# Presentation: 017583

## Genetics for EpidemiologistsStudy Designs: Family-based Studies

- Thomas A. Pearson, MD, PhD
- University of Rochester
- School of Medicine
- Visiting Scientist, NHGRI

## Genetics for Epidemiologists:Study Designs:  Family-based Studies

**Learning Objectives**
- Introduce study designs to generate or test genomic hypotheses.
- Describe the major study designs which involve genetically related individuals.
- Provide examples of family-based designs from the literature.
- Consider the advantages and disadvantages of family-based designs in the study of gene-disease associations.

## Identical Twins, 51 Year Old Males, with Myocardial Infarction* 

- 	_Characteristic__	_	         _EB_		_AB_
- 	Cigarette smoking		            1 ppd		1 ppd
- 	LDL Cholesterol (mg/dl)		151		151
- 	Blood pressure			Normal	             Normal
- 	Diabetes				None		None
- 	Coronary Arteriography		JHH		HFH
- 		Coronary Dominance	             Left		Left
- 		Right Coronary Lesions	             None		None
- 		Left Ant. Descending Lesions	None		None
- 		Left Circumflex Lesions	         >90% stenosis   >90% stenosis
- 						[Single lesion in OM branch]
- * Herrington DM, Pearson TA.  Am J Cardiol 1987; 59: 366-7.

## The Genetic Etiology of Disease

- 			         Gene Variant
- 			     Gene Expression
- 				Gene Product
- 			    Altered Physiology
-  			   Phenotype (Disease)

## Hierarchy of Questions Regarding a Genetic Etiology of a Disease

- Does it aggregate in families?
- Is it inherited from parent to offspring?
- Which chromosomes carry the gene(s)?
- Which gene(s) are associated with it?
- Which gene variant(s) are associated with it?
- What gene products are altered as a potential direct or indirect cause of it?

## Candidate Gene Approaches(Hypothesis-driven)

- 	Twin       	  Linkage       Other Family-
- 	Studies		  Analysis       based Designs
-                       Candidate Genes
- 			  Disease vs. No Disease
- 				    Replication

## Genome-wide Association(Agnostic)

- 				Entire Genome
- 			Disease vs. No Disease
- 				 Replication

## Familial Aggregation?Family History as an Independent  Risk Factor

- Definition of a positive family history
- Self-reported vs. verified
- Specific definitional elements
- Age of onset of disease
- Degree of relatedness of affected relatives
-    (1st, 2nd, 3rd degrees)
- Number of relatives affected
- _Family information bias_:  The flow of family information about exposures or illnesses may be stimulated by, or directed to, a new case in its midst .
-      (Sackett D.  J Chron. Dis. 1979; 32: 51-63)
- Relative risk ratio:  A measure of the strength of familial aggregation:
-        		       	     Prevalence of disease in
- 	Relative Risk Ratio (λ) =    _relatives of affected persons_
- 				     Prevalence of disease in
- 			                     the general population

## Risk Ratios for Siblings of Probands with Complex Diseases with Familial Aggregation* 

- 	_	__Disease__	_				     _λ_
- 	Schizophrenia				             12
- 	Autism						  150
- 	Bipolar Disorder 				      7
- 	Type 1 Diabetes Mellitus		             35
- 	Crohn Disease				             25
- 	Multiple Sclerosis				    24

- * Nussbaum et al: Thompson and Thompson’s Genetics in Medicine, 2007, p 153.

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## Studies of Familial Aggregation of Disease in Siblings

- Twins
- Monozygous (MZ) twins (0.3% of births)
- Dizygous (DZ) twins (0.2-1.0% of births)
- Twins reared apart
- Twins adopted and raised by unrelated foster parents
- Siblings

## Measures of Degree of Genetic Contribution to Disease in Family Studies

- Qualitative traits or diseases
- Concordance
- Quantitative traits
- Correlation
- Heritability

## Concordance

- Calculated as the number of twin-pairs with disease amongst those twin-pairs with at least one affected twin (Gordis):
-       _#twins with both affected_
-       # twins with both affected + # twins with only one affected
- Concordance < 100% in MZ twins is evidence for nongenetic etiological factors.
- Concordance in MZ twins > DZ twins is evidence for genetic etiological factors.

## Concordance Rates for Parkinson’s Disease in Twin Pairs *

-                        	        		 Number	Concordant Pairs                           _Types of Pairs _      		 _of Pairs_	_Number_	          _% _
- 	All twin pairs
- 		Monozygous		    71		     11             15.5
- 	        Dizygous		    90		     10	         11.1
- 	Onset <50 years
- 		Monozygous                         4                        4            100.0
-              Dizygous		     12                       2              16.7
- 	Onset >50 years
- 		Monozygous		     65		      7              10.8
- 		Dizygous                             76		      8              10.5
- *Tanner CH et al.  JAMA 1999; 281: 341-346 as cited in Gordis, 2004

## Concordance Rates in MZ and DZ Twins*

- 						_Concordance (%)_
- _Disorder_				   _ MZ _        _DZ_
- Nontraumatic epilepsy	    	   70.0	          6
- Multiple sclerosis		   	   17.8           2
- Schizophrenia			    40	          4.8
- Bipolar disorder		        	    62	          8
- Osteoarthritis			               32	        16
- Rheumatoid arthritis	      	               12.3         3.5
- Psoriasis				    72	        15
- Cleft lip				    30             2
- Systemic lupus erythematosus	    22             0

- Nussbaum et al.  Thompson and Thompson’s Genetics in Medicine, 2007

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## Measures of Degree of Genetic Contribution to Disease in Family Studies

- Qualitative traits or diseases
- Concordance
- Quantitative traits
- Correlation
- Heritability

## Correlation Among Relatives for Systolic Blood Pressure*

-  _Relatives Compared_	  _Correlation (r)_
- 	Monozygotic twins		0.55
- 	Dizygotic twins			0.25
- 	Siblings					0.18
- 	Parents and offspring		0.34
- 	Spouses				0.07
- * Feinlieb M et al  as cited in Gordis, 2007

## Heritability (h2)

- Defined as the fraction of total phenotypic variance of a quantitative trait that is caused by genes.
- Calculated from twin studies:
-             h2 = _Variance in DZ pairs-Variance in MZ pairs_
-                                             Variance in DZ pairs
-             Varies from 0.0 (no heritability) to
- 		     1.0 (strong heritability);  >.7 or .8 suggest       	      strong influence of heredity on trait.

## Limitations of Twin Studies

- Environmental exposures may not be identical even in MZ twins.
- MZ twins can have different gene expressions.
- The risk of the genotype may be heterogeneous between twin pairs.
- Ascertainment bias:  Co-twin with disease is more likely to participate in twin studies as compared to unaffected co-twin.

## Linkage Analysis:  Family-based Approach to Identification of Susceptibility Genes

- Linkage:  the tendency for alleles at loci that are close together to be transmitted together as an intact unit (haplotype).
- Recombinant fraction (Θ) varies 0.0-0.5:
-       0.0 = tightly linked, no recombination
- 	  0.5 = unlinked, independently assorting
- Map distance in centimorgans:  genetic length over which one recombinant cross-over will occur in 1% of meioses.

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## Determination of Linkage in Family Studies 

- Assume a mode of Mendelian inheritance.
- Identify markers with known positions to serve as references.
- In families, determine the number of 1st degree relatives who show recombination assuming various values of θ (0.0 to 0.5).
- Calculate ratio of liklihood of observing the family data for values of θ to the likelihood of observing the family data if the loci were unlinked (θ = 0.5).

## LOD Score (Z= Logarithm of Odds)

- Z = _Likelihood of the data if loci linked at a particular __θ_
- 		     Likelihood of the data if loci are unlinked (θ = 0.5)
- Best estimate of θ, the recombinant frequency between a marker locus and the disease locus.
- Magnitude of Z assesses strength of likelihood of linkage (LOD>3 is 1000/1 odds that loci are linked).
- LOD scores can be added across families.

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## Trios:  Study Design of Affected Offspring and Both Parents

- Phenotypic assessment only in affected offspring.
- Genotyping in both parents and affected offspring.
- Used in both discovery and replication GWAS.
- Advantage:  Not susceptible to population stratification due to sampling of cases and controls from populations of different ancestries.

## Parents and Offspring: Transmission Disequilibrium Testing (TDT)

- Tests whether an allele at given locus (linked to disease or trait) transmitted to affected offspring by parents more frequently than expected by chance.
- Heterozygous parents transmit alleles m1 and m2 at given locus with equal frequency (50%); affected offspring should receive disease-associated allele more frequently.
- Obviates need for control group.

## TDT in Type I Diabetes: Excess Transmission of D18s487 Allele 4(Merriman T et al.  Hum. Molec. Genet 1997; 6;1003-1010) 

| Families | Trans-mitted | Not Trans-mitted | % T | P-value |
| --- | --- | --- | --- | --- |
| Affected | 348 | 276 | 55.8 | 0.004 |
| Not affected | 101 | 98 | 50.8     |   NS |

## Comparison of GWAS Studies Using Case-Control and Trio Designs to Identify Associations Between Three SNP’s and Type 1 Diabetes Mellitus*

|  | rs2476601 | ra10255021 | rs2903652 |
| --- | --- | --- | --- |
| Case-Control |  |  |  |
|    Allele |         A |           A |          A |
|    Minor Allele Frequency |  |  |  |
|       Cases (N=561) |     .1471 |       .0667 |       .2834 |
|       Controls (N=1143) |     .0876 |       .1095 |       .3782 |
|    OR |        1.8 |          .58 |        .65 |
|    P Value |   1.3 x 10-7 |   1.2 x 10-4 |   4.8 x 10-8 |
| Trio |  |  |  |
|    Alleles |       A:G |         A:G |         A:G |
|    Trans : Untrans   |      137:64 |       18:57 |      160:228 |
|    TDT P Value |    2.6 x 10-7 |    6.7 x 10-6 |    7.9 x 10-5 |

-  *Hakonarson H, et al.  Nature 2007; July 15

## Limitations of Trios

- Difficult to assemble trios if late onset of disease in affected child.
- Sensitive to small degrees of genotyping errors which can distort transmission proportions between parents and offspring (Mitchell AA et al.  J Hum Genet 2003; 72: 598-610)
- Example in GWAS of schizophrenia (Kirov G et al: Molec Psych 2008; 1-8).

## Other Issues in Family-based Designs

- GWAS of Affected/Unaffected sibling comparisons
-       (Maraganore DM et al. Am J Hum Genet 2005; 77:685-693)
- Attribution of heritability or genetic risk.
- 1.Multivariate adjustment of disease association for susceptibility SNPs to determine if risk can be accounted for:
-       Y = β0 + β1(+FH) + β2(SNP1) + β3(SNP2) + etc.
- 2. Multiple adjustment for intermediary risk factors to identify excess risk in first degree relatives (Framingham Heart Study).

## Does the Framingham Risk Score Predict Risk in Siblings of Early Premature Coronary Patients?

- 784 sibs (30-59 yrs.) of 449 pts. With CAD with onset <60yrs.
- Ten year follow-up for incident CAD events.
- Ten year risk from FRS calculated at baseline.
- Excess risk in men (66.6%) and in women (12.7%).
-     Vaidya D et al, AJC 2007; 100:  1410-1415

## Conclusions

- Family-based studies have been the cornerstone of identification and quantification of the familial risk and heritability of human diseases.
- Linkage analysis identifies the location of genes relative to known markers and the alleles within a haplotype in linkage disequilibrium.
-  Trios provide a family-based design for candidate genes or for discovery or replication GWAS.