Genetic Linkage Analysis

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 disease 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 neurodegenerative 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 generated 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 (␪max) 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 ␪ =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

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. Three-generation pedigree segregating an autosomal dominant trait. Alleles at 3 marker loci designated A, B, and C are shown. Squares indicate males; circles, females; open symbols, normal phenotype; and solid symbols, disease phenotype.

specific chromosomal segments. Logarithm of the Odds Scores Calculated Using the MLINK Program5 Chromosomal regions with in- Under the Assumption of Autosomal Dominant Inheritance creased recombination are referred and a Disease Frequency of 1 in 100 000 to as “recombination hot spots.” The Recombination Fractions total human genome contains about 3700 cM.8 Genetic maps of the hu- Locus Penetrance 0 0.01 0.05 0.1 0.2 0.3 0.4 man genome have provided the A 1.0 −ϱ 1.0 1.5 1.6 1.4 1.1 0.6 backbone for the construction of 0.5 1.8 1.8 1.7 1.6 1.2 0.9 0.5 physical maps of human chromo- somes. Genetic maps are now avail- B 1.0 3.0 2.9 2.8 2.6 2.0 1.5 0.8 able to map disease traits in model 0.5 2.1 2.1 1.9 1.8 1.4 1.0 0.5 organisms such as Caenorhabditis el- C 1.0 −ϱ −5.0 −2.3 −1.3 −0.4 0.0 0.1 egans, zebra fish, and the mouse. The 0.5 −4.9 −2.5 −1.1 −0.6 −0.1 0.0 0.1 latest versions of human genetic and physical maps can be found elec- tronically.9 DNA polymorphisms based on the potential to detect hundreds of microsatellite repeats can be easily single-nucleotide polymorphisms IDENTIFICATION OF GENETIC detected. The DNA sequence flank- at a time. LOCI FOR NEUROLOGIC ing the repeat is used to synthesize DISEASES complementary oligonucleotide GENETIC MAPS primers (Figure 2, top). After PCR The localization of mendelian dis- amplification, the alleles of differ- The discovery of STRs made it fea- ease genes on genetic maps has pro- ent lengths are resolved by electro- sible to generate a large number of vided the first step toward the even- phoresis (Figure 2, bottom). For ge- markers and then map them by ge- tual isolation of these genes. Positive nome searches several STR markers netic linkage analysis. In contrast to linkage also provides the formal evi- can be amplified by differential fluo- the physical map that measures dis- dence that the particular disease is in- rochrome labeling and simulta- tances in base pairs or a karyotypic deed inherited as a mendelian trait. neously detected. map that assigns markers to chro- Even a couple of years ago it would Single-nucleotide polymor- mosomal bands, the genetic map have been possible to list the disease phisms have the disadvantage of gives distances in recombinational genes mapped by genetic linkage being bi-allelic and are thus not as units (the centiMorgan [cM]). On analysis, but in 1999, the number of highly polymorphic as STRs. average 1 cM corresponds to 1 mil- disease loci is far too great and in- However, they represent the most lion base pairs. Male and female re- cludes all classes of neurologic dis- frequent type of polymorphism combination rates are not identi- ease. After a disease gene is local- and their detection can be auto- cal, and there are significant changes ized to a specific chromosomal mated.7 Genotyping chips have in the rate of recombination along region, fine mapping is greatly facili-

ARCH NEUROL / VOL 56, JUNE 1999 669

©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 5′- C A T GG T T A------3′- A G C G T GG T A C C A A T A T GC T CGA C C T C A C A C A C A C A C A C A C A C A C AGA A CG T C AG T T GC A T T A CGCGA C AGC A T GC A T C CT- 5′

5′- T C G C A C C A T GG T T A T A CGAGC T GGAG T G T G T G T G T G T G T G T G T G T C T T GC A T CGA A CG T A A T GCGC T G T CG T A CG T AGGA-3′ ------GCATGCAT-5′

5′- C A T GG T T A------3′- A G C G T GG T A C C A A T A T GC T CGA C C T C A C A C A C A C A C A C A C A C A C A C A C AGA A CG T C AG T T GC A T T A CGCGA C AGC A T GCATCC T-5′

5′- T C G C A C C A T GG T T A T A CGAGC T GGAG T G T G T G T G T G T G T G T G T G T G T G T C T T GC AG T C A A CG T A A T GCGC T G T CG T A CGTAGGA-3′ ------G C A T GC A T-5′

123456 7 8 9 10 11 12

Figure 2. Top, Schematic of a microsatellite locus; 2 alleles containing different (CA/GT)n tracts and the location of oligonucleotides used to amplify the polymorphic CA tract are shown. The oligonucleotides serve as primers for a DNA polymerase and 2 new templates are synthesized. At the beginning of the second cycle, the primers, supplied in molar excess, anneal to 2 templates each, at the third to 4 each and so on, resulting in exponential DNA amplification. The dotted line denotes the newly synthesized DNA strand. Bottom, An example of DNA analysis with a microsatellite marker: oligonucleotide primers radioactively labeled with phosphorus 32 flanking a CA repeat marker (AFM164ze3 in the D22S275 locus) are used to generate polymerase chain reaction amplicons. These are separated on a polyacrylamide gel and visualized by autoradiography. Note the great heterozygosity of this marker system. Mendelian inheritance of the alleles can be seen by comparison with the pedigree symbols provided above the autoradiograph. Squares indicate males; circles, females; open symbols, normal phenotype; and solid symbols, disease phenotype.

tated by the density of markers on to- served in consanguineous matings Allelic and Nonallelic day’s genetic maps. This is in stark and in highly inbred or geographi- Heterogeneity contrast to just a few years ago when cally confined populations. As a re- a research team had to generate new sult, affected individuals likely carry Even before a disease gene is actu- markers, order them within the can- identical mutant alleles, including ally isolated, knowledge of its chro- didate region, and then use the newly marker alleles that tightly flank the mosomal location allows important generated markers to define recom- disease locus. This implies that the studies. In families of sufficient size, binational breakpoints. candidate region can be identified by linkage to the same locus (locus ho- Although Figure 1 illustrates searching for marker loci that are ho- mogeneity) may be established. Ex- the use of genetic linkage analysis for mozygous in affected individuals. amples of different phenotypes map- a dominant trait, recessive traits can The shared homozygous allele can ping to the same locus, such as be mapped in a similar fashion. Re- even be detected when DNA samples Machado Joseph disease and spino- cessive mutations, especially rare re- from affected individuals are cerebellar ataxia type 311 as well as cessive alleles, are commonly ob- pooled.10 identical phenotypes mapping to dif-

ARCH NEUROL / VOL 56, JUNE 1999 670

A 2 3 4 5 3 5 2 4

B 3 5 2 4 2 3 4 5 Figure 3. Inheritance of alleles in 2 affected sib pairs. For marker A, no alleles are shared, which indicates that the chromosomal segment does not likely contain a gene contributing to the disease. However, for marker B, 1 allele is shared in the left pedigree, and both alleles are shared in the right pedigree. Squares indicate males; circles, females; open symbols, normal phenotype; and solid symbols, disease phenotype.

II:1 II:2 II:1 II:2

A 2 4 3 5 5 2 3 4

B 3 2 3 4 2 4 2 4

ferent locations, abound.12 Diseases markers on chromosome 22 flank- cost-effective because it can ex- with differing phenotypes but map- ing the NF2 gene. clude non–gene carriers from fur- ping to the same locus are called However, indirect testing is not ther screening. As with all genetic allelic because they are caused by dif- limited to diseases in which the caus- testing involving asymptomatic in- ferent mutations in the same gene. Fa- ative mutation has not yet been iden- dividuals, counseling protocols milial hemiplegic migraine, episodic tified. Since tracking of the disease should be followed. ataxia type 2, and spinocerebellar chromosome (or more precisely the ataxia type 6 are allelic because they chromosomal region that contains LINKAGE DISEQUILIBRIUM are caused by different mutations in the gene) can be performed inde- the same gene, a calcium channel gene pendently of the precise mutation Linkage disequilibrium refers to the on chromosome 19. causing the disease, indirect test- occurrence of specific alleles at 2 loci For the demonstration that 2 ing can be used for diseases where with a frequency greater than ex- disorders are not allelic, a rela- the disease is caused by a great num- pected by chance. If the alleles at lo- tively small pedigree can be suffi- ber of different mutations in a large cus A are a1 and a2 with frequen- cient as long as 1 recombination gene. Examples are NF1 or cases of cies of 0.7 and 0.3, and alleles at event for 2 markers flanking the dis- Duchenne muscular dystrophy in locus B are b1 and b2 with frequen- ease locus is detected. For ex- which the mutations are not de- cies of 0.6 and 0.4, the expected fre- ample, exclusion of linkage to all tected by multiplex exonic PCR. Pre- quencies of haplotypes would be known spinocerebellar ataxia loci in dictive accuracy for indirect testing a1b1, 0.42; a1b2, 0.28; a2b1, 0.18; a family with ataxia provided the im- is very high (with the caveats men- and a2b2, 0.12. Even if the 2 loci are petus to search for additional ataxia tioned below), when intragenic or closely linked, unrestricted recom- genes.13 The presence of nonallelic closely flanking markers are used. bination should result in allelic com- or locus heterogeneity has impor- For a large gene such as the DMD binations in the general population tant implications for molecular ge- gene that covers a genetic distance that are close to the frequencies given netic diagnosis using linked ge- of close to 10 cM, recombination be- above. When a particular combina- netic markers. tween the genetic marker and the tion occurs at a higher frequency, for disease mutation needs to be ex- example a2b2 at a frequency of 0.45, Genetic Testing Using Linked cluded by markers that flank the this is called linkage disequilib- Genetic Markers gene. These markers must be infor- rium. Linkage disequilibrium may mative (heterozygous in the af- result from natural selection or by Even before gene isolation, mark- fected or carrier parent) so that a re- chance. When a disease mutation ers closely flanking a disease gene combination event occurring within arises on a founder chromosome and can be used to track the mutation in the gene can be excluded. not much time has elapsed since the a pedigree. For indirect testing, As soon as markers linked to a mutational event, the disease muta- samples from at least 2 affected first- disease have been identified, the dis- tion will be in linkage disequilib- degree relatives are required in ad- ease can be studied in its presymp- rium with alleles from loci close to dition to the proband’s DNA. The ac- tomatic stage where only subtle the gene. Thus, linkage disequilib- curacy of the molecular diagnosis is biochemical or morphologic abnor- rium can be a powerful tool for ge- dependent on the correctness of di- malities can be detected. This early netic mapping. agnosis in all individuals used for study provides the opportunity to testing. For example, if the diagno- determine penetrance of a particu- ASSOCIATION sis of neurofibromatosis (NF) type lar mutation in that asymptomatic 2 is missed in affected family mem- gene carriers can be identified.14 In Linkage and association should not bers and the diagnosis of NF1 is diseases where the identification of be confused with one another. Link- made, indirect testing would em- asymptomatic gene carriers can age refers to the relationship of loci, ploy markers on chromosome 17 avoid painful or costly screening pro- whereas association refers to the re- flanking the NF1 gene instead of cedures, molecular genetic testing is lationship of alleles at a frequency

ARCH NEUROL / VOL 56, JUNE 1999 671

©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 greater than predicted by chance. To THE FUTURE OF GENETIC Reprints: Stefan M. Pulst, MD, study association, one has to deter- LINKAGE ANALYSIS Division of Neurology and Rose Moss mine allele frequencies in unre- Laboratory for Parkinson’s and Neu- lated cases and compare them with “Are Linkage Studies Boring” was rodegenerative Diseases, Cedars- the allele frequencies found in con- the title of an editorial commenting Sinai Medical Center, the University trols. Association studies may point on the fact that linkage techniques of California, Los Angeles School of to genetic factors involved in the had evolved so rapidly that the Medicine, 8631 W Third St, 1145 E, pathogenesis or susceptibility of a scientific questions answered by Los Angeles, CA 90048 (e-mail: disease. One of the best-studied as- these methods appeared to have [email protected]). sociations of a protein allele and a become trivial.16 What the author common disease is the association concluded then remains valid 5 of the e4 allele at the apolipopro- years later: genetic linkage analysis REFERENCES tein E locus with age of onset in Alz- still remains the cornerstone of dis- heimer disease.12 Association can ease gene localization, and it will 1. Blumberg RB. Mendel Web Archive. Available at: http://www.netspace.org/MendelWeb/ also be seen with very close linkage not lose its utility even after the MWpaptoc.html. Accessed November 8, 1998. in the general population, given that human genome is sequenced in its 2. Bird TD, Ott J, Giblett ER. Evidence for linkage of Charcot-Marie-Tooth neuropathy to the Duffy lo- the disease mutation arose on a entirety. However, in the area of cus on chromosome 1. Am J Hum Genet. 1982; shared ancestral chromosome (see neurogenetic testing, it can be pre- 34:388-394. above). Great caution needs to be ex- dicted that indirect testing will be 3. Botstein D, White RL, Skolnick M, Davis RW. Con- struction of a genetic linkage map in man using ercised to exclude population strati- increasingly replaced by more effi- restriction fragment length polymorphisms. Am fication when individuals with the cient and less costly direct-testing J Hum Genet. 1980;32:314-331. disease belong to a genetically dif- methods. 4. Morton NE. Sequential tests for the detection of linkage. Am J Hum Genet. 1955;7:277-318. ferent subset of the population. Novel genetic markers, auto- 5. Terwiliger J, Ott J. Handbook for Human Genetic mated genotyping, and new math- Linkage. Baltimore, Md: Johns Hopkins Univer- sity Press; 1994. MODEL-FREE GENETIC ematical models now permit appli- 6. Weber JL, May PE. Abundant class of human DNA ANALYSIS cation of genetic linkage analysis to polymorphisms which can be typed using the poly- nonmendelian (complex) diseases. merase chain reaction. Am J Hum Genet. 1989; 44:388-396. For many complex diseases, a pre- But even for mendelian diseases, 7. Wang DG, Fan JB, Siao CJ, et al. Large-scale iden- cise genetic model cannot be speci- new questions can be answered, tification, mapping and genotyping of single- fied, which makes lod score analysis such as the identification of modi- nucleotide polymorphisms in the human genome. Science. 1998;280:1077-1082. invalid. Although less powerful than fying loci that explain variability 8. Dib C, Faure S, Fizames C, et al. A comprehensive lod score analysis, nonmendelian dis- in the phenotype, reduced pen- genetic map of the human genome based on 5,264 microsatellites. Nature. 1996;380:152-154. eases can be analyzed by studying af- etrance, or variability in age of 9. The Cooperative Human Linkage Center. Avail- fected sibling pairs. For a randomly onset. Genetic linkage analysis is able at http://www.chlc.org/ChlcMaps.html. Ac- chosen marker, sibs are expected to here to stay and has not lost any of cessed November 8, 1998. 10. Nystuen A, Benke PJ, Merren J, Stone EM, Shef- share both parental alleles 25%, 1 al- its luster. field VC. A cerebellar ataxia locus identified by DNA lele 50%, and no allele 25% of the pooling to search for linkage disequilibrium in an time (Figure 3). It is obvious that isolated population from the Cayman Islands. Hum Mol Genet. 1996;5:525-531. a large number of sib pairs must be Accepted for publication December 15, 11. Twist EC, Casaubon LK, Ruttledge MH, et al. studied before a statistically signifi- 1998. Machado Joseph disease maps to the same region of chromosome 14 as the spinocerebellar cant deviation from the expected al- The research for this article was ataxia type 3 locus. J Med Genet. 1995;32:25- lele distribution can be identified, es- supported by Friends of Research, In- 31. pecially when a large number of genes vestigation, Education of Neurologic 12. Tanzi RE, Blacker D, et al. The genetics of Alzhei- mer disease: current status and future pros- contribute to the phenotype or when Diseases, the Drown Foundation, the pects. Arch Neurol. 1998;55:294-296. genetic factors interact with environ- Warschaw Endowment for Neurol- 13. Zu L, Grewal R, Figueroa KP, et al. Mapping of a mental stressors. Although sib-pair ogy, Los Angeles, Calif, and grants new autosomal dominant ataxia (SCA 10) to chromosome 22. Am J Hum Genet. 1999;64:594-599. analysis was successful in identify- RO1 NS33123 and RO1 NS37883 14. Baser ME, Mautner VF, Ragge et al. Presymp- ing susceptibility loci in multiple from the National Institutes of Health, tomatic diagnosis of neurofibromatosis 2 using linked genetic markers, neuroimaging, and ocu- sclerosis, only a subset of loci was Bethesda, Md. lar examinations. Neurology. 1996;47:1269- shared by 3 independent studies, I gratefully acknowledge the 1277. which indicates the necessity to vali- members of the families who have con- 15. Bell JI, Lathrop GM. Multiple loci for multiple sclerosis. Nat Genet. 1996;13:377-378. date susceptibility loci in indepen- tributed to past and ongoing genetic 16. Bird TD. Are linkage studies boring? Nat Genet. dent patient groups.15 linkage studies. 1993;4:213-214.

ARCH NEUROL / VOL 56, JUNE 1999 672