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From Genetic Study to Clinical TrialsFrom Genetic Study to Clinical Trials

Chao Agnes Hsiung, Ph.D.

Division of Biostatistics and BioinformaticsNational Health Research Institutes

Taiwan

Chao Agnes Hsiung, Ph.DChao Agnes Hsiung, Ph.D..

Division of Biostatistics and BioinformaticsNational Health Research Institutes

Taiwan

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Pharmacogenetics/ Pharmacogenomics

Recent advances in molecular biology and pharmacogenetics have contributed substantially to understanding of the genetic control of the metabolism as well as the effect of drugs.As an example, Asian patients, in general, respond to lower doses of antidepressants than those recommended based on studies involving Caucasian subjects.One of the mechanism responsible for such differential treatment responses and side effect profiles lies in genetic differences that alter the activity of CYP450 that metabolize antidepressants.

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Pharmacogenetics/ Pharmacogenomics

Research data suggest that up to 70% Asian posses CYP2D6 and CYP2C19 unique alleles that slow down these enzyme activity (Lin et. Al. 1996,1994 ect).As another example, most patients with non-small-cell lung cancer (NSCLC) have no response to the tyrosine kinase inhibitor gefitinib (Iressa), which targets the epidermal growth factor receptor (EGFR). However, about 10% of patients have a rapid and dramatic clinical response.A subgroups of patients with non-small-cell lung caner have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the Iressa.

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Pharmacogenetics/ Pharmacogenomics

Screening for such mutations in lung cancers may identify patients who will have a response to Iressa. (NEJM 2004)Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan than in U.S..Somatic mutations of the EGFR were found in 15/58 unselected tumors from Japan and 1/61 for the US. (Science 2004)EGFR mutations in lung cancer: correlate with clinical response to Gefitinib therapy With progress in pharmacogenetics, some tools enable us to systematically examine factors that determine inter-individual as well as cross-ethnic variations in drug response

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Design of Future Clinical Trials

Before a sensible clinical trial based on pharmacogenomics studies is designed, genetic studies would be of some help.

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An International Collaborative Genetic Study

SAPPHIReNational Health Research Institutes (NHRI) and fourmedical centers (National Taiwan University Hospital, Veteran General Hospital- Taipei, VGH-Taichung and Tri-Service General Hospital) in Taiwan havecollaborated with Stanford University, Pacific HealthResearch Institutes at Hawaii on the study SAPPHIRe.

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遺傳研究遺傳研究 -- SAPPHIReSAPPHIRe

SStanford

AAsian

PPacific

PProgram in

HHypertension and

IInsulin

ReResistance

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Objectives

To map and identify major genetic loci underlying hypertension in Chinese and JapaneseTo study interactions between genetic and non-genetic determinants of hypertension in defined populations

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A Gene Mapping Study for Hypertension

Hypertension is known to be a complex

disease with genes and environmental

factors interacting to control risk of this

disease

We have built up an infrastructure to

facilitate the gene-mapping study

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Infrastructure of SAPPHIReInfrastructure of SAPPHIRe

SAPPHIReSAPPHIRe

Administrative Core

Administrative Core

DataCoordinating

Center

DataCoordinating

Center

Laboratory Center

Laboratory Center

GeneticsCenter

GeneticsCenter

ClinicalField

Center

ClinicalField

Center

StanfordCenter

StanfordCenter

TaiwanCenter

TaiwanCenter

TaiwanCenter

TaiwanCenter

HonoluluCenter

HonoluluCenter

San FranciscoBay Area CenterSan Francisco

Bay Area Center

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Study Population

Hawaii, San Francisco Bay Area, Taiwan

1318 sib pairs who are Japanese or Chinese, 35 to 60 years old and who both have hypertension or one is hypertensive and one is hypotensive

Pedigrees with hypertension

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Measured Variables

Demographic: age, gender, ethnicity, educationAnthropometry: height, weight, hip and waist circumferenceClinical/Biochemical: SBP, DBP, HDL, triglycerides, total cholesterol, OGTT, medicationsBehavioral: physical activity, smoking, alcohol intakeGenetic: 390 evenly spaced microsatellite markers for linkage analysis and many candidate genes for association studies

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Hypertension as a Complex Disease

Hypertension is a major health problem, and represents a major risk factor for stroke, myocardial infarction, and renal disease.Hypertension often occurs in combination with other metabolic complications such as hyperlipidemia, obesity and insulin resistance.The combined disorder is often called the metabolic syndrome.

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Some Study Topics

Try to identify chromosomal regions that may harbor genes influencing blood pressure, lipids or insulin resistanceIs the candidate gene studied (such as PPARγ, Alsosterone Synthase etc) associated with hypertension/insulin resistance? (How do the different genotypes affect blood pressure, insulin resistance?)

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Association Studies (1) —Sibling based control study

Eliminate some environmental confounding factors.PPAR γ2– Peroxisome proliferator activated receptor Pro12Ala

polymorphism, The most common variant is the C -> G substitution at codon 12 causing an Alanine substitution for Proline

– Localized at 3P25– “A“ variant siblings tend to have lower level of fasting

glucose (p=0.0018) and lower glucose levels at 1 hour (p=0.00258)

following oral glucose loading (after adjusting for age, gender and body mass index) to be more insulin sensitive

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Association Studies (2) — Genetic Heterogeneity

Test whether the nucleotide variation in HUT2 (SNPs) were

associated with BP variation.

HUT2 — kidney specific human urea transporter

– 2 SNPs result in Valine/Isoleucine and Alanine/Threonine

amino acid substitutions at position 227 and 357 in the

HUT2 ORF

– Isoleucine 227 and Alanine 357 alleles were associated

with low diastolic BP in men but not in women.

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Association Studies (3) — Gene-gene interaction

Adiponectin gene– T94G in exon 2– T allele of adiponectin gene was associated with a

higher insulin level at 1 hour in oral glucose tolerance tests (OGTT) (p=0.0008)

– Significant interaction between adiponectin & PPARγ2in fasting insulin level, insulin levels at 2 hours in OGTT.

– Adiponectin G allele and PPARγ2 Ala12 allele are more insulin sensitive.

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Candidate genes approachStudying SNP genotyping data for

promising spots

Genome-wide scan approachImproving the power of linkage

analysis in a sibpair design

Candidate genes approachCandidate genes approachStudying SNP genotyping data for

promising spots

GenomeGenome--wide scan approachwide scan approachImproving the power of linkage

analysis in a sibpair design

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Study DesignThe study design was based on “ ascertaining sibpairs with extremely discordant or highly concordant phenotypes to obtain greater power to detect linkage ” (Risch & Zhang 1995, 96)

We suggest a systematic way to decide how to select discordant sibpairs or concordant sibpairs in linkage analysis based on their phenotype trait values.

We also propose a multipoint linkage analysis method which outperforms existing methods under the situationof linkage disequilibrium.

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Genome-Scan Linkage Studies

There is a great ethnic heterogeneity of the genetic influence on metabolic variable levels.

It would be interesting to conduct a genome-wide scan to identify QTL influencing metabolic phenotypes in Chinese and Japanese population recruited in SAPPHIRe.

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Likelihood-ratio tests were used to test of no linkage (i.e., the variance component due to the QTL being 0).

LOD (log of odds) scores = log10(LR) LOD ≥ 3.3: genome-wide significance for evidence of linkageLOD ≥ 1.9: suggestive evidence for linkage

likelihood of the estimated variance component due to the QTLThe likelihood ratio(LR)

likelihood of the variance component = 0 =

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Linkage Studies

Linkage studies based on genome-wide scan data on ~400 microsatellite markers. LOD score > 4 was obtained for fasting insulin levels in Chinese.

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Linkage study -Genome-Wide Scan

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Candidate genes approachStudying SNP genotyping data for

promising spots

Genome-wide scan approachImproving the power of linkage

analysis in a sibpair design

Candidate genes approachCandidate genes approachStudying SNP genotyping data for

promising spots

GenomeGenome--wide scan approachwide scan approachImproving the power of linkage

analysis in a sibpair design

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Association Between Single Nucleotide Polymorphisms in Protein Tyrosine Phosphatase 1β and Hypertension

Protein Tyrosine Phosphatase-1β (PTP1B) is a primary target in the search for genetic variations in patients with metabolic syndrome.

PTP1B

Insulin and leptinsignaling pathway

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PTP-1β

The importance of PTP-1β for several metabolic pathways has been illustrated in mice deficient for this specific phosphatase.These animals had lower blood glucose concentrations and reduced circulating insulin levels when compared to their normal littermates.On a high-fat diet, PTP-1β deficient mice were resistant to weight gain, and remained insulin-sensitive, while their wild-type littermates became insulin resistant and obese.

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PTPN1

Investigate the possible role of genetic variation in PTPN1 on the development of hypertension, hyperlipidemia, and obesity.Whether genetic variation in the human PTPN1 gene was associated with blood pressure, plasma lipid levels, or body mass index (BMI) as a measure of obesity.

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Positions of all SNPs Identifies

1 2 3 4 5 6 7 8 9 1050,772 3,515 3,310 6,038 3,765

538 373 1,338 1,231

g.-7077G>C

g.-4022G>A

g.8235G>T

g.54281T>A

g.58585T>C

g.69220T>C

g.6930C>T

g.10985A>G

g.56860C>A

g.64840T>C

g.67543A>G

g.70139G>T

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SNP Allele Frequencies

Minor Allele Frequency SNP Name dbSNP Chinese Japanese

g.-7077 G>C 29.4% 24.9% g.-4022 G>A rs6012953 32.8% 27.6% g.8235 G>T 39.0% 25.6% g.54281 T>A 35.1% 35.7% g.58585 T>C rs2038526 31.8% 34.1% g.69220 T>C rs2282146 19.6% 20.4%

Obtained complete genotyping data from 1553 subjects from 672 families for these six SNPs

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Pairwise Linkage Disequilibrium (LD)| D´|: D(AB) = P(AB) - P(A)P(B)

standardized

g.-7

077G

>C

g.-4

022G

>C

g.82

35G

>T

g.54

281T

>A

g.58

585T

>C

g.69

220T

>C

g.-7077G>C 0.88 1.00 0.35 0.51 0.23 g.-7077G>C

g.-4022G>C 0.75 0.53 0.76 0.47 0.13 g.-4022G>C

g.8235G>T 0.86 0.63 0.42 0.49 0.35 g.8235G>T

g.54281T>A 0.70 0.92 0.33 0.92 0.55 g.54281T>A

g.58585T>C 0.63 0.53 0.27 1.00 0.70 g.58585T>C

g.69220T>C 0.38 0.33 0.23 0.60 0.79 g.69220T>C

g.-7

077G

>C

g.-4

022G

>C

g.82

35G

>T

g.54

281T

>A

g.58

585T

>C

g.69

220T

>C

Chinese

Japanese

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Estimated Common HaplotypesChinese Japanese

37.8% 47.9% Haplotype 1

11.3% 6.7% Haplotype 2

7.0% 6.2% Haplotype 3

6.1% 8.1% Haplotype 4

5.6% 5.5% Haplotype 5

5.3% 3.8% Haplotype 6

5.1% 6.7% Haplotype 7

5.0% 3.6% Haplotype 8

83.2% 88.5%

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Haplotype Analysis

1 2 3 4 5 6

g.-7077 G>C 0.66840.8238

g.-4022 G>A 0.9042 0.1410.5972 0.175

g.8235 G>T 0.7258 0.216 0.02820.173 0.0804 0

g.54281 T>A 0.0534 0.0084 0.31640.0092 0.2722

g.58585 T>C 0.7656 0.00360.0826

g.69220 T>C 0.0676

B. Chinese

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Haplotype Analysis

1 2 3 4 5 6

g.-7077 G>C 0.01620.1348

g.-4022 G>A 0.132 0.23080.081 0.212

g.8235 G>T 0.0126 0.0248 00.0064 0.2022 0.3106

g.54281 T>A 0.1214 0.0048 0.01280.2448 0.0296

g.58585 T>C 0.2516 0.0920.0494

g.69220 T>C 0.5112

C. Japanese

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Conclusion and Discussion

This analysis suggests a role of PTP-1β in essential hypertension and lipid profile in Asians.Examine the relationship of the different genes and their combined role in glucose and lipid metabolism as well as obesity.Given the size of the genomic interval spanned by this gene, it is conceivable that the large intronic regions harbor some regulatory elements that have not been uncovered and may be specific to humans.

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Future Work

Over the past years, some synthetic inhibitors of PTP-1βhave been reported that can be used to reduce or inhibit the activity of the phosphatase. These inhibitors may offer a new approach to treating hypertension or dyslipidemiain Chinese or Japanese.

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Design of Future Clinical Trials

Tailor made medicines Drug responder and non-responder Global study– Perspective clinical trials of the safety and efficacy of

test drugs may be conducted to study the inter-individual and cross-ethnic variation in drug response.