# Presentation: 511963

##  Understanding the Genetic Architecture of Common Disease: A Comparison of Genome Scans

- Dennis G. Ballinger, Ph.D.
- VP, bioinformatics
- Perlegen Sciences Inc.
- dballinger@perlegen.com

## Genes, through the proteins they encode, interact with challenges from the environment

**Genes, through the proteins they encode, interact with challenges from the environment**

## Whole-Genome Patterns of Common Human DNA Variation Have Recently Been Characterized

**...............****..October, 2005**

**Whole-Genome Patterns of Common Human DNA Variation Have Recently Been Characterized**

- 27 October 2005

- 18 February 2005

## Common Human SNPs

- Robust assays have been developed for more than 2.5 million of the total 9-10 million common SNPs estimated to be present in the humans.
- Although the vast majority of these SNPs are present in all human ethnic groups and geographic locations, the allele frequencies are more variable between than within ethnicities and geographies
- The correlation structure of more than 2.5 million SNPs has been empirically determined using small population samples from diverse geographic locations.
- The observed correlations between common SNPs reveal that by genotyping a selected subset of 300,000- 400,000 SNP (ie. TAG SNPs) in an individual, one obtains nearly the same genetic information as would be obtained by interrogating all 9 to10 million common human SNPs.

## Genetic Association

**Cases- drug toxicity**

**Controls- no toxicity**

**If a segment of the genome is “associated” with toxicity, cases**
**will have a different SNP allele frequency than controls.**

- 40% Green and 60% red

- 50% Green and 50% red

## Number of LD Bins (>1 SNP) Spanning the Genome in the Perlegen Sciences Study vs. the HapMap Study

| r2  threshold  | Afr-Am | Eur-Am | Han Chinese |
| --- | --- | --- | --- |
| r2  >= 0.8 | 540,533 | 296,313 | 256,766 |

| r2  threshold  | YRI | CEU | CHB +JPT |
| --- | --- | --- | --- |
| r2  >= 0.8 | 474,409 | 293,835 | 259,779 |

## Questions That Remain Unanswered

- What is the relative role of common versus rare genetic variation in complex human traits?
- What is the relative role of population specific versus global genetic variation in complex human traits?
- Which segments of the human genome play the most important role in human phenotype variation?
- What is the size of individual genetic effects?

## Small Sample Size Results in Large Variation in Observed Allele Frequency

| ALLELE FREQUENCY | 0.10 | 0.20 | 0.30 |  |
| --- | --- | --- | --- | --- |
| SAMPLE SIZE |                         95% confidence interval of allele frequency |  |  |  |
| 10 | +/-  0.13 | +/-  0.18 | +/-  0.20 |  |
| 20 | +/-  0.09 | +/-  0.12 | +/-  0.14 |  |
| 50 | +/-  0.06 | +/-  0.08 | +/-  0.09 |  |
| 100 | +/-  0.04 | +/-  0.06 | +/-  0.06 |  |
| 200 | +/-  0.03 | +/-  0.04 | +/-  0.04 |  |
| 400 | +/-  0.02 | +/-  0.03 | +/-  0.03 |  |
| 600 | +/-  0.02 | +/-  0.02 | +/-  0.03 |  |
| 800 | +/-  0.01 | +/-  0.02 | +/-  0.02 |  |
| 1000 | +/-  0.01 | +/-  0.02 | +/-  0.02 |  |

## Case/Control  Genetic Association Requires Hundreds of Samples to Detect Small Allele Frequency Differences

|                     Case/Control Allele Frequency Difference
 (3 Controls/Case, Control Allele Frequency = 0.20, Single SNP)

                                                      0.30                        0.20                         0.10                   0.08                       0.05                |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|     # of CASES |      Probability of observing allele frequency difference by chance  |  |  |  |  |
| 10 | 0.019399 | 0.106478 | 0.3887 | 0.4827 | 0.651109 |
| 20 | 0.000912 | 0.022023 | 0.2214 | 0.3193 | 0.521173 |
| 50 | 1.49E-07 | 0.000286 | 0.0527 | 0.1147 | 0.309503 |
| 100 | 1.05E-13 | 2.85E-07 | 0.0061 | 0.0256 | 0.150402 |
| 200 | 7.26E-26 | 3.82E-13 | 0.0001 | 0.0016 | 0.041908 |
| 400 | 4.81E-50 | 9.49E-25 | 4E-08 | 8E-06 | 0.004007 |
| 600 | 3.67E-74 | 2.71E-36 | 2E-11 | 4E-08 | 0.000424 |
| 800 | 2.97E-98 | 8.18E-48 | 9E-15 | 3E-10 | 4.7E-05 |
| 1000 | 2.5E-122 | 2.55E-59 | 4E-18 | 2E-12 | 5.36E-06 |

## Whole Genome Genetic Association Requires A Multiple Testing Penalty

|                     Case/Control Allele Frequency Difference
 (3 Controls/Case; Control Allele Frequency = 0.20; 325,000 SNPs)

                                                      0.30                        0.20                         0.10                   0.08                       0.05                |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|     # of CASES |      Probability of observing allele frequency difference by chance  |  |  |  |  |
| 10 | 1 | 1 | 1 | 1 | 1 |
| 20 | 1 | 1 | 1 | 1 | 1 |
| 50 | 0.049 | 1 | 1 | 1 | 1 |
| 100 | 3.43E-08 | 0.093 | 1 | 1 | 1 |
| 200 | 2.36E-20 | 1.242E-07 | 1 | 1 | 1 |
| 400 | 1.56E-44 | 3.08E-19 | 0.013 | 1 | 1 |
| 600 | 1.19E-68 | 8.80E-31 | 5.98E-06 | 0.015 | 1 |
| 800 | 9.66E-93 | 2.66E-42 | 2.81E-09 | 8.64E-05 | 1 |
| 1000 | 8.1E-117 | 8.29E-54 | 1.36E-12 | 5.28E-07 | 1 |

## Definitions of SNP Association

**Exploratory SNP association**** ****– non-significant association of an allele with the trait, when correctly adjusting for multiple tests**
**Valid SNP association**** ****-  significant association of an allele with the trait in a single sample corrected for multiple testing**
**Replicated SNP association**** ****– significant association of an allele with the trait in multiple independent samples from a population of interest**

## Slide 12

## The top 10 SNPs from joint analysis of all data

|   |   | Maraganore et al. (Tier 1 and Tier 2) |  |  |  |  | Meta-analysis, replication studies |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Marker | Alleles (high-risk allele) | cases  | controls | allele frequency (controls) | Odds Ratio  | p | cases  | controls | allele frequency (controls) | Odds Ratio  | p |
|  |  |  |  |  | (95% CI) |  |  |  |  | (95% CI) |  |
| rs10200894 | C/G (C) | 772 | 772 | 0.88 | 1.84 (1.38–2.45) | 1.70E-05 | 1566 | 1546 | 0.89 | 1.14 (0.96 - 1.35) | 0.125 |
| rs11737074 | G/A (A) | 764 | 764 | 0.19 | 1.50 (1.21–1.86) | 1.55E-04 | 1563 | 1542 | 0.21 | 1.02 (0.9 - 1.15) | 0.77 |
| rs16851009 | C/T (T) | 741 | 741 | 0.08 | 1.84 (1.36–2.49) | 4.17E-05 | 1539 | 1544 | 0.1 | 0.98 (0.83 - 1.16) | 0.853 |
| rs17329669 | A/G (G) | 768 | 768 | 0.12 | 1.71 (1.33–2.21) | 2.30E-05 | 1554 | 1525 | 0.12 | 1.13 (0.97 - 1.32) | 0.102 |
| rs2245218 | A/G (G) | 770 | 770 | 0.12 | 1.67 (1.29–2.14) | 4.61E-05 | 1571 | 1563 | 0.16 | 0.94 (0.82 - 1.08) | 0.369 |
| rs2313982 | C/T (T) | 740 | 740 | 0.07 | 2.01 (1.44–2.79) | 1.79E-05 | 1562 | 1554 | 0.09 | 0.88 (0.73 - 1.04) | 0.138 |
| rs7520966 | C/T (C) | 769 | 769 | 0.7 | 0.67 (0.55–0.81) | 2.96E-05 | 1563 | 1550 | 0.72 | 1.07 (0.96 - 1.2) | 0.242 |
| rs7702187 | T/A (T) | 761 | 761 | 0.81 | 1.74 (1.36–2.24) | 7.62E-06 | 1541 | 1541 | 0.83 | 1.07 (0.93 - 1.22) | 0.334 |
| rs7723605 | T/C (C) | 773 | 773 | 0.11 | 1.78 (1.35–2.35) | 3.30E-05 | 1567 | 1571 | 0.13 | 1.03 (0.89 - 1.19) | 0.684 |
| ss46548856 | G/C (G) | 765 | 765 | 0.9 | 1.88 (1.38–2.57) | 3.65E-05 | 1551 | 1528 | 0.9 | 1.12 (0.94 - 1.33) | 0.196 |

- Four replication studies published in AJHG August 2006

**Notes:**
Tier 1 443 discordant sibs
Tier 2 332 case / unrelated control

## Large-scale replication study in ~5,000 cases and controls

- Elbaz, A. *et al.*  Lancet Neurology Early Online Publication, 27 September 2006

## SNP Associations with HDL levels 

**“****Replication” Population- 83 Low HDL, 78 High HDL**

**Original Population- 345 Low HDL, 321 High HDL**

**All four CETP SNPs significantly associated with HDL level**
**in the original population are also significantly**
** ****associated with HDL level in the “replication population**

- Hinds et. al., Human Genomics 1:421-434, 2004

**4 SNPs, all of which are in the same gene (CETP), have**
**significantly different allele frequencies between high and**
** ****low HDL individuals**

## Four SNPs in the CETP gene are very significantly associated with HDL levels in both original and replicate study populations

## Associations of SNPs in the CETP gene with HDL levels

*****

- Haplotype pattern 2 in Haplotype block a is present at a 14% increased frequency in individuals with high HDL versus low HDL

## A genome-wide association study in breast cancer

- Douglas F Easton, Alison M Dunning, Karen Pooley, Paul DP Pharoah, David R Cox, Dennis G Ballinger, Deborah Thompson, D Gareth Evans, Diana Eccles, Nazneen Rahman, Michael R Stratton, Julian Peto, Olivia Fletcher, Bruce AJ Ponder, The Breast Cancer Association Consortium

## Breast Cancer Study Design

- “High-risk” breast cancer cases (n=440)

- Compare genotype frequencies P<.05?

- Phase I: 266,722 tagging SNP set

- Female controls EPIC (age>50) (n=400)

-  Prospective meta-analysis (n=20,000+, BCAC)
-  Detailed evaluation of loci

- Phase III: 12-100 SNPs

- P<.0001?

- 4,600 controls (EPIC)

- 4,600 breast cancers (ABC)

- Phase II: ~12,000 SNPs

**112 excess p values <0.05**

## SNP Associations with Breast Cancer

| Locus | Heterozygote
OR (95%CI) | Homozygote
OR (95% CI) | P-trend
(1df) | P-het (2df) |
| --- | --- | --- | --- | --- |
| A | 1.24 (1.12-1.36) | 1.56 (1.36-1.77) | 5x10-17 | 4.7x10-16 |
| B | 1.30 (1.14-1.47) | 1.38 (1.21-1.58) | 2x10-6 | 3.7x10-8 |
| C | 1.19 (1.05-1.36) | 1.45 (1.26-1.66) | 4.1x10-8 | 1.4x10-7 |
| D | 1.18 (1.06-1.31) | 1.33 (1.17-1.50) | 8.5x10-8 | 1.9x10-7 |
| E | 1.25 (1.12-1.39) | 1.55 (1.18-2.04) | 1.0x10-7 | 6.4x10-7 |
| F | 1.13 (1.03-1.24) | 1.42 (1.22-1.66) | 1.9x10-7 | 4.7x10-7 |
| G | 1.07 (0.97-1.18) | 1.38 (1.21-1.57) | 6.7x10-7 | 3.8x10-7 |
| H | 1.13 (1.03-1.23) | 1.27 (1.08-1.49) | 3.9x10-7 | 2.5x10-6 |
| I | 1.07 (0.97-1.18) | 1.35 (1.19-1.53) | 6.2x10-7 | 7.0x10-7 |
| J | 1.19 (1.08-1.32) | 1.43 (1.20-1.72) | 8.3x10-7 | 4.9x10-6 |

**SNP Associations with Breast Cancer**

- K, L and T also replicate in phase 3

**Notes:**
112 excess p<0.05

## Estimated Contribution to Breast Cancer

| Locus | Minor allele frequency  | Attributable risk  | Genetic variance explained (%) |
| --- | --- | --- | --- |
| A | 38% | 16% | 2.0% |
| B | 59% | 22% | 0.7% |
| C | 54% | 18% | 1.4% |
| D | 46% | 14% | 0.8% |
| E | 16% | 7% | 1.3% |
| F | 30% | 8% | 0.9% |
| G | 41% | 9% | 1.0% |
| H | 28% | 7% | 0.5% |
| I | 44% | 9% | 0.9% |
| J | 29% | 10% | 1.2% |

**Notes:**
7/10
10/19

## Conclusions

-  Genome-wide study successful at identifying novel
- 	breast cancer predisposition loci
-  No loci of major effect (RR>2)
-  Many loci of small effect (10s-100s)
*and many probably missed *
-  Little overlap with previously suggested candidate loci
-  Little evidence of dominance
-  Little evidence of age-dependent relative risks
-  No evidence yet of (non-multiplicative) interactions

## Olanzapine TreatmentEmergent Weight Gain

**25% Highest**
**Gainers**

**25% Lowest**
**Non-Gainers**

**N=255**

**N=258**

**Mean BMI change**** ****7.8**** **

**Mean **
**BMI change**
** ****-1.9**

**BMI Change**

**Number of Patients**

- BMI =

- _Weight in Kilograms_
-  (Height in Meters)^2

## Genetic Association with Treatment Emergent Weight Gain

|  |                       |  |
| --- | --- | --- |
| SNP |  P-VALUE | ALLELE FREQ. DIFFERENCE |
| 1 | 1.4E-06 | 0.15 |
| 2 | 5.4E-06 | 0.10 |
| 3 | 5.4E-06 | 0.12 |
| 4 | 5.8E-06 | 0.10 |
| 5 | 6.5E-06 | 0.11 |
| 6 | 6.9E-06 | 0.10 |
| 7 | 1.2E-05 | 0.14 |
| 8 | 1.3E-05 | 0.14 |
| 9 | 1.6E-05 | 0.13 |
| 10 | 2.1E-05 | 0.13 |
| 11 | 3.3E-05 | 0.13 |

## Follow-up of whole genome association results

- Test the hypothesis that some genes associated with olanzapine  emergent weight gain are also associated with familial obesity
- Perform a genetic association study using  samples from an independent set of individuals with familial obesity and 11 SNPs from the eight genes that had the strongest evidence for association with treatment-emergent weight gain in the genome-wide study

## Ascertainment of Familial Obesity

**	****Proband: BMI≥35**
**	****Normal Weight:**
**	**** ****Parent: BMI<27**
**	**
**	****Optional:**
**	****Obese Sibling (BMI>30)****	****Thin Sibling (BMI<25)**
**           ****348 Independent Families **

****

- Optional

**Ascertainment of Familial Obesity**

##  PKHD1 Genetic Association with Obesity

** ****PKHD1 Genetic Association with Obesity**

**Frequency of SNP allele predisposing to weight gain  = 0.52  **
** **
**50% of individuals in population have one predisposing allele**
**27% of individuals in population have two predisposing alleles**
**Relative risk of obesity with one copy of allele   = 1.30 (0.93 - 1.81)**
**Relative risk of obesity with two copies of allele   = 1.62 (1.05 - 2.51)**
**Fraction of obesity attributable to SNP allele   = 0.24 (0.027- 0.453)**

## PKHD1 Mutant Mouse Model

**(Greg Germino, JHU School of Medicine)**

**The mutant mouse PKHD1 gene lacks exons 3 and 4**
**Homozygote mutants develop kidney and pancreatic cysts**
**Heterozygous male mutants are obese, 50.7 +/- 2.9 g**
**versus age and strain matched controls, 36.3 +/- 1.3 g**
**(ANCOVA p= 0.0001)**
**Heterozygous mutants have increased abdominal and**
**retroperitoneal fat deposits**

## Mice heterozygous for a partial deletion of PKHD1

** ****have obesity and marked accumulation of visceral fat**

## Novel pathway for drug development?

**The PKHD1 gene product is a ciliary protein**
**Recent studies suggest that a primary cilium, present on most**
**vertebrate cells, plays an important role in intracellular**
**signaling by regulating the transport of transcription factors**
**to the nucleus**
**Several human single gene disorders, including Alstrom syndrome and Bardet-Biedl Syndrome, characterized by multiple abnormalities including obesity, are caused by genetic**
**defects in ciliary proteins**

## Lessons Learned From SNP

**Association Studies To Date**

**SNP associations can lead to novel biological insights**
**It is not possible to predict the  fraction of variation of a complex trait determined by a SNP prior to performing an association study**
**The majority of  SNP associations account for**
**a small fraction trait variability**
**Independent SNP associations, each accounting for a**
**small fraction of trait variability, can be used in combination to provide clinically useful information**

## 1 1

**Additive Genetic Variance Predicts A Subset of the Population At Increased Risk For An Adverse Response To Treatment**

**1**

**1**

**2**

**2**

**3**

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**4**

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**5**

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**2 2**

**3 3**

**4 4**

**5 5**

## Stratifier 

## Slide 34