cannot imagine how much you have occupied my thoughts all day and how very greatly the prospect of seeing you again very soon comforts me. On Monday evening I think it will be necessary for me to go to Avondale; afterwards I trust, if things are propitious on your side, to return to London on Tuesday or Wednesday.—Yours always, C. AVONDALE, RATHDRUM, October 22, 1880. I was very much pleased to receive your wire this morning, forwarded from Dublin, that you had received my note of last Saturday. I was beginning to fear that it had gone wrong. After I had finished at Roscommon and received your message in Dublin on Monday I decided upon coming here where I have been unexpectedly detained. If all goes well you will see me in London on Monday evening next.... I send you enclosed one or two poor sprigs of heather, which I plucked for you three weeks ago, also my best love, and hope you will believe that I always think of you as the one dear object whose presence has ever been a great happiness to me. Meanwhile the Government had been temporizing with the land question. They had brought in a very feeble Compensation for Disturbances Bill and they had allowed it to be further weakened by amendments. This Bill was rejected by the House of Lords, with the result that the number of
- 42. evictions in Ireland grew hourly greater and the agitation of the Land League against them; outrages, too, were of common occurrence and increased in intensity. Speaking at Ennis on September 19th Mr. Parnell enunciated the principle which has since gone by the name of "The Boycott." "What are you to do," he asked, "to a tenant who bids for a farm from which another tenant has been evicted?" Several voices cried: "Shoot him!" "I think," went on Mr. Parnell, "I heard somebody say 'Shoot him!' I wish to point out to you a very much better way—a more Christian and charitable way, which will give the lost man an opportunity of repenting. When a man takes a farm from which another has been unjustly evicted, you must shun him on the roadside when you meet him; you must shun him in the shop; you must shun him on the fair-green and in the market-place, and even in the place of worship, by leaving him alone; by putting him into a sort of moral Coventry; by isolating him from the rest of the country, as if he were a leper of old—you must show him your detestation of the crime he has committed." Forster, the Irish Secretary, who had some amount of sympathy for the tenants, was, however, a Quaker, and the outrages horrified him more than the evictions. Nor, strangely, was he able to connect the one with the other. Undoubtedly the evictions almost ceased, but, said he, they have ceased because of the outrages, and the outrages were the work of the Land League; and he pressed for the arrest of its leaders. This was unwise, considering that it was Parnell who had advocated the abandonment of violence for the moral suasion of the boycott. On November 3rd Forster decided to prosecute the leaders of the Land League, and among them Parnell, Dillon, Biggar, Sexton and T. D. Sullivan. Two days later, in a speech at Dublin, Parnell expressed his regret that Forster was degenerating from a statesman to a tool of the landlords. Biggar when he heard the news exclaimed, "Damned lawyers, sir, damned lawyers!
- 43. Wasting the public money! Wasting the public money! Whigs damned rogues! Forster damned fool!" DUBLIN,[1] November 4, 1880. MY DEAR MRS. O'SHEA,—I take advantage of almost the first moment I have had to myself since leaving you to write a few hasty lines. And first I must again thank you for all your kindness, which made my stay at Eltham so happy and pleasant. The thunderbolt, as you will have seen, has at last fallen, and we are in the midst of loyal preparations of a most appalling character. I do not suppose I shall have an opportunity of being in London again before next Thursday, but trust to be more fortunate in seeing Captain O'Shea then than the last time.—Yours very truly, CHAS. S. PARNELL. DUBLIN,[1] Saturday. MY DEAR MRS. O'SHEA,—I hope to arrive in London on Tuesday morning, and trust to have the pleasure of seeing you before I leave. Do you think you shall be in town on Tuesday? Kindly address 16, Keppel Street.—Yours very truly, CHAS. S. PARNELL. On November 5th that year the village was great on the subject of "gunpowder, treason, and plot," and during dinner that evening there was such a noise and shouting outside my house that I asked the maid who was waiting what all the excitement was about. She answered breathlessly that "the procession, ma'am, have got Miss Anna Parnell in a effigy 'longside of the Pope, and was waiting outside for us to see before they burnt 'em in the village."
- 44. This electrifying intelligence was received with grave indifference by Mr. Parnell till the disappointed maid left the room; then with a sudden bubble of laughter—"Poor Anna! Her pride in being burnt, as a menace to England, would be so drowned in horror at her company that it would put the fire out!" The cheering and hooting went on for some time outside the house, but, finding we were not to be drawn, the crowd at last escorted the effigies down to the village and burnt them, though with less amusement than they had anticipated. DUBLIN,[2] November 6, 1880. MY DEAR MRS. O'SHEA,—You can have very little idea how dreadfully disappointed I felt on arriving here this evening not to find a letter from either you or Captain O'Shea. I send this in hope that it may induce you to write in reply to my last letter and telegram, which would appear not to have reached you.—Yours very sincerely, CHAS. S. PARNELL. AVONDALE, Monday. MY DEAR MRS. O'SHEA,—I enclose keys, which I took away by mistake. Will you kindly hand enclosed letter to the proper person[3] and oblige,—Yours very truly, CHAS. S. PARNELL. DUBLIN, Wednesday night, November 11, 1880. MY DEAREST LOVE,—I have made all arrangements to be in London on Saturday morning, and shall call at Keppel Street for a letter from you. It is quite impossible for me to tell you just how very much you have changed my life, what a small interest I take in what is going on about me, and how I detest everything which has happened during the last few days to keep me away from you—I think of you always, and you must never believe there is to be any "fading." By the way, you must
- 45. not send me any more artificial letters. I want as much of your own self as you can transfer into written words, or else none at all.—Your always, C. S. P. A telegram goes to you, and one to W.,[4] to-morrow, which are by no means strictly accurate. DUBLIN, December 2, 1880. MY DEAR MRS. O'SHEA,—I succeeded in getting the train at Euston with just ten minutes to spare, and, arriving here this morning, found that my presence to-day was indispensable. I need not tell you how much I regretted leaving Eltham so suddenly; but we cannot always do as we wish in this world. My stay with you has been so pleasant and charming that I was almost beginning to forget my other duties; but Ireland seems to have gotten on very well without me in the interval. Trusting to see you again next week on my way to Paris.—Yours very sincerely, CHAS. S. PARNELL. I have been exceedingly anxious all day at not receiving your promised telegram to hear how you got home. [1] These letters were really written from London. [2] Sent to Dublin to be posted. [3] Myself. [4] Captain O'Shea.
- 46. CHAPTER X THE LAND LEAGUE TRIALS "The surest way to prevent seditions is to take away the matter of them."—LORD BACON. Through the whole of 1880 Parnell was determinedly organizing the Land League throughout Ireland, and during the winter, doubtless encouraged by the enormous distress that prevailed over the whole country, the force and power of the League grew with a rapidity that surpassed even the expectations of Parnell and his party. All through the vacation Parnell and his followers held meetings in carefully calculated areas of Ireland, and in his speeches Parnell explained the meaning and wide-reaching scope of the League's agitation, i.e. that tenant farmers were to trust in their own combination alone and "should give no faith to the promises of the English Ministers." During the early session that year Parnell had introduced a Bill called "Suspension of Ejectments Bill," and this first pressed upon the House the necessity of dealing with the Irish landlord troubles. Parnell's party urged this Bill with so united a front that Mr. Gladstone was obliged to consider the main substance of it, and he agreed to insert a clause in the "Relief of Distress Bill" which would deal with impending evictions of Irish tenants. But the Speaker of the House held that the interpolation of such a clause would not be "in order," and the Chief Secretary for Ireland (Mr. Forster) then, by Mr. Gladstone's direction, brought in his "Disturbances Bill," which was to all practical purposes Parnell's Bill under another name. In the course of the debate on this Bill Mr. Gladstone himself said that "in the circumstances of distress prevalent in Ireland (at that time) a sentence of eviction is the equivalent of a sentence of death." These absolutely true words of Gladstone's were used by Parnell very many times during his Land League tours both in speeches and privately, and many times he added—as so often he did to me at home—bitter comment upon the apathy of the English Government, upon the curious insensibility of the
- 47. English law-makers, who knew these things to be true in Ireland and yet were content to go on in their policy of drift, unless forced into action by those who saw the appalling reality of the distress among the Irish poor that was so comfortably deplored in London. In this connexion Parnell used to say that the fundamental failure in the English government of Ireland was: First, the complete inability of the Ministers in power to realize anything that was not before their eyes; and, secondly, their cast-iron conviction that Ireland was the one country of the world that was to be understood and governed by those to whom she was little but a name. In all this time of trouble and eviction Parnell went backwards and forwards between England (Eltham) and Ireland as occasion required, and so successful were his efforts in spreading the agitation and linking up the League that the Government became uneasy as to the outcome of this new menace to landlordism. Finally Parnell and fourteen of his followers were put on trial, charged with "conspiracy to impoverish landlords." Parnell, of course, went over to Ireland for these "State trials," but he considered the whole thing such a farce, in that it was an impotent effort of the Government to intimidate him, that he could not take it seriously in any way. No jury (in Ireland) would agree to convict him he was well aware, and he attended the trials chiefly, he said, for the "look of the thing," and to give the support of his presence to his colleagues. Incidentally he told me on one occasion that he had considerably hurried the jury when he was very anxious to catch a train in time for the night mail to England (Eltham) by "willing" them to agree (to disagree) without the long discussion of local politics with which all self-respecting Irish jurors beguile the weary ways of law. He observed that here, in the question of how far an unconscious agent can be "willed" into a desired action, he had discovered another and most entrancing study for us when we had more time to go into it thoroughly. Talking of the Land League's procedure against the interests of the Irish landlords, I may, I think, here pertinently remind those who have, among so many other accusations, brought against Parnell the charge of self-seeking in regard to money matters, that Parnell himself was an Irish landlord and of very considerable estates, and that this land campaign (really, of course,
- 48. directed against eviction), meant, to all practical purposes, the loss of his rents, and that not only for a time, as in other cases, but, with the very generous interpretation put upon his wishes by the "Chief's" tenants, for all time—or rather for all his lifetime. Captain O'Shea also had certain estates in Ireland, and naturally, not being in sympathy with Parnell's policy, but being at heart a thorough Whig and a strong advocate for Mr. Shaw, the ex- leader of the Irish party, he was furious at the League's anti-landlord work, and refused to have any hand in it. He considered that hapless as was the plight of those who had to pay in rent the money they did not possess, that of the landlord whose rent was his all was but little to be preferred. During this period the stories of the evictions brought home to me by Parnell himself made my heart sick, and often he sat far into the night at Eltham speaking in that low, broken monotone, that with him always betokened intense feeling strongly held in check, of the terrible cruelty of some of the things done in the name of justice in unhappy Ireland. How old people, and sometimes those sick beyond recovery, women with the children they had borne but a few hours before, little children naked as they had come into the world, all thrust out from the little squalid cabins which were all they had for home, thrust out on the roadside to perish, or to live as they could. I in my English ignorance used to say: "Why did they not go into the workhouse or to neighbours?" and Parnell would look wonderingly at me as he told me that for the most part such places were few and far between in Ireland, and "neighbours," good as they were to each other, were in the same trouble. There were instances where a wife would beg, and with none effect, that the bailiffs and police should wait but the little half-hour that her dying husband drew his last breath; and where a husband carried his wife from her bed to the "shelter" of the rainswept moor that their child might be born out of the sight of the soldiers deputed to guard the officials who had been sent to pull their home about their ears. And, remembering these and so many other tales of some of the 50,000 evictions that he afterwards calculated had taken place in Ireland, I have never wondered at the implacable hatred of England that can never really die out of the Irish heart. On December 4th, 1880, he wrote to me from Dublin:
- 49. I was exceedingly pleased to receive your letters; to say the truth, I have been quite homesick since leaving Eltham, and news from you seems like news from home. The Court refused our application to-day for a postponement of the trial (of the Land League), but this we expected, and it does not much signify, as it turns out that we need not necessarily attend the trial unless absolutely directed to do so by the Court. You will also be pleased to hear that the special jury panel, of which we obtained a copy last night, is of such a character as in the opinion of competent judges to give us every chance of a disagreement by the jury in their verdict, but we cannot, of course, form an absolute conclusion until the jury has been sworn, when we shall be able to tell pretty certainly one way or the other. Since writing Captain O'Shea it does not look as if I could get further away from Ireland than London, as Paris is inconvenient from its distance. I have no letter from him yet in reply to mine. And again on the 9th:— I returned from Waterford last night, and shall probably get through all necessary work here by Saturday evening so as to enable me to start for London on Sunday morning. I do not know how long I can remain in London, but shall run down and see you on Monday, and perhaps my plans will be more fixed by that time. I have decided not to attend any more meetings until after the opening of Parliament, as everything now can go on without me. Kindly inform Captain O'Shea that the meeting of Irish members will be in Dublin on the 4th January. On December 12th of that year Mr. Parnell wrote from Avondale to say that the jury panel was to be struck on the following Monday for the prosecution of the Land League.
- 50. ... And it will be necessary for me to see it before giving final directions. I have consequently postponed my departure till Monday evening. I have come here to arrange my papers and find a number which I should not like to destroy, and which I should not like the Government to get hold of in the event of their searching my house in the troublous times which appear before us. May I leave them at Eltham? And the next day:— I have just received a note from Healy, who is to be tried at Cork on Thursday, saying that his counsel thinks it of the utmost importance I should be present. This is very hard lines on me, as I had looked forward to a little rest in London before my own trial commences; but I do not see how it can be helped, as Healy's is the first of the State trials, and it is of the utmost importance to secure an acquittal and not merely a disagreement. I shall leave Cork on Thursday night and arrive in London Friday evening, and shall call to see you at Eltham Saturday. Your letters, one directed here and the others to Morrison's, reached me in due course, and I hope to hear from you again very soon. Parnell, now, always made my house his headquarters in England, and on his return from Ireland after the trials came down at once as soon as he had ascertained that I was alone. There were times when he wished to keep quiet and let no one know where he was; and, as it became known to the Government that Mr. Parnell frequented my house a good deal, it was somewhat difficult to avoid the detectives who were employed to watch his comings and goings. On one occasion in 1880 he was informed privately that his arrest for "sedition" was being urged upon the Government, and that it would be well to go abroad for a short time. I think his enigmatic reply, "I will disappear
- 51. for a few weeks," must have puzzled his informant. He came down to me at night, and when I answered his signal at my sitting-room window, and let him in, he told me with a deprecating smile that I must hide him for a few weeks. As I sat watching him eat the supper I always had ready for him at 3 a.m. I felt rather hopeless, as he was a big man, and I did not see how he could be hidden from the servants. He said the latter must not know he was there, as they would talk to the tradespeople, and they to the Government men. He did not wish to be arrested until later on, when it might be more useful than not. Then he awaited suggestions, and at length we decided that a little room opening out of my own must be utilized for him, as I always kept it locked and never allowed a servant into it—except very occasionally to "turn it out." It was a little boudoir dressing-room, and had a sofa in it. Mr. Parnell was then still feeling ill and run down, and enjoyed his fortnight's absolute rest in this room. None of the servants knew that he was there, and I took all his food up at night, cooking little dainty dishes for him at the open fire, much to his pleasure and amusement. He spent the time very happily, resting, writing "seditious" speeches for future use, and reading "Alice in Wonderland." This book was a favourite of his, and I gave it to him with the solemnity that befitted his grave reading of it. I do not think he ever thought it in the least amusing, but he would read it earnestly from cover to cover, and, without a smile, remark that it was a "curious book." In all this fortnight no one had the least idea that he was in the house, and the only comment I ever heard upon my prisoner's diet was that "the mistress ate much more when she had her meals served in her sitting- room." At the end of this fortnight he had arranged to go to Paris on some Land League business, and wanted me to go to see him off. He had brought certain political correspondence from Avondale and London and placed it in my charge, and this I kept in a box in this little private room, where I hid them. But there were two papers that he did not wish left even here, and, fearing arrest, could not carry on him. For these he had a wide, hollow gold bracelet made in Paris, and after inserting the papers he screwed the
- 52. bracelet safely on my arm; there it remained for three years, and was then unscrewed by him and the contents destroyed. The winter of 1880 was terribly cold, and as I let him out of the house in the bitterly cold morning I wished he did not consider it necessary to go to Paris by such a roundabout route as he had chosen. However, we drove off to Lewisham that morning, quite unobserved; from thence we went by train to New Cross, and drove by cab to London Bridge. At Vauxhall we started for Lowestoft; for Mr. Parnell had arranged to go to Paris via Harwich. I was anxious about him, for the cold was intense, and the deep snow over the large dreary waste of salt marshes seemed reflected in his pallor. Our train slowly passed through the dreary tract of country, feet deep in its white covering, and we could see no sign of life but an occasional seagull vainly seeking for food, and sending a weird call through the lonely silences. I wrapped Parnell up in his rugs as he tried to sleep. I loathed the great white expanse that made him look so ill, and I wished I had him at home again, where I could better fight the great fear that so often beset my heart: that I could not long keep off the death that hovered near him. A lady and gentleman in the carriage remarked to me—thinking he slept—that my husband looked terribly ill, could they do anything? And I noticed the little smile of content that flitted over his face as he heard me briskly reply that, No, he had been ill, but was so much better and stronger that I was not at all uneasy. It was the cold glare of the snow that made him look so delicate, but he was really quite strong. He hated to be thought ill, and did not see the doubt in their faces at my reply. Arrived at Lowestoft I insisted upon his resting and having a good meal, after which he felt so cheered up that he decided to return to London with me, and go to Paris by the usual route the next day! We had a new Irish cook at this time, from County Tipperary, and her joy exceeded all bounds when she learnt that the Irish leader was really in the house and she was to cook for him. I had to ask Mr. Parnell to see her for a moment, as she was too excited to settle to her cooking. Directly she got into the room Ellen fell down on her knees and kissed his hands, much to
- 53. his horror, for, although used to such homage in Ireland, he disliked it extremely, and he told me with some reproach that he had expected to be quite free from that sort of thing in my house. At Christmas he tipped my servants generously, and indeed Ellen and the parlourmaid Mary vied with each other in their attention to his comfort. The enthusiasm of the cook was so great that she bought an enormous gold locket, and, having inserted a portrait of Mr. Parnell in it, wore it constantly. Mary, not to be outdone, thereupon bought a locket of identically the same design, and wore it with an air of defiance, when bringing in tea, on New Year's Day. This was against all regulations, and I said laughingly to Mr. Parnell that he was introducing lawlessness into my household. He answered, "Leave it to me," and when Mary appeared again he said gently to her, "Mary, that is a magnificent locket, and I see you are kind enough to wear my portrait in it. Mrs. O'Shea tells me that Ellen has bought one also, but I just want you and Ellen not to wear them outside like that, for Mrs. O'Shea lets me come down here for a rest, and if people know I'm here I shall be worried to death with politics and people calling." So Mary promised faithfully, and Ellen came running in to promise too, and to threaten vengeance on "the others" if absolute silence was not observed. The lockets went "inside," and only a tiny bit of chain was allowed to show at the throat in evidence of homage continued, though hidden. Meanwhile, events were fusing in Ireland. Parnell had gone over there immediately after Christmas. From Dublin he wrote:— DUBLIN, Monday evening, December 27, 1880. MY DEAR MRS. O'SHEA,—I have been exceedingly anxious all day at not receiving your promised telegram to hear how you got home; trust I may have something to-morrow morning that it is all right.[1]— Yours in haste, C. S. P.
- 54. MORRISON'S HOTEL, Tuesday, December 28, 1880. MY DEAREST WIFE,—You will be delighted to learn that everything is proceeding first-rate so far. The jury sworn to-day cannot possibly convict us, and there is a very fair chance of an acquittal. I do not think the Government will attempt to prevent me from being present at the opening of Parliament, though I am not quite sure yet whether it will be prudent for me to leave until Wednesday evening. So far as I can see there is no necessity for the presence of any of the Traversers; one of them, Gordon, who has broken his leg, has not appeared at all, and his absence has not been even mentioned or noticed. I was immensely relieved by your letter this morning. You must take great care of yourself for my sake and your and my future.—Yours always, C. S. P. I have wired and written to Madrid[2] explaining situation lest my observations at yesterday's meeting as to doubt of my being in Parliament, intended to throw dust in eyes of Government, might be literally interpreted. DUBLIN, Thursday, December 30, 1880. MY DEAREST LOVE,—Your letters have reached me quite safely, and you cannot tell how much pleasure they give me. I fear I was very foolish to allow you to come with me the day of my departure; I felt sure it would do much harm, and until your first letter arrived I was in a continual panic lest some dreadful disaster had happened. That my poor love should have suffered so much makes my heart very sore, and she must take great care of herself for the sake of our future.... I enclose letter from W.S.[3]—Yours always affectionately, C. S. P. Will send you photo to-morrow.
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