American Journal of Medical Genetics 72:363–368 (1997) Limb Girdle Muscular Dystrophy in Manitoba Hutterites Does Not Map to Any of the Known LGMD Loci Tracey Weiler,1 Cheryl R. Greenberg,2,3 Edward Nylen,1 Kenneth Morgan,4,5 T. Mary Fujiwara,4,5,6 M. Joyce Crumley,4,5 Teresa Zelinski,2,3 William Halliday,7 Barbara Nickel,1 Barbara Triggs-Raine,1,2 and Klaus Wrogemann1,2,3* 1 Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada Department of Human Genetics, University of Manitoba, Winnipeg, Manitoba, Canada 3 Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada 4 Departments of Human Genetics and Medicine, McGill University, Montreal, Quebec, Canada 5 Montreal General Hospital Research Institute, Montreal, Quebec, Canada 6 Department of Pediatrics, McGill University, Montreal, Quebec, Canada 7 Department of Pathology, University of Manitoba, Winnipeg, Manitoba, Canada 2 Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of disorders affecting primarily the shoulder and pelvic girdles. Autosomal dominant and recessive forms have been identified; 8 have been mapped and 1 more has been postulated on the basis of exclusion of linkage. An autosomal recessive muscular dystrophy was first described in 1976 in the Hutterite Brethren, a North American genetic and religious isolate [Shokeir and Kobrinsky, 1976; Clin Genet 9:197–202]. In this report, we discuss the results of linkage analysis in 4 related Manitoba Hutterite sibships with 21 patients affected with a mild autosomal recessive form of LGMD. Because of the difficulties in assigning a phenotype in some asymptomatic individuals, stringent criteria for the affected phenotype were employed. As a result, 7 asymptomatic relatives with only mildly elevated CK levels were assigned an unknown phenotype to prevent their possible misclassification. Two-point linkage analysis of the disease locus against markers linked to 7 of the known LGMD loci and 3 other candidate genes yielded lod scores of <-2 at u=0.01 in all cases and in Contract grant sponsors: Medical Research Council of Canada; Muscular Dystrophy Association of Canada; Manitoba Medical Services Foundation; Canadian Genetic Diseases Network; Winnipeg Rh Institute Foundation; Children’s Hospital of Winnipeg Research Foundation. *Correspondence to: Klaus Wrogemann, MD, PhD, Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, MB, Canada R3E 0W3. E-mail: [email protected] UMANITOBA.CA Received 11 March 1997; Accepted 21 May 1997 © 1997 Wiley-Liss, Inc. most cases at u=0.05. This suggests that there is at least 1 additional locus for LGMD. Am. J. Med. Genet. 72:363–368, 1997. © 1997 Wiley-Liss, Inc. KEY WORDS: LGMD; exclusion; limb girdle; gene mapping; muscular dystrophy; Hutterite INTRODUCTION To date, 9 separate loci for limb girdle muscular dystrophy (LGMD) have either been mapped through linkage analysis or postulated to exist by exclusion of linkage. Two autosomal dominant LGMD loci (LGMD1A and LGMD1B) were mapped to chromosome regions 5q31-q33 [Speer et al., 1992; Yamaoka et al., 1994] and 1q11-q21 [Van der Kooi et al., 1997] respectively. Autosomal recessive loci (LGMD2A-2F) were mapped to 6 chromosome regions: 15q15.1-q21.1 [Allamand et al., 1995], 2p13.3 [Passos-Bueno et al., 1995a], 13q12-q13 [Ben-Othmane et al., 1992; Ben Othmane et al., 1995], 17q12-q21.33 [Roberds et al., 1994], 4q12 [Bönnemann et al., 1995; Lim et al., 1995], and 5q33-q34 [PassosBueno et al., 1996]; a 7th locus has been postulated to exist by exclusion to known loci [Passos-Bueno et al., 1996]. Recently, Miyoshi myopathy (MM) was mapped to chromosome region 2p12-p14 [Bejaoui et al., 1995], and we and others have suggested that mutations at the LGMD2B locus cause both MM and LGMD2B [Bejaoui et al., 1995; Weiler et al., 1996]. Genes have been identified for 5 of the 6 autosomal recessive loci: mutations in the gene encoding calpain 3 (CANP3) cause LGMD2A [Richard et al., 1995]; and mutations in the genes encoding 4 components of the sarcoglycan complex (a-, b-, g- and d-sarcoglycan) cause LGMD2D [Roberds et al., 1994], LGMD2E [Lim et al., 1995; Bön- 364 Weiler et al. nemann et al., 1995], LGMD2C [Noguchi et al., 1995], and LGMD2F [Nigro et al., 1996], respectively. Here we report the exclusion of 7 of the known LGMD loci as causing LGMD in 4 Canadian Hutterite families with an autosomal recessive form of LGMD. One of the patients included in this study was in the original description of muscular dystrophy in the Hutterites [Shokeir and Kobrinsky, 1976] (MIM [254110). MATERIALS AND METHODS Patients and Pedigree We reconstructed a detailed pedigree on the basis of information obtained from the initial publication [Shokeir and Kobrinsky, 1976], Schmiedeleut family records [Gross, 1996], our genealogical database (Fujiwara, Crumley, and Morgan, unpublished data), and confirmatory interviews with the family (Fig. 1). Personal interviews and musculoskeletal examinations were performed by CRG and consulting neurologists on available relatives included in this study. Blood samples were obtained from all consenting individuals for DNA banking, Epstein Barr virus transformation, creatine kinase (CK) analysis, and blood group serology. Electrophysiological studies, open muscle biopsies, and echocardiographic assessments were performed where feasible. Individuals were considered to be affected with LGMD if: (1) they exhibited signs and symptoms of proximal muscle weakness with CK levels ù4× normal in the absence of any other explanation for CK elevation; (2) they exhibited signs and symptoms of proximal muscle weakness and had a muscle biopsy consistent with LGMD; or (3) their CK levels were ù15× normal but they were asymptomatic. Individuals were considered to be unaffected if they were symptomfree, had a normal musculoskeletal exam, and a normal CK level. Individuals were assigned an unknown phe- notype if their CK levels were > normal but ø4× normal and they were asymptomatic. DNA Studies DNA was extracted from whole blood as previously described [Greenberg et al., 1987]. Oligonucleotide primers designed to amplify 36 microsatellite loci linked to 10 candidate loci, including DAG1, LGMD1A, LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, SNT2B1, and SNT2B2 [Weber et al., 1991; Ben-Othmane et al., 1992; Bashir et al., 1994; Fougerousse et al., 1994; Yamaoka et al., 1994; Allamand et al., 1995; Passos-Bueno et al., 1995b; Lim et al., 1995; Passos-Bueno et al., 1996], were obtained from Research Genetics, Inc. (Huntsville, AL). Markers linked to LGMD1B were not tested because the location of this disease gene only became known during review of this paper. The chromosomal locations were obtained from maps located in the Genome Database (web site: http:/ /gdbwww.gdb.org/). Genetic distances between candidate genes and linked markers were obtained from recent publications [Weber et al., 1991; Ben-Othmane et al., 1992; Bashir et al., 1994; Fougerousse et al., 1994; Yamaoka et al., 1994; Allamand et al., 1995; PassosBueno et al., 1995b; Lim et al., 1995; Passos-Bueno et al., 1996]. DNA samples were genotyped according to protocols reported elsewhere [Sirugo et al., 1992; Rodius et al., 1994] with minor modifications. Linkage Analysis Linkage analysis was performed on data obtained from microsatellite typing of 18 patients, their parents, and sibs available for study from 4 families using the LINKAGE programs (versions 5.1 and 5.2) [Lathrop and Lalouel, 1984] and the FASTLINK version (3.0P) of the LINKAGE programs [Cottingham et al., 1993; Schäffer et al., 1994]. MLINK was used for 2-point Fig. 1. Pedigree of 21 Hutterite patients exhibiting LGMD, 18 of whom participated in the study. The pedigree includes the closest cousin relationships between the parents of 4 LGMD families (A, B, C, and D) and the parents of patient 5, and at least 1 of the closest links between the families, thus not all genealogical relationships are shown. Affected individuals are designated with solid symbols, unaffected individuals are designated with open symbols, and 7 individuals with unknown phenotype are designated with grey symbols. Individuals whose DNA was used for microsatellite genotyping are indicated with asterisks. LGMD Does Not Map to Any Known Loci analysis of an autosomal recessive trait with complete penetrance. Disease allele frequency was estimated to be 0.05 based on the number of known cases of LGMD in Manitoba Hutterites. Marker allele frequencies were assumed to be equal. No consanguinity or marriage loops were used. RESULTS Pedigree and Clinical Description Figure 1 shows 21 individuals (13 males and 8 females) who are highly suspected or confirmed to have LGMD. Disease segregation is compatible with autosomal recessive inheritance. Clinical data from the 18 affected individuals assessed in this study are presented in Table I. Significant intra- and interfamilial variability is evident. In families A and B, 3 of 14 individuals (patients 4, 9, and 10) have grossly elevated CK levels (ù15× normal) but are asymptomatic and to date, their muscle strength is preserved. A dystrophic muscle biopsy was obtained on patient 4 confirming the assignment of an affected phenotype. In symptomatic individuals (patients 1–3, 5–8, 11–18), onset of muscle weakness and easy fatigability generally were noted from childhood to mid 30s and clinical progression tended to be slow. Typically, patients complained of 365 different degrees of leg weakness and had difficulty running, climbing stairs, and lifting objects. Six individuals indicated that they suffered neck and back pain. All symptomatic patients demonstrated slender proximal and distal muscle mass in their upper and lower limbs without contractures. There was no evidence of facial muscle weakness in contrast to the reports by Shokeir and Kobrinsky  and Shokeir and Rozdilsky . Neither cardiomyopathy nor cardiac conduction defects were present in the patients included in our study. Ataxia, fasciculations, muscle cramps, sensory impairment, and myotonia were not observed. All patients assessed had normal intellect, bladder, bowel, and swallowing functions, and none had an associated systemic illness or other disease. Electromyographic studies have been primarily myopathic with some neurogenic characteristics in several patients. Muscle biopsies were also compatible with a dystrophic muscle process. In 1 patient who underwent a muscle biopsy, grouping of small fibres raised the possibility of a neurogenic component. Genealogical Analysis The ancestry of almost all of the contemporary Hutterites can be traced back to 89 founders (Fujiwara, Crumley, and Morgan, unpublished data). Thus, the TABLE I. Clinical Data of Patients With Limb Girdle Muscular Dystrophy Patient no. (Family) Age at onset (yr) Age at presentation (yr) 1(A) 25 32 2(A) 25 32 3(A) 4(A) 15–16 * 27 * 5(B) mid 20s 53 6(B) 27 32 7(B) 8(B) 22 mid 20s 28 26 9(B) 10(B) 11(B) * * 18 * * 21 12(C) 27 30 13(C) 14(C) 15 20 26 23 15(D) 8 45 Proximal weakness, fatigue, falling Muscle wasting & weakness, back pain Proximal weakness Asymptomatic, past history of carpal tunnel syndrome Proximal weakness, waddling gait Difficulty climbing stairs, low back pain, waddling gait Weak legs Neck pain, wasting of shoulder girdle Asymptomatic Asymptomatic Intermittent neck pain Proximal weakness, fatigue Proximal weakness Proximal weakness, low back pain Back pain 16(D) 10 41 Proximal weakness 692 17(D) 11–13 34 943 18(D) 11–13 29 Proximal weakness, fatigue Proximal weakness, fatigue a Presenting symptoms CK (U/L)a Muscle biopsy 2,065 Present status (age in years) Ambulatory (37) 250 Dystrophic Ambulatory (36) 922 2,975 Dystrophic Ambulatory (27) Asymptomatic (22) 317b Myopathic 2,030 2,135 1,700 Myopathic 2,740 4,280 2,916 906 Myopathic Ambulatory (28) Ambulatory (26) Asymptomatic (25) Asymptomatic (23) Ambulatory (21) Dystrophic Neurogenic, myopathic Myopathic Ambulatory with difficulty (36) Ambulatory (33) Ambulatory (29) Dystrophic Myopathic Ambulatory with difficulty (45) Ambulatory with difficulty (41) Ambulatory (38) 797 3,160 1,092 Wheelchair (60) Ambulatory (32) Myopathic Dystrophic 897 Highest recorded value; normal values for females: 28–116 U/L; normal values for males: 52–175 U/L. CK reported 4× normal in 1976 [Shokeir and Kobrinsky, 1976]. *Asymptomatic, no data. b EMG Myopathic Ambulatory (35) 366 Weiler et al. contemporary population of >30,000 can be considered as 1 extended kindred. The Hutterite Brethren established 3 endogamous subdivisions, or leut (Dariusleut, Lehrerleut, and Schmiedeleut), when they immigrated to the US in the late 1870s. The Manitoba Hutterites belong to the Schmiedeleut. We estimated the average inbreeding coefficient of 10,693 Schmiedeleut considered to be in a 1981 census of our genealogical database as 0.0338. The kinship coefficient of the parents (or the inbreeding coefficient of a child) of Families A, B, C, and D is 0.0172, 0.0651, 0.0452, and 0.0589, respectively. The kinship coefficient is largely due to the closest cousin relationship between the parents, and in these families is 3rd cousins once-removed in 3 ways, 2nd cousins in 2 ways, 2nd cousins, and half-1st cousins once-removed, respectively (Fig. 1). Patient 5 has 2 affected sibs and is a parent of Family B. The parents of patient 5 are most closely related as 1st cousins onceremoved and their kinship coefficient is 0.0522. There are many more distant relationships that also contribute to the kinship coefficient. The total number of ways the parents are related as cousins is 187, 223, 267, and 154 different ways. The average kinship coefficient of the 24 pairs of parents who are not married to each other is 0.0364 (range 4 0.0098 to 0.0880). There are at least 10 ancestors born in the 1700s who could have contributed an allele to each of the 8 parents of the LGMD sibship and to the paternal grandparents of Family B. Linkage Analysis Four families were tested for linkage of the disease locus to 10 candidate loci on 9 chromosomes. These include 7 of the currently mapped LGMD loci (LGMD1A and LGMD2A–LGMD2F) as well as 3 genes for 4 members of the dystrophin associated protein complex (DAG1, SNT2B1, and SNT2B2) [IbraghimovBeskrovnaya et al., 1993; Ahn et al., 1996]. Lod scores ø−2 were obtained for 15 markers (at least 1 marker linked to each candidate locus) (Table II) suggesting that each of the 10 candidate loci can be excluded as the locus causing the disease in these families. DISCUSSION Given the genetic heterogeneity now clearly evident in LGMD, one strategy is to study large consanguinous kindreds where the parents of all affected individuals are likely to carry copies of the same disease allele identical by descent. The Hutterite families described in this report represent such a kindred. Genealogical analysis indicates that the parents of all patients in this kindred can be traced back to 10 ancestors, 6 to 9 generations back, allowing us to consider the possibility that the disease in each of the patients is caused by mutation(s) in the same gene. Physical and laboratory examinations of individuals from these 4 families have resulted in the identification of 21 individuals with some or all of the symptoms of LGMD, 3 of whom did not participate in this study. Many of our findings on physical examination of symptomatic individuals confirm those of Shokeir and Kobrinsky  and Shokeir and Rozdilsky , including a waddling gait and difficulty in rising from a squatting position (although we did not see any evidence of the facial muscle involvement that they had reported). Because of the mild nature of the disease in this kindred and the overlap between affected and normal individuals with respect to clinical phenotype and serum CK elevation, we found it difficult to determine reliably the clinical status of every individual. This is especially so because serum CK, the most useful biochemical criterion of a muscular dystrophy, is a nonspecific finding and varies in any given individual. High CK levels may also result, for example, from prolonged or weight-bearing exercise as well as from heatstroke, myocardial infarction, and acute renal failure [Noakes, 1987]. Phenotypes were therefore defined stringently to include only those individuals who had extremely elevated CK levels (ù15× normal), or those who were symptomatic either with CK ù4× normal or a positive muscle biopsy. Using these criteria, patients varied considerably in their clinical phenotype, from completely asymptomatic to limited ambulation with a walker. CK levels in our patients were also variable, from 2× to 25× normal. Seven asymptomatic individu- TABLE II. Lod Scores From Two-Point Linkage Analysis Between LGMD and Markers Linked to 10 Candidate Loci Recombination fraction (u) Candidate locus Marker locusa 0.00 0.01 0.05 0.10 0.20 0.30 0.40 LGMD1A LGMD2A CSF1R D15S182 D15S778 D2S291 D2S2109 D2S2111 D13S115 D17S806 D17S941 D4S1547 D4S1594 D5S470 D3S1766 D8S199 D16S266 −` −` −` −` −` −` −` −` −` −` −` −` −` −` −` −4.53 −5.32 −5.75 −3.97 −3.47 −2.45 −9.28 −4.95 −2.16 −2.06 −6.48 −4.69 −5.80 −5.44 −10.22 −1.91 −2.63 −2.45 −1.96 −1.51 −1.14 −4.58 −2.28 −0.91 −0.74 −3.13 −2.52 −2.99 −2.68 −4.87 −0.93 −1.56 −1.20 −1.17 −0.77 −0.66 −2.71 −1.25 −0.47 −0.25 −1.82 −1.58 −1.81 −1.56 −2.78 −0.20 −0.64 −0.23 −0.49 −0.22 −0.27 −1.11 −0.43 −0.16 0.09 −0.72 −0.70 −0.76 −0.62 −1.03 0.00 −0.24 0.05 −0.19 −0.05 −0.11 −0.41 −0.13 −0.06 0.13 −0.26 −0.28 −0.29 −0.23 −0.33 0.02 −0.05 0.05 −0.04 0.00 −0.03 −0.09 −0.02 −0.01 0.05 −0.06 −0.06 −0.07 −0.05 −0.06 LGMD2B LGMD2C LGMD2D LGMD2E LGMD2F DAG1 SNT2B1 SNT2B2 a Markers were chosen on the basis of reported significant positive lod scores to the respective disease loci. LGMD Does Not Map to Any Known Loci als with mildly elevated CK were defined as ‘‘unknown’’ to prevent their misclassification. The variation in phenotype may also be due to differences in the genetic background or the influence of modifier gene(s). In fact, the involvement of a 2nd locus in the determination of the clinical phenotype has been suggested to play a role in 3 of the currently mapped LGMDs (i.e., LGMD2A, LGMD2B, and LGMD2C) [Richard et al., 1995; Weiler et al., 1996; McNally et al., 1996; van Ommen, 1995; Beckmann, 1996]. Phenotypic variation has also been observed for LGMD2B and LGMD2C where severe and mild phenotypes are associated with a single haplotype [Weiler et al., 1996] and a single mutation in g-sarcoglycan, D521-T, respectively [McNally et al., 1996]. Using a conservative definition of the affected phenotype, 2-point linkage analysis of the 12 microsatellite loci linked to the known LGMD loci (LGMD1A, LGMD2A–2F) yielded lod scores ø−2 at a recombination fraction of 0.01 and in some cases 0.05 (Table II). This suggests that the disease in these families does not map to any of the known LGMD loci. Since most genes causing LGMD encode members of the dystrophin associated protein complex, we tested markers linked to 3 genes encoding other members of the complex (DAG1, SNT2B1, and SNT2B2). Two-point linkage analysis of the disease versus these markers has also yielded lod scores ø−2 which indicates that the disease in these families does not map to any of these loci either. Our study suggests that there is at least 1 more locus causing autosomal recessive LGMD, in agreement with the report by Passos-Bueno et al. . The portion of the pedigree illustrated here represents only 4 of the Manitoba families with LGMD in the Schmiedeleut. We know of 60 Hutterites exhibiting LGMD in Canada from all 3 subdivisions. The additional families from the other 2 subdivisions, who are more distantly related to the Manitoba families, will facilitate the mapping of the gene using an identity by descent approach. This approach was successfully used to map a recessive gene in the Mennonite population which has a population structure similar to that of the Hutterite population [Puffenberger et al., 1994]. The LGMD disease allele frequency in the Hutterite population appears to be relatively high since there is no strong clustering among the 4 Schmiedeleut families and the disease is present in all 3 subdivisions of the population. ACKNOWLEDGMENTS We are indebted to the patients and their families for their participation in this study. 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