Am. J. Hum. Genet. 50:1211-1217, 1992

Confirmation of Genetic Heterogeneity in Limb-Girdle Muscular Dystrophy: Linkage of an Autosomal Dominant Form to Chromosome Sq Marcy C. Speer,*,t,1 Larry H. Yamaoka,* James H. Gilchrist,§ C. P. Gaskell,* Jeffrey M. Stajich,* Jeffery M. Vance,* Alexey Kazantsev,* Anselmo A. Lastra, Carol S. Haynes, * Jacques S. Beckmann, # Daniel Cohen, # James L. Weber, * * Allen D. Roses,* T and Margaret A. Pericak-Vance*

*Division of Neurology, fUniversity Program in , and tDepartment of Zoology, Duke University, Durham, NC; §Department of Neurology, Rhode Island Hospital and Brown University, Providence; IlDepartment of Computer Science, University of North Carolina, Chapel Hill; #Centre d'Etude du Polymorphisme Humain, Paris; and **Marshfield Medical Research Foundation, Marshfield, WI

Summary Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogeneous group of disorders, with both recessive and dominant forms reported. Recently, a series of recessive LGMD families were linked to chromosome 1 5q. We report herein the results of our linkage studies in a previously reported large autosomal dominant family. The LGMD in this family was localized to chromosome 5q22.3-31.3 by using a series of CA(n) microsatellite repeat markers. Linkage to 15q was excluded. These findings confirm genetic heterogeneity in this clinically diverse syndrome.

Introduction Initial reports of LGMD described families with re- Limb-girdle muscular dystrophy (LGMD) is a progres- cessive inheritance (LGM2; MIM 253600) (Jackson sive muscular dystrophy with weakness beginning in and Strehler 1968; Shokeir and Kobrinsky 1976) and the hip and shoulder girdle and later progressing to the sporadic cases (Morton and Chung 1959). Later re- distal muscles. The LGMD diagnostic classification ports described families with dominant forms (LGM1; is ill-defined and heterogeneous (Morton and Chung MIM 159000) (Chutkow et al. 1986; Gilchrist et al. 1959; Brooke 1986). The diagnosis is clouded by tre- 1988). Thus, there is clearly heterogeneity in the mode mendous variability in expression, as well as by of genetic transmission in LGMD. Clinical heteroge- asymptomatic obligate heterozygotes in autosomal neity is also present, as evidenced both by the variation dominant families (DeCoster et al. 1974). The diagno- in age at onset and rate of progression and, to a lesser sis is further complicated in that other neurogenic/ extent, by the distribution of muscle weakness. Clini- myopathic disorders, particularly Becker muscular cal heterogeneity is present both within the dominant dystrophy (BMD) and Kugelberg-Welander syndrome class and between the dominant and recessive classes. (Kazakov et al. 1977; Coers et al. 1979), may clini- Recently, Beckmann et al. (1991) reported linkage cally mimic LGMD. Fascioscapulohumeral muscular of a series ofrecessive (LGM2) families, collected from dystrophy (FSHMD) should also be included in the the Isle of La Reunion, to the anonymous marker of LGMD (Chyatte et al. 1966). D1SS2S (pTHH114), with a maximum LOD score (Z) of 5.52 at recombination fraction (0) = 0. Studies ReceivedJuly 30, 1991; final revision received January 24, 1992. by Young et al. (1991) have subsequently supported Address for correspondence and reprints: Margaret A. Pericak- this localization in a large, highly inbred Amish family Vance, Ph.D., Division of Neurology, Box 2900, Duke University (Jackson and Strehler 1968). Linkage studies reported Medical Center, Durham, NC 27710. © 1992 by The American Society of . All rights reserved. by Gilchrist et al. (1988) and Speer et al. (1989) failed 0002-9297/92/5006-0008$02.00 to find linkage in this form of LGMD. We report, in

1211 1212 Speer et al. this family, evidence for linkage to chromosome 5q fied from those of Weber and May (1989). The stan- and exclusion of linkage to chromosome 15q. dard PCR contained 0.03 jg of genomic DNA and 0.06 Rg of each primer in a volume of 15 gl. Amplifi- cation (27 cycles) was carried out in microtiter plates Subjects and Methods (Stratagene SCS 96 thermal cycler) with denaturing for 940C (1 min), 550C (2 min), 72"C (1 min). The Family 39: Ascertainment and Diagnostic Criteria final extension was for 6 min at 720C. After amplifi- This large LGM1 family (fig. 1) was ascertained cation, the products were separated on 6.5% denatur- through the Duke University Medical Center Neuro- ing polyacrylamide gels (IBI STS-45). Markers used muscular Research and Muscular Dystrophy Associa- for linkage of LGM1 to chromosome 5 are listed in tion Clinic. A detailed clinical description ofthe family table 1. has been reported elsewhere (Gilchrist et al. 1988). The family has been substantially expanded since the Statistical Analysis original report. Blood from 218 individuals was ob- LGM1 was analyzed as an autosomal dominant tained for creatine kinase testing, DNA extraction, trait with age-dependent . The gene fre- and lymphoblastoid cell line establishment. quency for LGM1 was fixed at 1/10,000. Two-point The diagnostic criteria for LGM1 were proximal leg Z values were calculated with the computer program weakness with or without proximal arm weakness, LIPED (Ott 1974). Allele frequencies for the RFLP absent ankle deep-tendon reflexes, and elevated cre- markers used were those published elsewhere (Kidd et atine kinase values or creatine kinase MB fractions. al. (1989) but did not deviate from those in our sample Obligate heterozygotes were classified as affected, in population. Allele frequencies for the dinucleotide re- the linkage analysis. Individuals with normal exami- peat markers were generated from a series of at least nations and with creatine kinase levels within normal 110 unrelated individuals (table 1). To facilitate com- limits were assigned heterozygosity risks based on puter analysis, the genotype results for the dinucleo- their age at examination. The probability of carrying tide repeat markers D5S119, DSS210, D5S207, and the LGM1 gene was generated from a normal distribu- IL-9 were recycled as suggested by Ott (1978), to mini- tion by assuming that the mean age at onset in this mize the number ofalleles while preserving the linkage family was 27.1 years (a = 8.5 years). Family mem- information. Support intervals about the estimate of bers who had signs or symptoms suggestive of LGM1 0 were calculated using the "one LOD score down" but who did not meet the strictly defined diagnostic method (Conneally et al. 1985). The analyses were criteria were considered to be of unknown disease sta- repeated using a low-risk penetrance for all unaffected tus. All spouses were considered normal with respect family members (i.e., an affecteds-only analysis). For to clinical status. This study includes genotype results this analysis, genotype information was preserved on on 218 individuals, 49 of whom were affected, 103 of all unaffected family members while eliminating the whom had normal examination, 28 of whom were phenotypic information regarding disease status, in considered of unknown disease status for this evalua- order to evaluate the contribution of known affecteds. tion, and 38 of whom were unrelated spouses. A genetic map for the markers D5S119, D5S207, D5S210, IL-9, and D5S70 was not available. There- Genotype Analysis fore, we attempted to generate a map based on the DNA was isolated from leukocytes or from trans- available information within the family. Map genera- formed cell lines. After digestion with restriction en- tion via a full likelihood analysis on the intact pedigree zymes, the DNA was transferred to Gene Screen Plus by the ILINK subprogram ofthe LINKAGE computer (NEN) and hybridized in 50O formamide according package on a 4/370 Sun workstation was not feasible, to methods described elsewhere (Yamaoka et al. for computational reasons. Reducing the family to its 1990). Polymorphic DNA markers were obtained nuclear components to facilitate mapping efforts led from the American Type Culture Collection or from to the loss of a considerable amount of information the investigators who developed them. Paternity and (e.g., for D5S210-D5S70 and D5S70-LGMD, the sample integrity were confirmed with the anonymous peak Z values were reduced by 25% and 71 %, respec- probe D2S44 (pYNH24) (Nakamura et al. 1987). tively). Thus, multiple pairwise mapping techniques Dinucleotide (CA) repeat polymorphism studies (Morton and Collins 1989) were used to generate a were carried out according to methods slightly modi- genetic map of this region. U

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Table I Markers with Allele Frequencies (for Microsatellite Repeat Markers) Used for Linkage of LGMD to Chromosome S Locus Marker Chromosomal Localization Fragment Sizea (% observed) Reference

DSS119 ...... Mfd6 5q31.1-31.1 202 bp(2) 200 bp(6) 198 bp (2) Weber et al., submitted 194bp(4) 192 bp (63) 190 bp (23) J

D5S207 ...... Mfd43 5q31.1-31.3 141 bp(32) 139 bp (52) Weber et al., submitted 137 bp(8) 135 bp(8)J DSS210 ...... Mfdl22 5q31.1-31.3 230bp(5) 228 bp(3) 222 bp (30) L Weber et al., submitted 220 bp(20)F 218 bp(8) 216 bp (32)J IL-9 5q22.3-31.3 139 bp (4) 137 bp(9) 133 bp (26) Polymeropoulos et al. 1991 131 bp (21) 129bp(9) J

DSS70...... pTP5E 5q33-q35 As published Farber et al. 1988 a Included in the analysis are only those that appeared within the pedigree. Other fragment sizes were observed on the screening of random individuals but are not included in this table.

Results chromosome 5q. The resulting two-point Z values are LGM1 was excluded for 45 cM proximal to shown in table 2. These markers all demonstrate link- D15S25 and for 40 cM distal to D15S25, the marker age to the LGM1 locus in this family. The peak Z most tightly linked to LGM2 (data not shown). These value is obtained with DSS210, which is linked to data clearly exclude LGM1 from the LGM2 gene re- LGMD with Z(O) = 10.81 at = .12. Figure 2 dem- gion. Linkage was established, however, for LGM1, onstrates segregation of the D5S207/D5S210 haplo- by a series of microsatellite repeat markers located on type in a subsection of family 39.

Table 2 Two-Point Z Values for Chromosome S Markers versus LGMD Z AT 0 OF SUPPORT Locus MARKER .001 .05 .10 .15 .20 .30 .40 o Z(O) INTERVALa

DSS119...... Mfd6 -12.81 -1.70 .01 .79 1.13 1.11 .55 .24 1.21 NA D5S207 ...... Mfd43 - 2.55 3.56 4.15 4.10 3.79 2.74 1.39 .11 4.16 .02-.25 D5S210...... Mfd122 - 3.64 9.47 10.76 10.66 9.89 7.16 3.49 .12 10.81 .06-.21 IL-9 .25 9.61 10.30 9.94 9.07 6.48 3.49 .11 10.31 .04-.18 D5S70 ...... pTP5E -1.95 6.51 7.17 6.92 6.28 4.37 2.03 .11 7.19 .03-.21 a NA = not applicable. Linkage of LGM1 to Chromosome Sq 1215 Family 39

B10 010

/ AC BD BC BD AD CD CD CB CB Figure 2 Portion of family 39 that demonstrates segregation of the D5S207/D5S210 haplotype with LGMD. Marker order is that of the DSS210/D5S207 haplotype. These individuals are enclosed within the box in fig. 1. Results in brackets are inferred.

Results of the affecteds-only analysis are summa- approximately equal likelihood. Additional informa- rized in table 3. Results for DSS210, D5S70, and IL-9 tion with regard to the genetic map of this region is remained significant, although the peak Z values are necessary before a meaningful multipoint analysis of reduced. Comparison of the full-pedigree analysis LGM1 in this family can be performed. with the affecteds-only analysis gives a peak Z value 10.31 at 0 = versus a Z value of 8.48 of .11, peak Discussion at = .06. These data indicate evidence for more recombination within the full pedigree and suggest These studies demonstrate linkage of LGM1 to that there are older unaffected individuals in the analy- chromosome 5q22.3-31.3. Genetic heterogeneity in sis who carry the LGM1 chromosome 5 haplotype. LGMD is already defined by the presence of both a Peak Z values for the chromosome 5 marker-to- dominant and a recessive form of the disease. The marker linkage analysis are presented in table 4. In advantage of working with a single large pedigree is order to define flanking markers for the LGM1 gene, evident, as difficulties with potential genetic heteroge- we attempted to generate a genetic map from the markers D5S210, D5S70, and IL-9 by using multi- Table 4 ple pairwise analysis (Morton and Collins 1989). Although we could confidently exclude the order Chromosome S Marker-to-Marker Two-Point Z Values DSS210-IL-9-DSS70, the other two orders were of MARKERS 0 Z(6) Support Interval

Table 3 DSS119/D5S207 ...... 13 5.16 .06-.22

D5S119/D5S210 ...... 15 6.40 .07-.23 for LGMD versus in Two-Point Z Values Markers D5S119/IL-9 ...... NA NA NA Affected Family Members Only D5S119/D5S70 ...... 30 .67 NA . MARKER Z(6) D5S207/D5S210 ...... 00 24.73 .00-.03 D5S207/IL-9 ...... 13 6.62 .07-.22

DSS119 ...... 23 .57 DSS207/DSS70 ...... 02 11.62 .001-.09

DSS207 ...... 09 2.38 DSS210/IL-9 ...... 15 10.13 .09-.21

D5S210 ...... 09 8.21 D5S210/D5S70 ...... 03 22.40 .001-.04

IL-9 ...... 06 8.48 IL-9/DSS70 ...... 11 7.15 .07-.21

DSS70 ...... 09 4.55 NOTE. -NA = not applicable. 1216 Speer et al. neity are circumvented. The limitation of studying a IL-9 were significant, and they remained so even after single large pedigree, however, is the inability to gen- the Z criterion level of significance for multiple analy- eralize from the significance of this one family to the ses was increased as suggested by Goldin (1990). diagnostic entity as a whole. Family 39 is unusual These results indicate that the linkage ofLGM1 in this in its clinical presentation, because of the dysarthria. family is robust, no matter what the clinical status. Whether this is evidence of additional heterogeneity The possibility of homology between chromosomes within the autosomal dominant form is not known. 15q and Sq has been raised (Donlon and Malcolm We are presently studying several other LGM1 fami- 1991). LGM2 and have been local- lies to assess the extent of heterogeneity. Worthy of ized to a small region of chromosome 1Sq, while additional note is a study by Morton (1959) which LGM1 and congenital contractural suggested that there were two loci either of which, (Lee et al. 1991), which is phenotypically similar to when homozygous, could lead to the phenotype of Marfan syndrome, have been localized to Sq. Al- LGMD. Ifthis proves to be the case, then genetic heter- though these similarities may be purely coincidental, ogeneity will be demonstrated even within the reces- they are intriguing nonetheless and warrant further sive form of LGMD. investigation. With the linkage of LGM1 to chromosome 5, the A limitation of the present study is the inability, present study demonstrates locus heterogeneity for a because of the unavailability of a well-supported ge- set of similar clinical entities of different inheritance netic map, to define flanking markers tightly linked to patterns. Clinically, family 39 and the recessive fami- LGM1. Future efforts will be focused on assessing the lies from the Isle of La Reunion are similar in that extent of heterogeneity within other autosomal domi- affected individuals have elevated creatine kinase lev- nant LGMD families, as well as on producing a well- els, proximal weakness beginning in the hip and shoul- supported genetic map from reference pedigrees, in der girdle, and a slowly progressive course. The pri- order to localize LGM1 on chromosome Sq more ac- mary difference, in affected individuals, between curately. family 39 and these recessive families is in the age at onset. Affected individuals in the Isle of La Reunion Acknowledgments families generally have onset at age 8-15 years and are usually ambulatory until age 30 years. Another large, This project was supported in part by NIH Program Proj- inbred, recessive, Amish family (Jackson and Strehler ect grants NS26630 (to M.A.P.-V.) and NS19999 (to 1968) demonstrates a clinical presentation similar to A.D.R.), by a grant from the Muscular Dystrophy Associa- that in the Isle of La Reunion with tion Task Force on Genetics, by Joseph and Kathleen Bryan families, juvenile Alzheimer's Disease Research Center grant AG05128 (to onset (average onset is at age 7 years). In contrast, the A.D.R) by the Leadership and Excellence in Alzehimer's mean age at onset in family 39 is 27.1 years. The Disease award (AG07922), and by NIH grant HG00248 (to majority of family members are ambulatory for their J.L.W.). The authors wish to thank Dr. Lindsay Farrer for adult life. helpful comments and suggestions; Peggy Pate and Helen The diagnostic complexities in LGM1 are under- Harbett for technical assistance; Eddie Hanson and Mike scored by the significant proportion (16% ) of individ- Wilkinson for preparation of DNA samples; and Susan Car- uals in family 39 who are of unknown diagnostic clas- ter, Bill Arana, Nigel Walker, Petra Koza-Taylor, and John sification. Considering them to be of unknown Pufky for RFLP and PCR analysis. Two of the authors diagnostic status in the analysis could bias the result (J.S.B. and D.C.) also wish to thank the Association fran- by masking potential recombination events. There- caise contre les for support. We are most grate- ful to members of family 39 for their continued interest and fore, all two-point analyses were also performed after for this varying the clinical status of the individuals of uncer- support project. tain diagnosis. Studies were performed after assuming that all uncertain individuals were either (1) affected, References (2) affected, but with allowance for 20% misdiagno- Beckmann JS, Richard I, Hillaire D, Broux 0, Antignac sis, or (3) unaffected but at risk and therefore assigned C, Bois E, Cann H, et al (1991) A gene for limb-girdle a probability of being affected that was based on age muscular dystrophy maps to by linkage. at examination. Regardless of the clinical status as- CR Acad Sci [III] 312:141-148 signed to these status-unknown individuals, all two- Brooke MH (1986) A clinician's view of neuromuscular dis- point results for the markers DSS210, DSS70, and ease. Williams & Wilkins, Baltimore Linkage of LGM1 to Chromosome Sq 1217

Chutkow JG, Heffner RR, Kramer AA, Edwards JA (1986) Lee B, Godfrey M, Vitale G, Hori H, Mattei MG, Sarfarazi Adult-onset autosomal dominant limb-girdle muscular M, Tsipouras P, et al (1991) Linkage ofMarfan syndrome dystrophy. Ann Neurol 30:240-248 and a phenotypically related disorder to two different fi- Chyatte SB, Bignos PJ, Watkiuns M (1966) Early muscular brillin . Nature 352:330-334 dystrophy: differential patterns ofweakness in Duchenne, Morton NE, Chung CS (1959) Formal genetics of muscular limb-girdle and facioscapulohumeral types. Arch Phys dystrophy. Am J Hum Genet 11:360-379 Med Rehabil 47:499-503 Morton NE, Collins A (1989) MAP, an expert system for Coers C, Telerman-Topper N (1979) Differential diagnosis multiple pairwise linkage analysis. Ann Hum Genet 53: of limb-girdle muscular dystrophy and spinal muscular 263-269 atrophy. Neurology 29:957-972 Nakamura Y, Gillilan S, O'Connell P, Leppert M, Lathrop Conneally PM, Edwards JH, Kidd KK, Lalouel J-M, Mor- GM, Lalouel J-M, White R (1987) Isolation and mapping ton NE, Ott J, White R (1985) Report of the Committee of a polymorphic DNA sequence pYNH24 on chromo- on Methods of Linkage Analysis and Reporting. Cyto- some 2 (D2S44). Nucleic Acids Res 15:10073 genet Cell Genet 40:356-359 Ott J (1974) Estimation of the recombination fraction in DeCoster W, DeReuck JD, Thiery E (1974) A late autoso- human pedigrees: efficient computation of the likelihood mal dominant form of limb-girdle muscular dystrophy. for human linkage studies. AmJ Hum Genet 26:588-597 Eur Neurol 12:159-172 (1978) A simple scheme for the analysis of HLA Donlon T, Malcolm S (1991) Report on the genetic constitu- linkages in pedigrees. Ann Hum Genet 42:225-257 tion of chromosome 15. Cytogenet Cell Genet 58:624- Polymeropoulos MH, Xiao H, Merril CR (1991) Dinucleo- 642 tide repeat polymorphism at the human interleukin 9 Farbear RA, Phalen T, Neuman WL, LeBeau MM, Was- gene. Nucleic Acids Res 19:688 muth JJ (1988) An anonymous DNA segment pTP5E Shokeir MHK, Kobrinsky NL (1976) Autosomal recessive (D5S70) maps to the long arm of chromosome 5 and muscular dystrophy in Manitoba Hutterites. Clin Genet identifies a TaqI polymorphism. Nucleic Acids Res 16: 9:197-202 2360 Speer MC, Pericak-Vance MA, GilchristJM, Yamaoka LH, Gilchrist JM, Pericak-Vance MA, Silverman L, Roses AD Hung W-Y, Roses AD (1989) Linkage studies in autoso- (1988) Clinical and genetic investigation in autosomal mal dominant limb-girdle muscular dystrophy. Cytogenet dominant limb-girdle muscular dystrophy. Neurology 38: Cell Genet 51:1083 5-9 Weber JL, May PE (1989) Abundant class of human DNA Goldin LR (1990) The increase in type I error rates in linkage polymorphisms which can be typed using the polymerase studies when multiple analyses are carried out on the same chain reaction. Am J Hum Genet 44:388-396 data: a simulation study. Am J Hum Genet 47 [Suppl]: WeberJL, Polymeropoulos MH, May PE, Kwitek AE, Xiao Al 80 H, McPhersonJD, WasmuthJJ. Mapping ofhuman chro- Jackson CE, Strehler DA (1968) Limb-girdle muscular dys- mosome 5 microsatellite DNA polymorphisms (sub- trophy: clinical manifestations and detection ofpreclinical mitted) disease. 41:495-503 Yamaoka LH, Pericak-Vance MA, Speer MC, Gaskell PC, Kazakov VM, Kovalenko TM, Skorometz AA, Mikhailov StajichJ, Haynes C, Hung W-Y, et al (1990) Tight linkage EP (1977) Chronic spinal muscular atrophy simulating of creatine kinase (CKMM) to myotonic dystrophy on facioscapulohumeral type and limb-girdle type of muscu- . Neurology 40:222-226 lar dystrophy. Eur Neurol 16:90-98 Young K, Williams P, Foroud T, Jackson CE, Beckmann J, Kidd KK, Bowcock AM, Schmidtke J, Track RK, Ricciuti Cohen D, Conneally PM, et al (1991) Confirmation of F, Hutchings G, Bale A, et al (1989) Report of the DNA linkage oflimb-girdle muscular dystrophy to chromosome committee and catalogs of cloned and mapped genes and 15. Cytogenet Cell Genet 58:1996 DNA polymorphisms. Cytogenet Cell Genet 51:622-947