Lesson 5 Haplotypes & Consanguinity 23-24 pre-lesson.pdf

Haplotypes & Consanguinity MD210 – GGE – Genetics Lesson 5 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Understand why consanguineous mating can increase the risk for hereditary disorders • Demonstrate how molecular “bar coding” of haplotypes using microsatelli...

Haplotypes & Consanguinity MD210 – GGE – Genetics Lesson 5 1 Essential Learning Outcomes By the end of this lesson you should be able to: • Understand why consanguineous mating can increase the risk for hereditary disorders • Demonstrate how molecular “bar coding” of haplotypes using microsatellites is used to track hereditary disease in consanguineous lineages • Understand the principles behind the coefficient of relationship and coefficient of inbreeding and use them to calculate inheritance risk for autosomal recessive disorders in children of consanguineous couples • Understand the Hardy-Weinberg principle/equation and use it to calculate allele and genotype frequencies in a population in Hardy-Weinberg equilibrium 2 Choudhary Family • Parents Mumtaz (F) and Aadnan (M) • Daughter Nasreen is deaf since birth Family History • Parents are 1st cousins (consanguineous) • Mumtaz parents are also first cousins • Mumtaz is one of 5 siblings: 2 female and 3 male ( 2 male siblings are deaf) 3 Consanguinity • Being descended from the same ancestor as another person • Globally 8.5% of children have consanguineous parents • Endogamy is widely practiced across the greater Middle East Clinically: • Consanguineous marriage = matrimony (breeding) between two family members who are second cousins or closer • Associated with increased risk of autosomal recessive disorders 4 Consanguinity • Consanguinity increases risk of genetic disease • From about 2% • To about 4% • Probability of having a child without a constitutional congenital defect reduced from 98% to 96% • Advantages: preservation of identity, of property, of culture and tradition, partnerships with people from similar background, stronger family ties, financial advantages, bride protection 5 Choudhary Family Pedigree GGF GGM • The pedigree suggests what pattern of inheritance ? • Autosomal recessive deafness • Accounts for about 65% of congenital deafness • (infection is also important) Benazir Aadnan Mumtaz 10/40 6/40 Waleed Nasreen 6 INHERITED DEAFNESS • Syndromic & Non-syndromic • The pattern of inheritance can be: • • • • • Dominant (DFNA) – 20-25% Recessive (DFNB) – 75-80% X-linked (DFNX) – 1-2% matrilineal (mitochondrial) - <1% http://davinci.crg.es/deafness/index.php • Autosomal recessive patterns of inheritance are usually related to homozygous state for a defective allele Credit: bloginonline.com • It is believed that >100 genes could be implicated • So for this family where do you start ? 7 Gene Mapping • Assumption: it is likely that there is an inherited determinant of deafness in this family (original concept of a “bad gene”) • Mapping is about finding the DNA sequence that corresponds to this “gene” • Why is that important ? • For family planning 8 Hereditary Deafness-Common Genes • A single locus, DFNB1 (13q) accounts for a high proportion • The gene is GJB2 (connexin 26 protein)(Cx26) – 2 exons • c.35delG common mutation in Europeans • c.del235C common mutation in Chinese • How do we check it? • Short gene (2 exons) so amplification and sequencing is practical • BUT - In this family GJB2 is OK 9 Hereditary Conditions in a Family • We started by looking for 1 common mutation • We can continue to look for a small number of other common mutations that are described and technically relatively easy to test for • If no mutation detected in any of these what next ? • To try to amplify and sequence all the >100 known genes associated with hereditary deafness is quite a job (was nearly impossible) 10 Another look at Pedigree of Choudhary Family • Assume that one of Nasreen’s great grandparents was heterozygous for a gene critical to hearing GGF GGM • One of the 2 alleles was defective • We do not know which chromosome pair this hypothetical gene is on Benazir Aadnan Mumtaz Waleed 10/40 6/40 Nasreen 11 Pedigree of Choudhary Family GGF • Note all of this family will inherit at least one of only 4 versions of each autosome: 2 from GGM and 2 from GGF GGM • Children of consanguineous couples are at risk of inheriting 2 copies of the same defective chromosome (2 identical homologs) Benazir Aadnan Mumtaz Waleed • Assume Nasreen and her deaf uncles have each got 2 copies of the defective allele because of consanguineous matings 10/40 6/40 Nasreen • Homozygous 12 For Example GGF Chr 1 So Suppose: • Great grandfather (GGF) has 2 homologs of chromosome 1 (maternal - m and paternal - p) and p has a deletion associated with deafness • He is not affected because 1m works mp Nasreen Chr 1 • The basic hypothesis is that Nasreen has ended up with two copies of GGF’s defective Chromosome 1p pp 13 Pedigree of Choudhary Family • If life was simple (and it isn’t) and the hypothesis is correct • All 3 deaf individuals should have 2 practically identical copies of the relevant ancestral chromosome that carries the defective allele • However, crossing over between maternal and paternal means that many offspring do not inherit a full intact version • Should all be homozygous for a particular chunk (haplotype) of ancestral Chr that carries the defective allele Crossing over in Meiosis I • But which Chr/haplotype and how do we find it? 14 Tracking Chromosomes or Chunks of Chromosome by Molecular Barcoding • Using microsatellites and SNPs you can construct a “barcode” for each Chr or piece of Chr (haplotype). If the barcodes match in affected individuals – likely that the pathogenic mutation is in that general area 15 The Choudhary Family: What Next ? • Identify deafness-related haplotype by linkage analysis: • Perform genomic analysis of polymorphic DNA markers associated with deafness for all family members • See which markers are carried only by deaf members and never by healthy members (segregate with the disorder) • High probability that deafness causing gene is linked to this/these marker(s) (same location on chromosome) 16 Autozygosity mapping: 1. Identify Microsatellite Sequences Associated with deafness loci using dbSNP database https://www.ncbi.nlm.nih.gov/snp/ 2. Amplify DNA from all of the loci of all family members 3. Determine the size of the PCR products in each individual – affected individuals should all be homozygous for the same haplotype (set of markers) 17 Microsatellite polymorphism example – one locus Deafness Microsatellite Locus D2S174 (Chr 2) • Nasreen and her 2 uncles all 3 yield a single 99 bp product Mumtaz • Single products suggests that they are all homozygous for a particular Chr2 haplotype with same no. of repeats (13) at that specific micro-satellite locus 81 • May have 2 copies of an ancestral Chromosome 2 from a specific grandparent 81 90 99 108 Nasreen 90 99 108 Waleed • Mumtaz (Nasreen’s mother – not deaf) 81 • May have 2 different products (heterozygous) 1. A 99 bp product (ancestral Ch1) 2. Maybe an 81 bp product (7 repeats) from the corresponding locus on the other Ch1 homolog 90 99 108 Mohammed 81 90 99 108 18 Choudhary Family – Autozygosity Mapping Results Homolog 1 Homolog 2 Marker Chr Nasreen Waleed Mohammed H1 H2 H1 H2 H1 H2 D10S537 10q22 2 4 4 4 4 1 D10S1432 10q22 2 1 2 1 4 2 D2S174 2p22 6 6 6 6 6 6 D2S158 2p23 2 2 2 2 2 2 No. of repeats 19 Choudhary Family Results - Interpretation • Affected individuals are homozygous for a particular Chromosome 2 haplotype - a chunk of chromosome on the short arm (p) of Chr 2 • Suggests the defective allele is in the p22-p23 region of Chr 2 • The deafness associated locus there is OTOF (otoferlin) – 2p23.3 • DFNB9 – The 9th identified type of autosomal recessive non-syndromic hearing loss 20 Association is not causation NB – Linkage of a trait or disorder with a gene/DNA sequence does not imply that the gene/DNA sequence associated with the trait is important in the pathogenesis of the condition (association is not causation) • In this example the deafness associated locus was OTOF (otoferlin) – 2p23.3 and not it’s linked microsatellite marker Choudhary Family – Risk calculation • Questions • If we [Mumtaz (F) and Aadnan (M)] have more children how likely is it they will be deaf? • If Benazir (sister of Aadnan) and Waleed (brother of Mumtaz) plan to marry – what is the risk for their children? GGF GGM Benazir Aadnan Mumtaz Waleed 10/40 6/40 Nasreen 23 What we know • The family deafness is DFNB9 and is associated with OTOF • All 3 affected are homozygous • What is the risk that Nasreen’s sib will also be homozygous? 24 What proportion of genes do relatives share? Coefficient of Relationship (COR) (Sewall Wright): • COR = The proportion of alleles that 2 people share by virtue of having one or more definable common ancestors Coefficient of Inbreeding (COI): • The proportion of loci at which the person is expected to be homozygous because of the consanguinity of the parents or • The probability that at any given locus the person receives two alleles that are identical by descent • COI = half the coefficient of relationship of the parents 25 How can we calculate the COR? • For outbred families the calculation is easy: • Parents share 50% of alleles with their children • Full siblings share 50% • Grandparents share 25% with grandchildren • Uncle/Aunt share 25% with Nephew/Niece also • Cousins share 12.5% • For consanguineous lineages like the Choudhary family, it is trickier • (beyond the scope of this course) 26 By Citynoise - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=37723128 Allele Frequency • The incidence of a gene variant in a population Number of times the allele of interest is observed in a population Total number of copies of all the alleles at that locus in the population • Allele frequencies are a reflection of genetic diversity 28 Allele Frequency • Note this is not the same a saying 40% of the population have a copy of the allele • For example: • If 10% of 100 people are homozygous for allele R1 (20 copies), • and 20% are heterozygous and for allele R1 – (20 copies) • allele frequency is? 29 Hardy-Weinberg Equilibrium • The Hardy–Weinberg principle, also known as the Hardy–Weinberg equilibrium, model, theorem, or law, states that allele and genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary influences Hardy-Weinberg equation: (p + q)2 = 1 or p2 + 2pq + q2=1 • Where p = frequency of allele “A” and q = frequency of allele “a” 31 Hardy-Weinberg Equilibrium • Alleles distribute randomly only under certain assumptions including the absence of selection and random mating (a panmictic population) • When these conditions apply, a population is said to be in Hardy– Weinberg equilibrium. • Does this apply to the Choudhary family? 32 Hardy-Weinberg Equilibrium In clinical genetics it is mainly used to determine the frequency of heterozygosity (carrier frequency) in autosomal recessive heritable disorders where only the disease frequency (q2) is known p2 + 2pq + q2=1 or f(AA) + f(Aa) + f(aa) = 1 NB p+q=1 33 Hardy-Weinberg Equilibrium • Take as example gene R on Ch1 • Allele frequency (p) for R1 = 40% (0.4) • What proportion of the population have allele R1? p = 0.4, q = 0.6 (0.4)(0.4) + 2(0.4)(0.6) + (0.6)(0.6) = 1 (p + q = 1) (p2 + 2pq + q2=1) 0.16 + 0.48 + 0.36 = 1 (R1,R1) + (R1,Rx) +(Rx,Rx) = 1 16% are (R1,R1) homozygous and 48% are (R1,Rx) heterozygous Therefore 64% of the population have R1 34 Menti Problem – breakout groups Allele X on Ch 1 has allele frequency of 20% 1 in 5 of all copies of chromosome 1 have allele X In a population in Hardy-Weinberg Equilibrium what proportion of the population have allele X? 35 Things to Remember 1. Consanguineous mating is valued in some cultures and is associated with some increased risk of hereditary disease 2. Linkage of a trait or disorder with a gene/DNA sequence does not imply that the gene/sequence associated with the trait is important in the pathogenesis of the condition (association is not causation) 3. The Coefficient of Relationship (COR) is the proportion of alleles that 2 people share by virtue of having one or more definable common ancestors 4. The Coefficient of Inbreeding (COI) is the probability that at any given locus the child receives two alleles that are identical by descent because of parental consanguinity and is ½ the COR of the parents 5. Hardy Weinberg Law states that allele and genotype frequencies in a panmictic population will remain constant from generation to generation in the absence of other evolutionary influences (incl. selection) 37