Am. J. Hum. Genet. 51:428-431, 1992

Linkage Localization of Facioscapulohumeral Muscular Dystrophy (FSHD) in 4q35 K. D. Mathews,* K. A. Mills,* E. P. Bosch,t V. V. Ionasescu,* K. R. Wiles,* K. H. Buetowt andJ. C. Murray* Departments of Pediatrics and tNeurology, University of Cancer Center, Philadelphia

Iowa

College of Medicine,

Iowa

City; and tDivision of Clinical Research, Fox Chase

Summary Fasioscapulohumeral muscular dystrophy (FSHD) has recently been localized to 4q35. We have studied four families with FSHD. Linkage to the 4q35 probes D4S163, D4S139, and D4S171 was confirmed. We found no recombinants helpful in detailed localization of the FSHD gene. Two of our families include males with a rapidly progressive muscle disease that had been diagnosed, on the basis of clinical features, as Duchenne muscular dystrophy. One of these males is available for linkage study and shares the haplotype of his FSHDaffected aunt and cousin.

Introduction

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with an incidence of 1-5 in 100,000 births (Leth 1985; Lunt 1991). FSHD has variable expression within families and 95% penetrance in heterozygotes over 20 years of age (Lunt 1989). Patients typically present in the second decade of life, with symptoms of facial and shoulder girdle weakness. The course is slowly progressive, with approximately 20% of affected individuals confined to a wheelchair by middle age (Upadhyaya 1989). Rare individuals with more rapidly progressive weakness with infantile onset have been described, and recognition of these families has raised questions about the homogeneity of the FSHD syndrome (Bailey et al. 1986; Lunt et al. 1989). FSHD was recently found to be linked to the polymorphic marker D4SI71 (Wijmenga et al. 1990) on 4q35. We have studied four multigeneration families with FSHD, by using additional markers from 4q35 as part of the collaborative effort to further localize FSHD. Two of these families include males with Received July 15, 1991; final revision received April 14, 1992. Address for correspondence and reprints: Katherine D. Mathews, M.D., Department of Pediatrics, University of Iowa Hospitals, Iowa City, IA 52242. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-0025$02.00

428

a rapidly progressive, early-onset form of muscle disease. Material and Methods Families Four families with dominant FSHD were available for linkage analysis. The four families include 52 individuals, 19 of whom are affected. All except three affected individuals have been evaluated by neuromuscular clinics at the University of Iowa, and all have typical clinical features. At least one member of each

pedigree has been studied with electromyography (EMG), nerve-conduction velocities, and muscle biopsy. The three individuals not available for personal examination are adults in one well-studied family who report typical facial weakness and inability to lift their arms over their head. The unaffected members of each pedigree have been examined clinically and show no signs of FSHD. The youngest unaffected individual included in the analysis is 20 years old. Two of our families include males with the clinical diagnosis of Duchenne muscular dystrophy (DMD). In family 3, a 15-year-old male (BG) presented at 22 mo of age with lower-extremity weakness manifested by frequent falls. On examination, he had calf hypertrophy and generalized hypotonia. Serum creatine phosphokinase (CPK) was 27,200 IU (normal 35-200

Linkage Localization for FSHD

429

IU). A muscle biopsy done in 1977 showed extensive inflammation with relatively little degeneration/regeneration, findings that may be more consistent with a severe variant of FSHD than with DMD. He has a normal karyotype. His course has been as expected for DMD. He required a wheelchair at age 7 years. His family reports that facial weakness has been apparent since at least age 11 years. He is now quite wasted, with contractures and diffuse weakness. Muscle from his previous biopsy is no longer available, and attempts to do immunohistochemical labeling of dystrophin, using available tissue sections, were unsuccessful. His mother has a maternal half-sister with FSHD confirmed by muscle biopsy. BG's mother has not been available for examination, although she did consent to have her blood drawn and mailed to us. Neither BG nor his mother has been included in the FSHD linkage analysis, because of the uncertainty regarding diagnosis. Family 4 includes a male child of a father who had a consistently elevated CPK (2-4 times the upper limit of normal). This father has a cousin with wellestablished FSHD who is included in our studies. The child presented in 1962 at 3 years of age with muscle weakness particularly affecting the upper extremities. Initial history reports that he always walked with a "high-stepping, slapping gait." Examination revealed a transverse smile, muscle atrophy and decreased tone-which was particularly striking around the shoulders-and mild calf hypertrophy. EMG was abnormal with a myogenic pattern. CPK was strikingly elevated (9 times the upper limit of normal). Muscle biopsy showed small collections of phagocytes, prominent fibrosis, and mild fatty infiltration, findings that are not strongly supportive of the diagnosis of DMD but that are consistent with the diagnosis of FSHD. Intelligence testing showed a Wechsler Intelligence Scale for Children IQ of 77. He was confined to a wheelchair at age 8 years. This child died in adolescence with the diagnosis of DMD, and DNA is not

available for study. One sister of this child had one elevated CPK at the age of 13 years, but she did not return for further evaluation. Other siblings and his mother had normal muscle enzymes. Immediate family members have been unavailable for the ongoing linkage study. Linkage Analysis

Radiographic films were scored by at least two independent investigators. Two-point lod-score analysis was performed using the MLINK option of LINKAGE (Lathrop and Lalouel 1988) to determine pairwise recombination estimates. FSHD was modeled as a partially penetrant autosomal dominant locus with a heterozygote penetrance of 95% (Lunt et al. 1989). The model also assumed a .0001 phenocopy frequency and a gene frequency of .0001. Probes used in this analysis are summarized in table 1. DNA was obtained from lymphocytes by using the rapid salt method (Miller et al. 1988), and RFLPs were analyzed according to standard protocol (Sambrook et al. 1989). A dinucleotide repeat polymorphism for D4S171 and a single-strand conformational polymorphism for D4S1 87 were analyzed according to the protocol described by Mills et al. (1992). Results

Results of linkage analysis are shown in table 2. A peak lod score (2) of 8.23 was obtained with D4S139 at a peak recombination fraction (0) of 0. We found no recombinants by using D4S139 or D4S163. FSHD could be excluded from residing within 1 cM of D4S171 by odds of greater than 1,000:1. Two additional markers, D4S187 and F11 (Kato et al. 1989; Mills et al. 1992), were studied in our families but were minimally informative or uninformative (data not shown). The marker D4S187 is derived from a breakpoint clone from a patient with a t(x;4) (p21 ;q35) transloca-

Table I Markers Used in Studies of FSHD Families

Locus D4S163

..........

D4S139

..........

D4S171 ..........

Enzymes

Probe Type

Reference

HindIII PvuII HindIII PvuII

VNTR

VNTR

Neuweiler et al. 1990 Altherr et al. 1991 Milner et al. 1989

(CA),

Weber and May 1990

...

Mathews et al.

430 Table 2 Pairwise Lod Score Analysis for FSHD and Three Markers Located in 4q35

SEX-AVERAGED DATA

LOD SCORES AT 0 OF

FSHD VS.

0

.001

.01

D4S171 .... D4S163 .... D4S139 ....

-4.43 5.89 8.23

- 3.37 5.89 8.22

- 2.09 5.83 8.14

.05 - .47 5.49 7.68

tion (Bodrug et al. 1990). The clinical description of this patient suggests that D4S1 87 may be a candidate locus for FSHD. In these families, we have found no evidence of deletion or rearrangement, on Southern blots probed with the D4S187. As noted previously, the patient with a DMD phenotype (BG) was excluded from formal analysis, because of the uncertainty about his diagnosis. Visual inspection of his haplotype data, shown in figure 1, reveals that he shares the "affected" haplotype with his FSHD-affected aunt and cousin. Discussion FSHD has shown linkage to D4S171 (Wijmenga et al. 1990) and to D4S139 (Upadhyaya et al. 1990; Wijmenga et al. 1991) on 4q35. Using probes recently mapped in 4q35 (Mills et al. 1992) in four families with FSHD, we have confirmed the previous linkage results to D4S171 and D4S139 and have shown tight linkage to D4S163. Although lack of information precluded establishing significant linkage to D4S187, its 3 2

0~~~150

z

0

3 4 l32 a a A BF F C

25

l 53 aaa _12 BG

l2 2

5

-AH G 2 3 GF

34 [

a A

(

I G

*33 a A BH

| 13 4

a A 1 7 BG

~~~D4S171

MM18

D4S163

MMD4S139

Abbreviated family 3 pedigree (omitting several unFigure I affected individuals). Typing of informative markers D4S171, D4S187, D4S163, and D4S139 is shown. * = Affected with FSHD; o = unaffected with FSHD; * = not examined; and 1 = clinical diagnosis of DMD. A slash (I) through the symbol indicates that individual is deceased.

.1

.2

.3

0

Zmax

.36 4.94 6.97

.88 3.63 5.32

.7 2.18 3.42

.22 .0 .0

.89 5.89 8.23

position, between D4S171 and D4S139, in the multipoint map of 4q35 (Mills et al. 1992) suggests it will be linked as well. The D4S187-containing region is interesting as a candidate locus for FSHD, on the basis of both the map localization to 4q35 and the clinical features in the patient in whom it originated. The patient with the t(x;4) (p21;q35) translocation was identified by prenatal amniocentesis. She has had an elevated CPK since birth. At 4 years of age, she was described as intellectually normal, with some weakness of the lower extremities that was evident when she climbed stairs. She had weak facial and shoulder muscles. Her X-chromosome breakpoint was mapped to an intron in the dystrophin gene, and findings were attributed to her heterozygosity at the DMD locus (Bodrug et al. 1990). It seems possible that the breakpoint on chromosome 4 disrupts the FSHD gene or its regulatory elements, thus explaining the early onset of facial weakness. Unfortunately, our results do not allow this locus to be confirmed or excluded as a candidate, because of the few informative meioses available. The recently available collaborative map localization of FSHD to the telomeric side of D4S1 63 makes D4S1 87 less likely to be involved in FSHD (Sarfarazi et al. 1992). Results for the two males whom we describe who have severe muscle disease of childhood onset and clinical diagnoses of DMD emphasize the importance of the recent advances in linkage and gene-based diagnosis. The implications for genetic counseling of these families are clearly quite different if the children have FSHD rather than DMD. A similar counseling problem was recently described by Reardon et al. (1991) when a father thought to have Becker muscular dystrophy had a daughter with typical features of FSHD. Because the boys seen in our clinics had atypical muscle-biopsy findings for the diagnosis of DMD and had clinical features similar to previously reported

Linkage Localization for FSHD

children with probable infantile FSHD, we suspect that the correct diagnosis is FSHD. The results of the ongoing collaborative search for the FSHD gene should allow resolution of this diagnostic dilemma in the child available for study.

Acknowledgments The study was supported in part by an American Health Care Advisory Association Foundation grant (to K.D.M.) and by National Institutes of Health grants RO1HGO035S and P50HG00206 (to J.C.M.) We thank Ann Muilenberg, R.N., and Sandra Hirsch, R.N., whose many telephone hours facilitated sample collection. Janice Widmer's careful manuscript preparation is appreciated. A special thanks goes to the family members who agreed to participate in this

study.

References Altherr MR, Wasmuth JJ, Nakamura Y, White R (1991) A highly polymorphic VNTR locus on the long arm of chromosome 4. Nucleic Acids Res 19:1168 Bailey RO, Marzulo DC, Hans MB (1986) Infantile facioscapulohumeral muscular dystrophy: new observations. Acta Neurol Scand 74:51-58 Bodrug SE, Roberson JR, Weiss L, Ray PN, Worton RG, Van Dyke DL (1990) Prenatal identification of a girl with a t(X;4) (p21 ;q35) translocation: molecular characterisation, paternal origin, and association with muscular dystrophy. J Med Genet 27:426-432 Kato A, Asakai R, Davis EW, Aoki N (1989) Factor XI gene (F1i1) is located on the distal end of the long arm of human chromosome 4. Cytogenet Cell Genet 52:77-78 Lathrop GM, Lalouel J-M (1988) Efficient computations in multilocus linkage analysis. Am J Hum Genet 42:498505

Leth A, Wulff K, Corfitsen M, ElmgreenJ ( 1985) Progressive muscular dystrophy in Denmark: natural history, prevalence and incidence. Acta Paediatr Scand 74:881-885 Lunt PW, Compston DAS, Harper PS (1989) Estimation of age dependent penetrance in facioscapulohumeral muscular dystrophy by minimizing ascertainment bias. J Med Genet 26:755-760 Lunt PW, Harper PS (1991) Genetic counseling in facioscapulohumeral muscular dystrophy. J Med Genet 28:655664

431 Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1945 Mills KA, Buetow KH, Xu Y, Ritty TM, Mathews KD, Bodrug SE, Wijmenga C, et al (1992) Genetic and physical mapping on chromosome 4 narrows the localization of the gene for fascioscapulohumeral muscular dystrophy (FSHD). Am J Hum Genet 51:432-439 Milner ECB, Lotshaw CL, Willems van Dijk K, Charmley P, Concannon P, Schroeder HW Jr (1989) Isolation and mapping of a polymorphic DNA sequence pH30 on chromosome 4 [HGM provisional no. D4S139] Nucleic Acids Res 17:4002 Neuweiler J, Ruvolo V, Baum H, Grzeschik KH, Balasz I (1990) Isolation and characterization of a hypervariable region [D4S163] on chromosome 4. Nucleic Acids Res 18:691 Reardon W, Temple 1K, Harwood G, Baraltser M (1991) Atypical facio-scapulo-humeral muscular dystrophy: a counseling dilemma. Clin Genet 39:172-177 SambrookJ, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Sarfarazi M, Wijmenga C, Upadhyaya M, Weiffenbach B, Hyser C, Mathews K, Murray J, et al (1992) Regional mapping of facioscapulohumeral muscular dystrophy gene on 4q35: combined analysis of an international consortium. Am J Hum Genet 51:396-403 Upadhyaya M, Sarfarazi M, Lunt PW, Broadhead W, Harper PS (1989) A genetic linkage study of facioscapulohumeral (Landouzy-Dejerine) disease with 24 polymorphic DNA probes. J Med Genet 26:490-493 Upadhyaya M, Lunt PW, Sarfarazi M, Broadhead W, DanielsJ, Owen M, Harper PS (1990) DNA marker applicable to presymptomatic and prenatal diagnosis of facioscapulohumeral disease. Lancet 336:1320-1321 Weber JL, May PE (1990) Dinucleotide repeat polymorphism at the D4S171 locus. Nucleic Acids Res 18:2022 Wijmenga C, Frants RR, Brouwer OF, Moerer P, Weber JL, Padberg GW (1990) Location of fasciocapulohumeral muscular dystrophy gene on chromosome 4. Lancet 336: 651-653 Wijmenga C, Padberg GW, Moerer P, Weigant J, Liem L, Brouwer OF, Milner ECB, et al (1991) Mapping of fasciocapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization. Genomics 9:570-575