Genetic epidemiology

Genetic epidemiologyin psychiatry Dr. Sherlyn E. MammenDNB resident, LIBSCHAIR: Dr. Ramachandrankutty MDHOD, LIBS

OutlineBasics in genetics

Genetic epidemiology

Study designs in GE

Methods of analysis in GE

Clinical implications

Future directives

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Basic Organization of the Human Genome

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3

Chromosomes 23 pairs

1 through 22 in order of size

Centromere

q & p

Base - pair positions numbers

Nucleotides

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DNA

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Central dogma of genetics

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Structure of gene and flow of information from DNA to protein

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Simple Mendelian diseasesSingle gene (monogenic disorder)

Simple pattern of inheritance (D/R)

No influence of the environment

Relatively rare (1 % of live births)

HD(AD), CF(AR), Retts syndrome (X-D), Hemophilia A (X-R), male infertility (Y-linked)12/16/20168

Complex diseasesMulti-factorial diseases

Several genes which may interact together

Environment also play a role

Environment Genes interactions

Cardiovascular diseases, cancer, psychiatric disorders...12/16/20169

Epigenetic mechanismsAny heritable change in the genome that does not change the nucleotide sequence

Mechanisms:Chemical modification of DNA (methylation)Chemical changes to the protein complexes of histones (phosphorylation, acetylation, methylation, ubiquitination)RNA interference (siRNA or microRNA non coding RNA bind to mRNA and slows protein synthesis)Prions 12/16/201610

Epigenetic process12/16/201611

Common Genetic terminologies1. PENETRANCE (f): Probability that a person with a given genotype will manifest the illness

2. HERITABILITY: is defined as the proportion of the trait variation directly attributable to genetic differences among individuals relative to the total variation in a population

A high heritability constitutes circumstantial evidence for genetic control of a trait

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3. CONCORDANCE RATES: Proportion of twin-ships in the population who are concordant (both are affected)MZ twins > DZ twins genetic basisDZ twins > full sibs role for shared environmental factors

4. CANDIDATE GENE: A known gene suspected to be associated with the disease of interest on the basis of the biological function of its protein

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5. CO-SEGREGATION: The tendency of two traits to be jointly inherited

6. LINKAGE : The phenomenon whereby phenotypes and alleles at one or more marker alleles tend to be inherited together more often than expected

7. PHENOCOPY : An environmentally caused phenotype that mimics a genetic trait12/16/201614

8. GENETIC HETEROGENITY : Different genes may independently produce the phenotype 9. GENOMIC IMPRINTING: Depending on the gene, either the copy from mother or father is epigenetically silenced ( DNA methylation)Prader Willi syndrome: dads copy is missingAngelman syndrome: moms copy is defective or missing

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HardyWeinberg principleAlleleand genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary influencesThese influences includemate choice, mutation, selection, genetic drift, gene flow etc12/16/201616

In Hardy Weinberg equilibrium, the equation always equals to 1

If the equation does not equal to 1, evolution is occuring12/16/201617

Hardy Weinberg equilibriumEvolution = change in allele frequencies in a population

Very large population size( no genetic drift)No migration ( no gene flow in or out)No mutation ( no genetic change)Random mating ( no sexual selection)No natural selection( everyone is equally fit)

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Genetic epidemiology - Definition

Includes understanding the causes, distribution and control of disease in groups of relatives and the multi-factorial causes of disease in populations12/16/201619

Why study genetic factors?Understand biological process leading to a disease

Diagnostic and prognostic

Prevention: screening & genetic counseling

Treatment: develop new treatments, personalized medicines

Strengthen or confirm epidemiological inference for modifiable environmental factors12/16/201620

GE QUESTIONS 12/16/201621

12/16/201622Familial aggregation studiesHeritability studiesSegregation analysisLinkage, association studiesVariant frequency, risk magnitude, environmental interactions

Study designs for genetic research on mental disorders

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Flow of research

2. Familial clustering: family aggregation studies

3. Segregation analysis

4. Find disease susceptibility loci: linkage analysis

5. Find disease susceptibility markers: association analysis12/16/201624

Phenotype definitionFirst step in GE process:

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Phenotyping strategies1. Categorical phenotypes( affected): DSM5 criteria

2. Continous traits( affectedness): Endophenotypes

Neurocognitive functionPersonality & temperamentNeuroanatomy & physiologyPharmacological responseNeuroendocrine/ immuneGene expression patterns

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Endophenotypes Gottesman and Gouldhighlighted five desirable characteristics of a putative Endophenotype

It is associated with illness in the populationIt is heritableIt is primarily state-independentIt co-segregates with illness within familiesThe endophenotype found in affected family members is found in non-affected relatives more frequently than in the general population

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Population studiesEtiological homogeneity

Homogeneity of cultural and environmental factors

Variations in prevalence and incidence provides clues to the causes and the balance between genetic and environmental contributions

Population Incidence rates : baselines for estimating heritability in family genetic studies

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GE: Flow of research1. Phenotype definition

3. Segregation analysis4. Find disease susceptibility loci: linkage analysis5. Find disease susceptibility markers: association analysis12/16/201629

What is familial aggregation ?

Based on phenotypic data only

Demonstrate that the disease tends to run in families more than what would expect by chance

Examine how that familial tendency is modified by the degree or type of relationship, age or environmental factors

Familial aggregation does not separate genetic from environment12/16/201630

Family studiesA disorder that runs in families may indicate

1. a genetic etiology

2. non-genetic reasons (shared environmental factors)

A disorder may not run in families and still have a genetic etiology

The design is similar to case-control studies A higher prevalence among relatives of affected probands is evidence that the disorder aggregates in families

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Measuring FAMILIALITY.Recurrence risk: The risk to relatives of affected probands One index of the strength of familiality is the recurrence risk ratio for first-degree relatives (1), defined as the ratio of the risk of the disorder in a first-degree relative of an affected individual to the prevalence in the general population

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Twin studiesTwin and adoption studies can be used to assess, to a degree, the contribution of genetic and environmental causes of familial aggregation

Twin studies compare concordance rates between MZ twins and DZ twins

A twin pair is concordant if both co-twins have the phenotype

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Twin studies

MZ concordance rate < 100% suggests that environmental factors influence the phenotype

Twin studies provide an estimate of theheritabilityof the disorder

VP=VG+VE

VP = The total variance in phenotypes in a population

Heritability = VG/VP12/16/201634

Adoption studiesAdoption studies can disentangle, to a degree, genetic and environmental influences on family resemblance by comparing rates of a disorder in biological family members with those in adoptive family members

Adoption studies provided the first convincing evidence that genes play an important role in the development of schizophrenia12/16/201635

GE: Flow of research1. Phenotype definition

2. Familial clustering: family aggregation studies

4. Find disease susceptibility loci: linkage analysis

5. Find disease susceptibility markers: association analysis12/16/201636

Segregation analysis

Is the aggregation due to environmental, cultural or genetic factors?

What proportion of the trait is due to genetic factors?

What mode of inheritance best represents the genetic factors?

Does there appear to be genetic heterogeneity?12/16/201637

Segregation

In the mid 1800's, Gregor Mendel demonstrated the existence of genes based on the regular occurrence of certain characteristic ratios of dichotomous characters among the offspring of crosses between parents of various characteristics and lineages12/16/201638

Segregation ratios

The analysis of segregation ratios remains an important research tool in human genetics

The demonstration of such ratios for a discrete trait among the offspring of certain types of families constitutes strong evidence that the trait has a simple genetic basis12/16/201639

GE: Flow of research1. Phenotype definition2. Familial clustering: family aggregation studies3. Segregation analysis

5. Find disease susceptibility markers: association analysis12/16/201640

Linkage studies basic ideaWhere in the genome, a disease susceptibility locus may reside

The degree to which alleles at two or more genetic loci are co-inherited within families

The likelihood that two loci on a chromosome will co-segregate is inversely proportional to the distance between them

This principle is due to the phenomenon of recombination between homologous chromosomes that occurs during meiosis

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Recall: recombination

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Gene mapping

12/16/201643Localize the disease gene with respect to the genetic markersCoarse mapping( > 1cM)

Linkage analysisTwo main types:

1. Parametric linkage analysis: estimate the recombination fraction between the causal locus (to be located) and the marker locus (known location)

2. Non-parametric linkage analysis: test whether affected relatives share more alleles identical by descent than expected by chance12/16/201644

Linkage analysisPhenomena that can attenuate the direct relationship between genotype and phenotype in LA

Phenocopies Incomplete penetranceVariable expression or a spectrum of phenotypesGenetic heterogeneity12/16/201645

Measuring Linkage analysis In parametric linkage analysis, the strength of the evidence in favor of linkage is calculated as alogarithm of the odds(LOD) score The LOD score is calculated as follows: LOD = Z = log10 probability of birth sequence with a given linkage value probability of birth sequence with no linkage12/16/201646

Flow of research 1. Phenotype definition

2. Familial clustering: family aggregation studies

3. Segregation analysis

4. Find disease susceptibility loci: linkage analysis

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Association studiesAssociation analysis examines the co-inheritance of alleles and phenotypesacrossunrelated families

While linkage analysis asks where a susceptibility gene resides, association analysis asks which specific genetic variants influence a phenotype

More powerful than linkage analysis for detecting small genetic effects

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CANDIDATE GENE STUDIES

Two classes of candidates have been studied:

Biological candidates (selected based on prior evidence that the gene or pathway is involved in the biology or treatment of a disorder)

Positional candidates (selected based on evidence from linkage or cytogenetic studies that a genomic region harbors susceptibility genes)

Common biological candidate genes: those involved in monoaminergic neurotransmission

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Genome-Wide Association Studies

GWAS are unbiased in that they do not require a pre-specified hypothesis about which genes are important; as such, they offer the opportunity to uncover novel molecules and molecular pathways in the biology of these disorders12/16/201650

GWAS Large number of statistical tests involved

GWAS usually examine relatively common polymorphisms, so that effects of rare susceptibility variants may be missed

GWAS have identified genetic loci associated with autism, bipolar disorder and schizophrenia12/16/201651

Whole-Exome Sequencing

Whole-exome sequencing can reveal rare single nucleotide variants (point mutations) that may be associated with a disorder

These point mutations can bede novomutations, arising in the offspring and not present in the parents

Whole-exome sequencing studies of autism have been recently published

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12/16/201653STUDYUNIT OF ANALYSISGOALPOPULATIONSUBJECTS IN GENERAL POPULATIONLIFETIME CUMULATIVE INCIDENCEFAMILYPEDIGREESESTABLISH FAMILIALITY, MODES OF TRANSMISSION & RISKS TO RELATIVE CLASSESTWINMZ & DZ TWINSGENETIC Vs ENVIROMENTAL EFFECTSADOPTIONADOPTEES, ADOPTIVE & BIOLOGICAL RELATIVES OF ADOPTEESGENETIC Vs ENVIRONMENTAL EFFECTSLINKAGENUCLEAR AND OR EXTENDED PEDIGREESCHROMOSOMAL LOCATION OF A DISEASE LOCUSASSOCIATIONUNRELATED AFFECTED INDIVIDUALS & CONTROLSIDENTIFY A SPECIFIC DISEASE LOCUSTRANSGENICGENE EXPRESSION IN MODEL SYSTEMS:WORM, FLY, MOUSEIMPLICATE GENES, MOLECULAR PATHWAYS & NEURAL CIRCUITS

Methods of genetic analysis

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METHODDATA SOURCEGOALPATH ANALYSISTWIN, ADOPTIONDISTINGUISH TRANSMISSIBLE ENVIRONMENT FROM POLYGENESSEGREGATION ANALYSISPEDIGREEDISTINGUISH A MAJOR LOCUS FROM POLYGENES OR TRANSMISSIBLE ENVIRONMENTLINKAGE ANALYSISPEDIGREEESTABLISH CHROMOSOMAL LOCALIZATION OF A PUTATIVE SUSCEPTIBILITY LOCUSASSOCIATION ANALYSISUNRELATED AFFECTS, CONTROLSIMPLICATE A SPECIFIC GENE AS A DISEASE SUSCEPTIBILITY LOCUS, GIVEN LD

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Linkage DisequilibriumLD is a phenomenon that is used to evaluate the genetic distance between two loci in populations rather than in families

When alleles at 2 loci occur together in the population more often than would be expected given the allele frequencies at the 2 loci, those alleles are said to be in LD

r2 : measures LD: a measure of difference between observed and expected haplotype probabilities12/16/201657

Gene mapping

Aims to identify genes implicated in heritable diseases

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Gene mapping strategies1. Linkage analysis - pedigree analysis - affected sib pair analysis

2. Genome wide association - case-control - family trios12/16/201659

Pedigree analysisSTUDY SUBJECTS: multigenerational families with multiple affected individuals

BASIC IDEA: identify genetic markers that co-segregate with disease phenotype

STRENGTHS: can detect rare variants of large effect, gains power by incorporating information about familial relationships into the model

LIMITATIONS: limited power to identify common variants of small effect, cost intensive12/16/201660

Affected Sib pair analysisSTUDY SUBJECTS: two or more affected siblings

BASIC IDEA: identify chromosomal regions shared by siblings concordant for disease

STRENGTHS: robust to differences in genetic composition of study population, allows incorporation of environmental data

LIMITATIONS: limited power to identify common variants of small effect12/16/201661

Case-controlSTUDY SUBJECTS: affected individuals and matched unaffected controls sampled from population

BASIC IDEA: test for statistical association of alleles and disease in cases Vs control

STRENGTHS: can detect common variants of small effect

LIMITATIONS: requires large sample sizes, increased false positive rates in the presence of population stratification12/16/201662

Family-TriosSTUDY SUBJECTS: affected individuals and parents

BASIC IDEA: tests for association using non-transmitted parental chromosome as control

STRENGTHS: can detect common variants of small effect, robust to problems of population stratification

LIMITATIONS: about 2/3rds as powerful as case-control designs, difficult to collect samples for late onset diseases12/16/201663

Genetic markers

Microsatellite markers: simple tandem repeats( STR)

Simple sequence length polymorphisms( SSLP)

Single nucleotide polymorphism ( SNP)

Indels : small insertion or deletion polymorphisms (1-30 base pairs)

CNV: copy number variations 12/16/201664

Copy Number Variants

Deletions, duplications, insertions, and inversions of regions of the genome

Over 1,000 CNVs in the human genome

Some CNVs are common allelles, while others are rare

Rare CNVs have been associated with psychiatric disorders (schizophrenia)

The deletion of 22q11.2, in velocardiofacial syndrome, has been associated with schizophreniaand the duplication of 15q11-13 with autism

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Association Studies

Population-based Cases and unrelated population controls from the same study baseFamily-based Child-family trios and TDT design is the most common

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Family-based association study designs

Haplotype Relative Risk (HRR) Method

Affected Family-Based Controls (AFBAC) Method

Transmission Disequilibrium/Distortion Test (TDT)12/16/201667

Variation in SNPs and VNTRs

1. SNPs (single-nucleotide polymorphism) Parent 1: AACCTCTACACParent 2: AACCTTTACACG= Possible genotypes: CC, CT, TT

2. VNTRs (variable number tandem repeat)3 repeats: CCACTCAGG CCACTCAGG CCACTCAGG2 repeats: CCACTCAGG CCACTCAGG = Possible genotypes: 3R/3R, 3R/2R, 2R/2R

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Clinical implications

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Heritability estimates

Schizophrenia 80 % Bipolar disorder 80 % Major depression 40 % GAD 30 % Panic disorder 40% Phobia 35 % Alcohol abuse/ ADS 60%12/16/201670

ADMZ: 50%

Familial forms: early onset, AD, High penetrance

APP gene- chr.21 amyloid deposits

Presenilin 1 (PS1) - 14q24.3

Presenilin2 (PS2) 1q12/16/201671

Trans-membrane proteins

ADSporadic forms: genetic contribution is similar to other NP disorders

Apolipoprotein E gene: 3 alleles: e2, e3, e4

e4 most common

GAB2: additional risk allele in e4 apoE carriers

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AUTISMMZ: 80-92%, DZ: 1-10%

Sibling recurrence risk : 2-6%

Synapse formation & maintainence

FMR1 (Xq27.3 m RNA translation at synapse)TSC1 (9q34), TSC2 (16p13)- GTPase inactivation dendritic spine structure)MeCP2 (Retts syndrome, methylated DNA binding protein gene expression)NLGN3, 4 ( chr.X, Defective trafficking and synapse induction)SHANK3 ( chr.22q13, structural organization of dendritic spines)12/16/201673

2. cell migration

7q22 RELN gene- codes for reelin role in neuronal migration & neural connections

7q31 WNT2 gene- signaling proteins in regulation of cell fate and patterning during embryogenesis severe language abnormalities12/16/201674

3. Neurotransmittor systems

GABA : 15q11-13 duplication in ASD ( Angelman & Prader Willi)12/16/201675

BPADMZ: 65-100%, DZ: 10-30%

Family studies: models of multiple interacting genes

Linkage studies: Chr: X, 11

Chr: X: co-segregate with color blindness & G6PD deficiency

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BPADsusceptibilty loci : one on 18pone on 18q(22-23)One on 13qOne on 22qOne on 6qOne on 8q5q31-334q31, 6q249p21, 10p14-21

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Costa Rican pedigrees

Meta-analytic studies

Colombian, Costa Rican

Spanish, Romanian & Bulgarian

UK, Ireland

Major Depressive Disorder

MZ - 30%50% , DZ - 12% to 40%

GWAS: functional polymorphism modulating the serotonin transporter (5-HTTLPR) showed that this genetic polymorphism was a risk factor for developing MDD only in the setting of individuals with prior stressful life events12/16/201678

Schizophrenia Candidate genes:1. Dysbindin - DTNB1 (Chromosome: 6p24-22 Irish family studies)Implicated in synaptic structure & signalling

2. DISC 1, DISC 2( Chr 1q21-22 & Chr. 1q32-42 Scottish family studies, t 1 & 11 : A/W serious mental illness)DISC 1: NEURITE FORMATION12/16/201679

3. Neuregulin 1 NRG 1, CHR. 8p21-22, Icelandic families - neuronal growth factor, influences synapse formation, neuronal migration & neurotransmission, glutaminergic hypofunction through erbB4 disruption( postsynaptic target of neuregulin)4. AKT1 on chr. 145. COMT on chr. 22

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Pharmacogenetics

Pharmacogeneticsrefers to the study of the role of inheritance in inter-individual variation in drug response.

Tailor made therapy for an individual patient, maximizing the likelihood of response while minimizing the risk of toxicity

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Pharmacological-relevant genes

1. Pharmacokinetics-relevant genes: eg: cytochrome P450 enzymes 2D6 gene

Genotype-specific antidepressant and antipsychotic guidelines for dosing or drug selection for PMs and ultra-rapid metabolisers (UMs) have been proposed12/16/201682

2. Pharmacodynamics-relevant genes: serotonin receptor gene

The short allele of the functional promoter polymorphism (5HTTLPR) has been associated with reduced or delayed therapeutic benefit and increased adverse effects with SSRI treatment

Associations between dopamine receptor gene variants and antipsychotic response

Association of variants in the serotonin 2C receptor gene with antipsychotic-induced weight gain12/16/201683

Genetic counselingThis process integrates:

1. Interpretation of family and medical histories to assess the chance of disease occurrence or recurrence

2. Education about inheritance, testing, management, prevention, resources

3. Counseling to promote informed choices and adaptation to the risk or condition.

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Reasons for genetic testing:

1. Family history of a genetic condition or chromosome abnormality or cancer

2. Molecular test forsingle gene disorder

3. Increased maternal age (35 years and older)

4. Increased paternal age (40 years and older)

5. Abnormal maternal serum screening results or USG findings

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FUTURE DIRECTIVESUse of Endophenotypes for classification

Pharmacogenomic testing to identify ultra rapid metabolizers and poor metabolizers of cyt P450 genes (2D6)

Gene therapy & personalized medicines

HDAC inhibitors: IMIPRAMINE increases histone acetylation AD action VALPROIC ACID is a potent inhibitor of class 1 & 2 HDACs12/16/201686

5. DNMT inhibitors: AZACITIDINE, ZEBULARINE, DOXORUBICIN inhibit DNMT1 & DNMT3 and decrease DNA methylation of the reelin promoter in neural progenitor cells12/16/201687

Conclusion

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12/16/201689THANKYOU