BASIC SCIENCE SEMINARS IN NEUROLOGY SECTION EDITOR: HASSAN M. FATHALLAH-SHAYKH, MD Genetic Linkage Analysis Stefan M. Pulst, MD enetic linkage analysis is a powerful tool to detect the chromosomal location of dis- ease genes. It is based on the observation that genes that reside physically close on a chromosome remain linked during meiosis. For most neurologic diseases for which the underlying biochemical defect was not known, the identification of the chromo- Gsomal location of the disease gene was the first step in its eventual isolation. By now, genes that have been isolated in this way include examples from all types of neurologic diseases, from neu- rodegenerative diseases such as Alzheimer, Parkinson, or ataxias, to diseases of ion channels lead- ing to periodic paralysis or hemiplegic migraine, to tumor syndromes such as neurofibromatosis types 1 and 2. Arch Neurol. 1999;56:667-672 With the advent of new genetic markers tin gene, diagnosis using flanking mark- and automated genotyping, genetic map- ers requires the analysis of several family ping can be conducted extremely rap- members. idly. Genetic linkage maps have been gen- erated for the human genome and for LINKAGE OF GENES model organisms and have provided the basis for the construction of physical maps When Mendel observed an “independent that permit the rapid mapping of disease assortment of traits” (Mendel’s second traits. law), he was fortunate to have chosen traits As soon as a chromosomal location that were not localized close to one an- for a disease phenotype has been estab- other on the same chromosome.1 Subse- lished, genetic linkage analysis helps quent studies revealed that many genes determine whether the disease pheno- were indeed linked, ie, that traits did not type is only caused by mutation in a assort or segregate independently, but that single gene or mutations in other genes traits encoded by these linked genes were can give rise to an identical or similar inherited together. Studying Drosophila ge- phenotype. Often it is found that similar netics, T. H. Morgan showed that the de- phenotypes can be caused by mutations gree of linkage increased with physical in very different genes. Good examples proximity of the genes and that the 4 ge- are the autosomal dominant spinocer- netic linkage groups actually corre- ebellar ataxias, which are caused by sponded to the presence of 4 chromo- mutations in different genes but have somes in Drosophila. very similar phenotypes. The first trait in humans linked to a In addition to providing novel, geno- chromosome was actually sex itself. This type-based classifications of neurologic dis- was followed by linkage of the Duffy lo- eases, genetic linkage analysis can aid in cus to chromosome 1 after the observa- diagnosis. However, in contrast to direct tion that certain Duffy blood group alle- mutational analysis such as detection of les were linked to a microscopically visible an expanded CAG repeat in the Hunting- chromosome 1 polymorphism. Interest- ingly, the Duffy locus was also the first pro- From the Division of Neurology and Rose Moss Laboratory for Parkinson’s and tein polymorphism linked to a neuro- Neurodegenerative Diseases, Cedars-Sinai Medical Center, University of California at logic disease, the Charcot-Marie-Tooth Los Angeles School of Medicine, Los Angeles. locus on chromosome 1, now called ARCH NEUROL / VOL 56, JUNE 1999 667 ©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 missing family members and re- Glossary duced penetrance assumptions, Allele: one of several forms of a DNA sequence at a specific computer programs such as MLINK 5 chromosomal locus. or LIPED are necessary. Genotype: the 2 types of alleles at a given chromosomal locus in an A lod score higher than 3.0 is individual. generally accepted as evidence for linkage, when the human genome is Haplotype: the string of alleles at linked (adjacent) loci on a single screened with 100 to 300 markers. chromosome. Although a lod score of 3 would trans- Locus: unique chromosomal location of a DNA sequence or gene. late into odds of 1000:1 favoring link- Lod score: statistical measure of the likelihood of genetic linkage age, the corresponding significance between two loci. level is closer to P = .05 owing to the Marker: polymorphic protein or DNA sequence at a specific calculation of linkage for multiple chromosomal locus. markers with the concomitant Morgan: unit of genetic distance. increase in observing a positive lod Phenocopy: Phenotype identical to the one caused by gene under study, score by chance. A lod score lower but caused by another gene or environmental factors. than −2 is accepted as evidence Phenotype: physical or biochemical characteristics of an organism or cell. against linkage. Lod score analysis requires the assumption of precise Polymorphism: presence of 2 or more alleles at a locus. genetic models, including pen- Recombination fraction: percentage of offspring who are recombinant. etrance, disease gene frequency, and the clear classification of individuals as affected or unaffected. Thus, the CMT1B.2 With the ability to detect LOGARITHM OF THE ODDS lod scores in the Table undergo sig- DNA polymorphisms, the study of SCORE ANALYSIS nificant changes when penetrance is genetic linkage blossomed, be- reduced to 0.5. Similarly, the misdi- cause polymorphisms were not lim- It is intuitively obvious that the ob- agnosis of 1 individual or the pres- ited to the relatively rare protein servation of linkage in 4 meioses is ence of phenocopies can drastically polymorphisms.3 less significant than the observation affect the lod score and the calcu- The segregation of an autoso- of linkage in 20 meioses. A measure lated location of the disease gene. mal dominant disease trait and alle- for the likelihood of linkage is the Instead of calculating lod scores les at 3 marker loci is illustrated in logarithm of the odds (lod) score. The between 2 loci at a time, it is also Figure 1. The markers are per- lod score Z is the logarithm of the possible to calculate maximum like- fectly informative, since individual odds that the loci are linked divided lihood estimates for multiple loci at II-1 is heterozygous at the 3-marker by the odds that the loci are un- a time. Thus, it is possible to order loci. Comparison of haplotypes of in- linked.4 Expression of the likeli- loci and to place a disease locus on dividuals I-1 and II-2 indicates that the hood as a logarithm allows summa- a map of ordered genetic marker loci. 1-2-4 haplotype marks the chromo- tion of likelihoods observed in This analysis also compensates for some with the disease mutation. The different pedigrees. noninformativeness of markers in 2 allele of marker B shows perfect co- Since the true genetic distance specific meioses.5 segregation with the disease trait, between 2 loci is frequently un- whereas the 1 allele cosegregates with known, the lod score is calculated GENETIC MARKERS the wild-type (normal) phenotype. for different recombination frac- Marker A shows one recombina- tions providing a maximum likeli- Before the advent of the polymer- tion event in the unaffected indi- hood estimate for the recombina- ase chain reaction (PCR), the most vidual III-10, whereas marker C tion fraction (umax) at which the commonly used procedure to de- detects multiple recombination greatest lod score (Zmax)isob- tect DNA sequence differences in- events. Thus, the disease trait shows served. Pairwise lod scores for the volved the use of DNA restriction linkage to markers A and B, but it 3 markers in Figure 1 and the dis- enzymes and Southern blotting (re- is unlinked to marker C. It is also in- ease trait are shown at fixed recom- striction fragment length polymor- teresting to examine the recombi- bination fractions (Table). Since no phisms).3 With the discovery of the nation occurring between the marker recombination events occurred PCR, a novel class of short tandem loci. Three recombination events are between marker locus B and the dis- repeat (STR) polymorphisms was 6 observed between markers A and B: ease, the Zmax is observed at u =0. discovered. The STRs are repeated 2 occurred on the maternal chro- Due to the 1 recombination event dinucleotide, trinucleotide, or tet- mosome, 1 on the paternal. Thus, the observed in III-10, the most likely ranucleotide sequences such as maternal recombination fraction is distance between marker A and the (CA)n or (GATA)n (Figure 2, top); 20%; and the paternal, 10%, giving disease locus is calculated to be at a STR loci are multiallelic, which in- a sex-averaged recombination frac- recombination fraction of 10%. For creases the probability that a given tion of 15%. Between marker loci B perfect families, lod score calcula- individual is heterozygous and thus and C, 8 recombination events are tions can be carried out by hand. that the paternal and maternal al- detected. However, for real-life pedigrees with leles can be distinguished. ARCH NEUROL / VOL 56, JUNE 1999 668 ©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 I:1 I:2 A 1 3 A 2 3 B 2 3 B 1 4 C 4 4 C 2 4 II:1 II:2 A 1 2 A 3 4 B 2 1 B 5 4 C 4 2 C 3 1 III:1 III:2 III:3 III:4 III:5 III:6 III:7 III:8 III:9 III:10 A 1 3 A 2 4 A 1 4 A 2 3 A 1 3 A 2 3 A 1 4 A 2 4 A 1 4 A 1 3 B 2 5 B 1 5 B 2 4 B 1 4 B 2 5 B 1 5 B 2 4 B 1 4 B 2 4 B 1 5 C 4 3 C 2 3 C 2 1 C 4 3 C 4 3 C 4 3 C 2 3 C 2 3 C 4 3 C 2 3 Figure 1.