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Revealing the Mode of Inheritance in Genetic Association Studies

UNIVERSITY OF THESSALY School of Medicine Laboratory of Biomathematics. Revealing the Mode of Inheritance in Genetic Association Studies. Genetics background Genetics is the science that studies the heredity of traits Genetic information is contained in DNA which consists of nucleotides

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Revealing the Mode of Inheritance in Genetic Association Studies

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  1. UNIVERSITY OF THESSALY School of Medicine Laboratory of Biomathematics Revealing the Mode of Inheritance in Genetic Association Studies

  2. Genetics background • Genetics is the science that studies the heredity of traits • Genetic information is contained in DNA which consists of nucleotides • Gene is a sequence of nucleotides that translates a protein • Genes (through proteins) determine traits (phenotypes) • A gene may have different forms called alleles • An allele can be mutant type-mt (change in nucleotudes) or wild type-wt • For each gene there are two alleles due to diploidy of humans (homologous chromosome pairs) • In an individual the genotype distribution of gene can be homozygous (wtwt or mtmt) or heterozygous (wtmt) • The multiple alleles of a gene is called polymorphism or variant (preserved mutations), they usually expressing different phenotypes

  3. Genetic association studies (GAS) • The evaluation of possible associations between phenotypic traits (diseases) and genetic variants (gene polymorphisms) is carried out using GAS • In the case of a genetic variant with two alleles (mutant type-mt and wild type-wt), where mt is thought to be associated with a disease, GAS will collect information on the numbers of diseased subjects and control subjects with each of the three genotypes (wt/wt, wt/mt, mt/mt)

  4. In an illustrative example of GAS with 8261/4374 cases/controls investigated the association between ACED/I (wt/mt) and CAD, the genotype distribution was Genotype Cases with CAD Controls mt/mt 1788 874 mt/wt 4145 2165 wt/wt 2328 1335 The association between disease status and the genetic variant is tested using a chi-squared (x2) test with (3-1)x(2-1)=2 df

  5. When the association is significant, various genetic models of genotypes are tested by merging genotypes These models include: • additive model: homozygous for mt vs. homozygous for wt • recessive model: homozygous for mt vs. wt-carriers • dominant model: mt-carriers vs. non-mt-carriers • co-dominant model: heterozygous vs. all homozygotes

  6. The significance of the genetic model is assessed using the respective odds ratio (OR) and its 95% confidence interval (CI). The OR for the additive model is For OR>1: an mt subject has greater chance of being diseased than a wt subject If the 95% CI does not include 1, then, the OR is significant (P<0.05) (i.e. the variant is associated with the disease).

  7. ACED/I (wt=D/mt=I) vs. CAD Genotype Cases with CAD Controls mt/mt 1788 874 mt/wt 4145 2165 wt/wt 2328 1335 • x2=9.42, P<0.05 (http://people.ku.edu/~preacher/chisq/chisq.htm) There is significant association between ACE D/I gene variant and development of CAD

  8. But, what is the mode of inheritance, or what is the real genetic model?

  9. Recessive model: Genotype Cases with CAD Controls mt/mt 1788 874 mt/wt+wt/wt 4145+ 2328=6473 2165+1335=3500 • Since “1” is not included in the 95% CI, we conclude that the OR is significant (P<0.05). • Since OR>1, we conclude that homozygous for the mt allele have 11% greater risk for CAD than wt-carriers

  10. Dominant model: Genotype Cases with CAD Controls mt/mt+mt/wt 1788+4145=5933 874+2165=3039 wt/wt 2328 1335 • Since “1” is not included in the 95% CI, we conclude that the OR is significant (P<0.05). • Since OR>1, we conclude that carriers of the mt allele have 12% greater risk for CAD than homozygous for the wt allele

  11. Additive model: Genotype Cases with CAD Controls mt/mt 1788 874 wt/wt 2328 1335 • Since “1” is not included in the 95% CI, we conclude that the OR is significant (P<0.05). • Since OR>1, we conclude that homozygous for the mt allele have 17% greater risk for CAD than homozygous for the wt allele

  12. Co-dominant model: Genotype Cases with CAD Controls mt/wt 4145 2165 mt/mt+wt/wt 1788+2328=4116 874+1335=2209 • Since “1” is included in the 95% CI, we conclude that the OR is not significant (P≥0.05).

  13. Recessive model: OR=1.11 (1.01, 1.21), significant Homozygous for the mt allele have greater risk than wt-carriers • Dominant model: OR=1.12 (1.03, 1.21), significant Carriers of the mt allele have greater risk than non-carriers • Additive model: OR=1.17 (1.06-1.30), significant Homozygous for the mt allele have greater risk than homozygous for the wt allele • Co-dominant model: OR=1.03 (0.96, 1.11), non-sign

  14. Is the genetic model, recessive, dominant or additive? Mess!

  15. The source of the problem The ORs of the genetic models (recessive, dominant, additive, co-dominant) are not independent the testing of association between genotype distribution and outcome (disease/controls) is based on 2 df (the df for the Chi-squared test)

  16. How can we avoid the hash of possible genetic models making the interpretation of the results straightforward at the same time?

  17. Zintzaras (2010, Stat Appl Genet) introduced the concept of a generalized odds ratio (ORG) as a metric for describing the association between disease status (disease vs. healthy or disease progression) and genotype (biallelic or multiallelic)

  18. The ORG is a single statistic that utilizes the complete genotype distribution and provides an estimate of the overall risk effect General definition: The ORG is the probability of a subject being more diseased relative to the probability of being less diseased, given that the more diseased subject has a higher mutational load

  19. Definition for bi-allelic variant and binary phenotype: ORG is the probability of a subject being diseased relative to probability of being free of disease, given that the diseased subject has a higher mutational load than the non-diseased When ORG>1 then an increased genetic exposure (mutational load) implies disease

  20. “ORGGASMA”: a software for implementing the generalized odds ratio methodology for the analysis and meta-analysis of GAS • The software “ORGGASMA” (together with instructions how to operate it) is freely available and it can be downloaded form the web site http://biomath.med.uth.gr

  21. ACED/I (wt=D/mt=I) vs. CAD Genotype Controls Cases with CAD mt/mt 1788 874 mt/wt 4145 2165 wt/wt 2328 1335 • Assumption: Subjects who are homozygous for I allele have the highest mutational load, those homozygous for D allele have the lowest, and heterozygous have an intermediate level. • ORG=1.13 with 95% CI: (1.08-1.19),

  22. ORG=1.13 with 95% CI: (1.08-1.19) For any two subjects, diseased and healthy, the probability of being diseased is 13% higher (relative to the probability of being non-diseased) given that the diseased subject has higher mutational load than the healthy one.

  23. Disease progression ADH2 *2/*1 Controls Alcoholics Alcoholicswith liver disease *1/*1 188` 874 321 *2/*1 145 265 456 *2/*2 238 135 231 ORG =1.37 (1.10-1.72): Risk of disease progression is related to mutational load A subject has 37% higher risk of being more diseased (relative to the risk of being less diseased) given that the subject has a higher mutational load. Multiallelic variant APOE Controls CAD e2/e2 23` 44 e2/e3 45 65 e2/e4 28 35 e3/e3 32 21 e3/e4 87 45 e4/e4 34 44 ORG=1.13 (1.07-1.19): Mutational load of APOE plays a role in disease susceptibility Diseased subjects with higher mutational load than healthy ones have13% higher risk for disease susceptibility.

  24. The ORG is a good solution, but, it is not enough! • Zintzaras and Santos (2010, Stat Med) provided the whole solution! Problem: The ORs of the genetic models (recessive, dominant, additive, co-dominant) are not independent Solution: Inferences should be based solely on the additive and co-dominant models

  25. Instead of talking for recessive, dominant, additive, co-dominant models we could talk for Dominance and Co-Dominance or even better for Degree of dominance

  26. Co-dominance In the extreme case where there is co-dominance (i.e., perfect additivity), the heterozygote wtmt “lies” exactly in the middle of the two homozygotes, with mtmt having the maximum susceptibility of being diseased and wtwt having the least • Co-dominant model is non-significant (P>0.05) • Additive model is highly significant (P<0.01)

  27. Dominance The heterozygote wtmt lies towards mtmt or wtmt Co-dominant model is significant (P<0.05) Additive model can be significant (P<0.05) or non-significant (P>0.05)

  28. Degree of dominance The degree of dominance could be derived from the ratio of the logarithms of the OR of co-dominant vs. the OR of the additive model the sign of ln(θco) determines the direction of dominance, and the value of ln(θco) relative to the absolute value of ln(θa)the magnitude of dominance deviation (i.e. deviation from the middle)

  29. -1<h<0: wtmt is expected to have a risk of being diseased somewhere in between the middle of the two homozygotes and towards to wtwt • 0<h<1: wtmt is expected to have a risk of being diseased somewhere in between the middle of the two homozygotes and towards to mtmt • h>1:wtmt has a higher risk of being diseased than mtmt • h<-1: wtmthas least chance of being diseased than wtwt

  30. Once significance in dominance is detected (i.e. co-dominant model has P<0.05) and h is obtained, the degree of dominance is inferred as follows:

  31. To summarize, inferences regarding any degree of dominance are obtained from the following order: • If the co-dominant model is non-significant and the additive model is significant (i.e. co-dominance), the risk of disease for the heterozygote is in the middle of the two homozygotes. • If the co-dominant model is significant (i.e. dominance), we then test for the direction of dominance. • If 0<|h|<1,wtmt has a risk of disease closer to mtmt or wtwt according to the sign (+ or -, respectively) of h • If |h|>1 is significant then, there is over- or under-dominance.

  32. ACED/I (wt=D/mt=I) vs. CAD Genotype Cases with CAD Controls mt/mt 1788 874 mt/wt 4145 2165 wt/wt 2328 1335 • The co-dominant model is not significant (P≥0.05) and the additive model is significant (P<0.05) the risk of disease for the heterozygote is in the middle of the two homozygotes.

  33. A GAS investigating the association between the alleles ADH2*1 and ADH2*2 with alcoholism produced the following genotype distributions: • Both co-dominant and additive models are significant. Since the co-dominant model is significant, we proceed to inquiry about the degree of dominance, which here is • indicating that the risk-associated allele *1 is dominant, or that dominance exists.

  34. In other words, the homozygous *1/*1 (mt/mt) has a greater risk of being alcoholic than the homozygous *2/*2 (wt/wt), and the heterozygote *2/*1 has a risk of alcoholism closer to the homozygote *1/*1 than to the midpoint between the two homozygotes.

  35. Ancient Theater of Larissa

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