Are Linkage Studies Boring? by Thomas D. Bird Nature Genetics July 1993

Transcription

1 6/2/10 Are Linkage Studies Boring? by Thomas D. Bird Nature Genetics July 1993 A colleague told me recently that linkage studies are boring. By this he meant they are easy to do, tedious and produce little information. I disagree. I find genetic linkage studies to be challenging, fascinating and valuable. The two linkage studies concerning the hereditary ataxias in the present issue of Nature Genetics are a case in point. Some historical perspective is helpful. As recently as 1980, only foolish faculty and fellows with extra time on their hands dabbled in (human) linkage studies. There were so few genetic markers that the likelihood of a hit was very small Then Botstein and colleagues announced the potential use of restriction fragment length polymorphisms for linkage.. 1

2 Why map genes? Some reasons: 1. Genetic mapping provides basic information about the organization of a chromosome and the physical context of a gene. Who are its neighbors? This basic information a. may be critical to understanding the regulation of a gene s expression (transcription) b. may be critical for strain construction and for designing good experiments 2. Genetic mapping provides basic information about the arrangement of the genome that is critical for the study of gene, gene family and genome evolution 3. Genetic mapping has been a critical first step in the cloning of (that is the biochemical purification of) many genes by a process called positional cloning 2

3 Using linkage analysis as a first step in cloning a gene Analysis of genes at the molecular level typically requires that the gene or at least portions of the gene be available in a purified form. Cloning (in a molecular context) means identifying and purifying a specific gene HOW to find a specific gene among the 3 billion pairs & twentysomeodd thousand genes that comprise the human genome? A number of strategies are available for cloning specific human genes What strategy you use depends to a great extent on the type of information you have at the outset Do you know what protein the gene produces? Do you have a clone of a related gene from another organism? 3

4 How do you clone a gene of interest if you have essentially no information about the gene except the mutant phenotype? In other words, you know what happens to the organism if a mutant allele is present but you don t have any idea what kind of protein the gene specifies don t have any information on the DNA sequence of the gene don t have an animal model This was the case for the geneticists who wanted to clone the genes responsible for cystic fibrosis, for Huntington disease and for many other human disease states: they had no idea what kind of protein the gene coded for 4

5 Used a strategy called positional cloning: See pg 735 in textbook Positional Cloning is Phenotype Driven: Genetic disease (or other trait) with specific phenotype Find map position of the Gene: Link to markers on a specific chromosome Then locate to specific site on this chromosome Isolate clones of genomic DNA that correspond to the map position of the gene Find candidate genes in the region closely linked to the disease phenotype Look for mutations in the candidate genes in individuals with the disease state 5

6 1cM = 1 centimorgan = 1 map unit = 1% of gametes are recombinant for the two genes/loci under consideration] LOD = log of odds = ratio of probability of linkage with stated map distance over probability of independent assortment. For example, a LOD = 3 means that linkage is a 1000 X more likely than independent assortment (OPTIONAL but recommended: see pg for a nice discussion of how LOD is calculated using the basic rules of probability and linkage) 6

7 Rather than looking at linkage or independent assortment of two organismal phenotypes, these studies look linkage between a disease or variant phenotype and molecular markers that tag specific sites on each chromosome FIRST STEP: random genome-wide screen with STR & SNP markers on all 22 autosomes to determine approximate location of gene of interest Useful molecular markers: STR fingerprint loci: highly polymorphic and easy to assay SNP = single nucleotide polymorphism (biallelic if using restriction enzymes to assay) 7

8 Detecting SNPs with restriction enzymes RFLP= restriction fragment length polymorphism P1 B1 GAATTC CTTAAG P2 P1 B2 GACTTC CTGAAG P2 SNP detected by PCR with primers 1 and 2 followed by digestion with the restriction enzyme EcoRI which only cuts at GAATTC 8

9 Figure 4-20 in text: Chromosome 1: 356cM & 247 Mbps 1 cm = 1 map unit Our genome is studded with STRs and SNPs: 9

10 Does the dominant disease phenotype appear linked to SNP A (located on chromosome 2) or SNP B (located on chromosome 15) or neither? NOTE:For the study to be useful, the polymorphic loci must be heterozygous in the individuals carrying the mutant allele A1A2 A1A1 A1 & A2 = SNP A1A2 A2A2 A1A2 A1A2 A2A2 A2A2 A2A2 A1A2 A2A2 A1A2 10

11 B1B2 B2B2 B1 & B2 = SNP B1B2 B1B1 B1B1 B2B1 B1B1 B2B1 B2B1 B2B1 B2B1 B1B1 Using LOD (logarithm of odds) scores to assess assess linkage in human pedigrees (see pg of text and end of this lecture) 11

12 Linkage to chromosome 2 STR loci was established using PCR and STR primers 12

13 Haplotype: a chromosomal segment defined by the specific alleles that it carries a group of closely linked alleles that tend to be inherited together The alleles may be SNPs (detected as RLFPs), STRs (fingerprint loci) or other polymorphic loci As illustrated in the next few figures, haplotype analysis can be used to map human disease genes very accurately by finding polymorphisms that flank the gene of interest Figure 4.16 Using haplotypes to deduce gene position The mutant allele remains in linkage disequilibrium only with SNPs 4 and 5. Hence, the gene must be in the vicinity of 4 and 5. 13

14 Each of eight different chromosome 2 STR loci were genotyped for each person How do these data confirm linkage to autosome 2? How do these data define the interval on chromosome 2 that must contain the disease gene? 14

15 The data are displayed in a standard haplotype format For example, individual V-1 in the third generation is heterozygous for alleles 6 and 2 for STR locus ---, for alleles 4 and 3 for STR locus ---- The loci are listed top to bottom in the order that they are found on chromosome 2 All alleles on the left are on one homolog of 2 and all alleles on the right are on the homolog 15

16 Remember that the STR or SNP markers used for these haplotype studies are linked to but otherwise unrelated to the disease mutation and so can be uncoupled from the disease phenotype by crossing over D allele P1 B1 GAATTC CTTAAG D= dominant disease allele d allele P1 B2 GACTTC P2 d = recessive wild-type alelle CTGAAG P2 SNP detected by PCR with primers 1 and 2 followed by digestion with the restriction enzyme EcoRI which only cuts at GAATTC NOTE: the SNP sequence variation is linked to but otherwise unrelated to the disease gene 16

17 EXAMINE DATA ON NEXT PAGE Taking the segregation data from all of the families together, in what portion of the haplotype is the gene mutated in erythermalgia located? Examine the haplotypes of heterozygotes in different families: why is the STR haplotype of the chromosome carrying the dominant mutant allele different in different families? 17

18 ~1 X 10 6 bp (1 Mb)/ centimorgan (cm) 18

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21 A previous study established the linkage of primary erythermalgia to a 7.94 cm interval on chromosome 2q. We report here the linkage of primary erythermalgia in a Chinese family to chromosome 2q24.2 q24.3. Critical recombination events in two patients in this family further defined the genetic region to 5.98 cm between D2S2370 and D2S2345. This genomic interval contains a cluster of sodium channel genes. We then identified two missense mutations in SCN9A in the family and a sporadic patient with primary erythermalgia. SCN9A, encoding a voltage-gated sodium channel alpha subunit mainly expressed in sensory and sympathetic neurones, may play an important role in nociception and vasomotor regulation. Our data suggest that mutations in SCN9A cause primary erythermalgia. 21

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23 (refers to no pain families see end of lecture) 23

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26 Positional Cloning is Phenotype Driven Remember this family? From article titled: Dominant modifier DFNM1 suppresses recessive deafness DFNB26 Nature Genetics 26: 431 December, % of childhood deafness is genetically determined About 30 different genes have for recessive deafness have been defined (complementation and other pieces of evidence) AS of 2000, only six of these genes had been characterized at a molecular level ie knew the protein product specified and the molecular nature of the defective alleles Researchers set out to identify the specific gene that was mutated in this family 26

27 141 members of a large consanguineous Pakistani family segregation deafness Black symbols: affected individual (deaf) and by DNA tests homozygous for a mutation in the gene DFNB26 Green symbols: these individuals have dodged the genetical bullet: they are homozygous (by DNA tests) for the DFNB26 mutation but have normal hearing 27

28 The researcher found that all normal individuals carrying the deafness mutation (d) also were heterozygous for another allelic variation (S) in a different gene called DRNM1 d + d + s + s + hearing d d s + s + deaf d d S s + hearing 28

29 BioEd Online ( December 13, 2006 The mutation that takes away pain by Michael Hopkin Imagine being unable to feel any pain at all. For a tiny handful of people, that is the reality and medical researchers have now pinpointed the mutation that removes their ability to perceive painful sensations. The study began when doctors in northern Pakistan examined a remarkable group of related families in which several individuals seem entirely unaffected by pain. Their attention was first attracted by one member of the clan, a locally famous boy who performed street theatre involving walking on burning coals and stabbing his arms with knives. Although it sounds like a party trick, the condition is devastating, as sufferers don't learn to know their limits. The street-performing boy killed himself on his fourteenth birthday after jumping off a house roof. The researchers studied six of his relatives, aged between 4 and 14 years. All had suffered many cuts and bruises, and injuries to lips and tongue caused by biting themselves; several had fractured bones without noticing. This shows the importance of pain for our health and survival, notes Geoffrey Woods of the Cambridge Institute for Medical Research, UK, who led the study. "Pain is there for a jolly good reason it stops us damaging ourselves," he says. For example, the pain from a broken arm or sprained ankle encourages us to rest that body part while it recovers. The children in the study had no such safety check, causing them to be both graceless and reckless. "One girl was continually knocked down in the playground and just didn't mind at all," Woods says. Nerve condition The researchers compared DNA samples from the six children and found that they all share a mutation in a gene called SCN9A, which is strongly expressed in nerve cells. They report their results in Nature. The SCN9A gene encodes a 'sodium channel': one of the structures that allows electrical charge to flow into nerve cells, triggering a signal, the researchers explain. Without this particular type of sodium channel, the brain does not receive any signal that the body has encountered a pain-causing stimulus. The discovery answers the question of whether the Pakistani subjects are truly unable to perceive pain or simply indifferent to it. 29

30 When the first ever pain-free patient was examined in the early twentieth century, some doctors thought that perhaps the condition involved a malfunction in the brain, rather than in the nerves. The Pakistan patients did seem to understand the concept of pain, and had picked up a knowledge of situations in which other people experience it. While being observed in a game of soccer, some of the older children in the study even acted as if in pain after being tackled. "Often, if you don't look as if you feel pain people think you're odd," Woods says. Oversensitive: Mutations in SCN9A are also involved at the other end of the pain spectrum, another recent study shows. Mutations that enhance, rather than inhibit, the protein's activation are at the root of paroxysmal extreme pain disorder, report researchers led by Mark Gardiner of University College London in the journal Neuron. It therefore seems that SCN9A acts as a "major trigger" of pain, says Woods. "It's amazing that we missed it for so long and then out of the woodwork come these two disorders," he says. This could offer potential new ways to treat severe pain. Current methods, such as local anaesthetics, are impractical, and constantly taking opiate painkillers can lead to addiction. Targeting SCN9A, perhaps through gene therapy, could also help sufferers of constant extreme pain from injuries, arthritis, spinal conditions or cancer. But, as the Pakistani subjects showed, the new discovery is of no use in tackling that perennial human condition, emotional pain. "They can blush and cry, and when they have flu they feel unwell," he says. "And they are hurt by rejection just the same as anyone." 1 Cox J. J., et al. Nature, (2006). An SCN9A channelpathy causes congenital inability to experience pain 2 Fertleman C. R. Neuron, (2006). Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia 30

31 Using LOD (logarithm of odds) scores to assess assess linkage in human pedigrees (see pg of text) Logarithm of odds score (LOD): a way of supporting a linkage hypothesis with small sample size (typical in human pedigree analysis.) Are these loci linked? 2 of 6 offspring are recombinants a recombination frequency of 33%. Are these loci unlinked? Maybe small sample size has skewed our interpretation so calculate the probability of obtaining 2 recombinant and 4 parental configurations under the condition that the RF=50% When you calculate a LOD score, you express the odds of linkage under a certain RF and the odds of getting a particular combination of progeny under independent assortment as a ratio, and take the logarithm of that ratio. Logarithms are exponents of base 10, so a LOD score of 3 means an RF value that is 10 3 as likely as no linkage. LOD = log10 of [likelihood if linked]/[likelihood if the loci are unlinked] 31

32 Lod score calculation Assume you have a testcross family with 8 non-recombinant offspring and zero recombinants between genes B and N Then, start with a hypothesis about linkage: maybe they are 10 cm apart What is the probability of no recombination for each gamete? 1-r = prob(no recombination) = 0.90 Remember there are two different parental genotypes. If you keep track of individual genotypes, then each has probability (1-r)/2 so, (1-r)/2 = 0.45 Probability of 8 non-recombinants if genes are linked (at 10 cm) is: Probability if not linked is remember, r=0.5 when loci are not linked Lod score = log10 (0.458/0.258) = log10 (110.19) = 2.04 The pattern of 8 non-recombinants is 110 times more likely if the genes are linked at 10 cm than if they were unlinked (LOD must be >3.0 to be significant ) 32