Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Zmax) of 17.28 at a recombination fraction theta of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.     

Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromosome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Z_max) of 17.28 at a recombination fraction θ of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.     

Genetic linkage map of facioscapulohumeral muscular dystrophy and five polymorphic loci on chromosome 4q35-qter

A genetic map of five polymorphic markers in the area of the facioscapulohumeral muscular dystrophy (FSHD) gene on chromosome 4q35-qter has been constructed. With these five markers, a number of recombinants have been identified that allow ordering of the marker and the disease loci. The most likely locus order and the relative position of the FSHD gene supported by the recombinants is centromere-D4S171-F11-D4S187-D4S163-D4S139-FS HD-telomere. However, at least one recombination event appears to be inconsistent with this order and suggests a location of FSHD proximal to D4S139.     

Linkage analyses of five chromosome 4 markers localizes the facioscapulohumeral muscular dystrophy (FSHD) gene to distal 4q35

The genetic locus for facioscapulohumeral muscular dystrophy (FSHD) has been mapped to chromosome 4. We have examined linkage to five chromosome 4q DNA markers in 22 multigenerational FSHD families. Multipoint linkage analyses of the segregation of four markers in the FSHD families and in 40 multigenerational mapping families from the Centre d'Etude du Polymorphisme Humaine enabled these loci and FSHD to be placed in the following order: cen-D4S171-factor XI-D4S163-D4S139-FSHD-qter. One interval, D4S171-FSHD, showed significant sex-specific differences in recombination. Homogeneity tests supported linkage of FSHD to these 4q DNA markers in all of the families we studied. The position of FSHD is consistent with that generated by other groups as members of an international FSHD consortium.     

Regional mapping of facioscapulohumeral muscular dystrophy gene on 4q35: Combined analysis of an international consortium

Members of an international consortium for linkage analysis of the facioscapulohumeral muscular dystrophy (FSHD) gene have pooled data for joint analyses, in an attempt to determine the precise location of the FSHD gene and the order of four DNA markers on 4q35 region. Six laboratories determined a total of 3,078 genotypes in 65 families, consisting of a total of 504 affected subjects and 559 unaffected subjects. For each marker, a mean of 648 meioses were informative. D4S139 and D4S163 were identified as the closest linked markers to the FSHD locus, with 99% upper confidence intervals of recombination fractions of .08 and .10, respectively. We have used the CRI-MAP program to construct the most likely order of cen-D4S171-F11-D4S163-D4S139-FSHD-tel, with favorable odds of 10(8)-10(114) over all other orders except that in which F11 and D4S171 are reversed, for which the odds ratio was 191:1. With this order, the genetic map of this region extends 25.5 cM in males and 13.8 cM in females (averaging 19.5 cM for sexes combined); the sex difference was statistically significant (P = .0013). Comparison between families for the two-point and multipoint lod scores involving FSHD showed no evidence for heterogeneity of this disorder. However, after the completion of this analysis, one large family which might show heterogeneity was identified. In view of this and the fact that all of the linked markers reside on the same side of the FSHD locus, the clinical application of these markers is not recommended at this time.     

Evidence for heterogeneity in facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is a slowly progressive primary disease of muscle which is usually inherited as an autosomal dominant disorder. FSHD has been localized to the long arm of chromosome 4, specifically to the 4q3.5-qter region. Initially published linkage studies showed no evidence for heterogeneity in FSHD. In the present study we have examined individuals in seven FSHD families. Two-point lod scores show significant evidence for linkage for D4S163 (lod score 3.04 at recombination fraction .21) and D4S139 (lod score 3.84 at recombination fraction .20). D4S171 also gave a positive score (lod score 2.56 at recombination fraction .24). Significant evidence for heterogeneity was found for each of the three markers. Multipoint linkage analysis in this region resulted in a peak multipoint lod score of 6.47. The multipoint analysis supported the two-point studies with odds of 20:1 showing linkage and heterogeneity over linkage and homogeneity. Five of the seven families gave a posterior probability of >95% of being of the linked type, while two families appeared unlinked to this region of 4q (P < .01%). Individuals in the two unlinked families met the clinical criteria for the diagnosis of FSHD, including facial weakness, clavicular flattening, scapula winging, proximal muscle weakness, and myopathic changes on muscle biopsies without inflammatory or mitochondrial pathology. This study demonstrates genetic heterogeneity in FSHD and has important implications for both genetic counseling and the elucidation of the etiology of FSHD.     

Linkage localization of facioscapulohumeral muscular dystrophy (FSHD) in 4q35.

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 FSHD-affected aunt and cousin.     

Genetic and physical mapping on chromosome 4 narrows the localization of the gene for facioscapulohumeral muscular dystrophy (FSHD).

We have used a combination of classical RFLPs and PCR-based polymorphisms including CA repeats and single-strand conformation polymorphisms to generate a fine-structure genetic map of the distal long arm of chromosome 4q. This map is now genetically linked to the pre-existing anchor map of 4pter-4q31 and generates, for the first time, a complete linkage map of this chromosome. The map consists of 32 anchor loci placed with odds of greater than 1,000:1. The high-resolution map in the cytogenetic region surrounding 4q35 provides the order 4cen-D4S171-F11-D4S187-D4S163-D4S139-4q ter. When we used somatic cell hybrids from a t(X;4)(p21;q35) translocation, these five markers fell into three groups consistent with the genetic map-D4S171 and F11 in 4pter-4q35, D4S163 and D4S139 in 4q35-4qter, and D4S187 as a junction fragment between these two regions. These markers are in tight linkage to the gene for facioscapulo-humeral muscular dystrophy (FSHD) mapped to this region by several collaborating investigators and provide a framework for further detailed analysis of this region.     

Chromosome 4q35 haplotypes and DNA rearrangements segregating in affected subjects of 19 Italian families with facioscapulohumeral musculatur dystrophy (FSHD)

Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810) detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new small fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.

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The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus

Facioscapulohumeral muscular dystrophy (FSHD) is a relatively common autosomal dominant neuromuscular disorder. The gene for FSHD has recently been assigned to chromosome 4q35. Although abnormal mitochondrial and biochemical changes have been observed in FSHD, the molecular defect is unknown. In addition to the FSHD gene, the human muscle adenine nucleotide translocator gene (ANT1) is located on chromosome 4. Interestingly, biochemical studies recently showed a possible defect of ANT1. In order to evaluate the potential role of ANT1 in the etiology of FSHD, the human ANT1 gene was isolated by cosmid cloning and localized to 4q35, in the region containing the FSHD gene. However, in situ hybridization and physical mapping of somatic cell hybrids localized the ANT1 gene proximal to the FSHD gene. In addition, a polymorphic CA-repeat 5 kb upsstream of the ANT1 gene was used as a marker in FSHD and Centre d'Etude du Polymorphisme Humain families to perform linkage analysis. These data together exclude ANT1 as the primary candidate gene for FSHD. The most likely order of the loci on chromosome 4q35 is cen-ANT1-D4S171-F11-D4S187-D4S163-D4S139-FSHD-tel.     

Germline mosaicism in 4q35 facioscapulohumeral muscular dystrophy (FSHD1A) occurring predominantly in oogenesis

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominantly inherited neuromuscular disorder affecting facial and shoulder girdle muscles with subsequent progression to the pelvic girdle and lower extremities. The major gene involved has been localized to chromosome 4q35 (FSHD1A). The 4q35 DNA marker p13E-11 (D4F104S1) detects a de novo EcoRI DNA rearrangement of < 30 kb in isolated and familial cases. The intrafamilial size of the fragment is constant, inversely correlated with the severity, and directly correlated with the age of onset of the condition. There has been evidence of parental mosaicism in FSHD1A for the D4F104S1 locus. Four female and three male clinically unaffected parents have been described to be carriers of EcoRI fragments of the same size as their affected offspring, but with a markedly less intensive hybridization signal (semi-quantitative evidence). In our total sample of 42 FSHD1A families, we found semi-quantitative evidence of parental D4F104S1 mosaicism in 11 families (EcoRI fragment size range: 12–27 kb). On analysis with adjacent 4q35 probes (D4S163, D4S139), additional qualitative evidence of germline mosaicism could be obtained in two families. In our mosaic families and in the families reported in the literature, a female predominance of mosaicism carriers (13 females versus 5 males) could be noted. In our sample, mosaicism was observed in multigeneration families, in families with isolated cases, and in families with two and three affected children from seemingly unaffected parents. A short EcoRI fragment once having emerged in a mosaicism carrier was found to be transmitted autosomal dominantly to subsequent generations. Of all reported sporadic patients, 19% have a mosaic parent. Finding evidence of parental mosaicism in all our families with more than one affected child of seemingly unaffected parents suggests that there is no autosomal recessively inherited form of FSHD1A. Received: 5 March 1996 / Revised: 14 May 1996     

Large-scale population analysis challenges the current criteria for the molecular diagnosis of fascioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4–8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1–8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.     

Correlation analysis of clinical parameters with epigenetic modifications in the DUX4 promoter in FSHD

The aim of our study was to identify relationships between epigenetic parameters correlating with a relaxed chromatin state of the DUX4 promoter region and clinical severity as measured by a clinical severity score or muscle pathologic changes in D4Z4 contraction-dependent (FSHD1) and –independent (FSHD2) facioscapulohumeral muscular dystrophy patients. Twenty primary fibroblast (5 control, 10 FSHD1 and 5 FSHD2) and 26 primary myoblast (9 control, 12 FSHD1 and 5 FSHD2) cultures originating from patients with FSHD and controls were analyzed. Histone modification levels were determined by chromatin immunoprecipitation. We examined correlations between the chromatin compaction score (ChCS) defined by the H3K9me3:H3K4me2 ratio and an age corrected clinical severity score (CSS) or muscle pathology score (MPS). Possible relationships were investigated using linear regression analysis and significance was tested by Pearson’s product-moment coefficient.   We found a significant difference of the ChCS between controls and patients with FSHD1 and between controls and patients with FSHD2. Tissue specific differences in ChCS were also observed. We also found a near-significant relationship between ChCS and the age corrected CSS in fibroblasts but not in myoblasts. Surprisingly, we found a strong correlation between the MPS of the vastus lateralis and the CSS. Our results confirm the D4Z4 chromatin relaxation previously shown to be associated with FSHD in a small number of samples. A possible relationship between clinical and epigenetic parameters could be established in patient fibroblasts, but not in myoblasts. The strong correlation between the MPS of the vastus lateralis and the CSS suggests that this muscle can be used to study for surrogate markers of overall disease severity.     

Correlation analysis of clinical parameters with epigenetic modifications in the DUX4 promoter in FSHD

The aim of our study was to identify relationships between epigenetic parameters correlating with a relaxed chromatin state of the DUX4 promoter region and clinical severity as measured by a clinical severity score or muscle pathologic changes in D4Z4 contraction-dependent (FSHD1) and –independent (FSHD2) facioscapulohumeral muscular dystrophy patients. Twenty primary fibroblast (5 control, 10 FSHD1 and 5 FSHD2) and 26 primary myoblast (9 control, 12 FSHD1 and 5 FSHD2) cultures originating from patients with FSHD and controls were analyzed. Histone modification levels were determined by chromatin immunoprecipitation. We examined correlations between the chromatin compaction score (ChCS) defined by the H3K9me3:H3K4me2 ratio and an age corrected clinical severity score (CSS) or muscle pathology score (MPS). Possible relationships were investigated using linear regression analysis and significance was tested by Pearson’s product-moment coefficient.

We found a significant difference of the ChCS between controls and patients with FSHD1 and between controls and patients with FSHD2. Tissue specific differences in ChCS were also observed. We also found a near-significant relationship between ChCS and the age corrected CSS in fibroblasts but not in myoblasts. Surprisingly, we found a strong correlation between the MPS of the vastus lateralis and the CSS. Our results confirm the D4Z4 chromatin relaxation previously shown to be associated with FSHD in a small number of samples. A possible relationship between clinical and epigenetic parameters could be established in patient fibroblasts, but not in myoblasts. The strong correlation between the MPS of the vastus lateralis and the CSS suggests that this muscle can be used to study for surrogate markers of overall disease severity.     

Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of the D4Z4 repeat in the subtelomere of chromosome 4q. Two allelic variants of chromosome 4q (4qA and 4qB) exist in the region distal to D4Z4. Although both variants are almost equally frequent in the population, FSHD is associated exclusively with the 4qA allele. We identified three families with FSHD in which each proband carries two FSHD-sized alleles and is heterozygous for the 4qA/4qB polymorphism. Segregation analysis demonstrated that FSHD-sized 4qB alleles are not associated with disease, since these were present in unaffected family members. Thus, in addition to a contraction of D4Z4, additional cis-acting elements on 4qA may be required for the development of FSHD. Alternatively, 4qB subtelomeres may contain elements that prevent FSHD pathogenesis.     

Mechanism and timing of mitotic rearrangements in the subtelomeric D4Z4 repeat involved in facioscapulohumeral muscular dystrophy

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD1A) is associated with contractions of the polymorphic D4Z4 repeat on chromosome 4qter. Almost half of new FSHD mutations occur postfertilization, resulting in somatic mosaicism for D4Z4. Detailed D4Z4 analysis of 11 mosaic individuals with FSHD revealed a mosaic mixture of a contracted FSHD-sized allele and the unchanged ancestral allele in 8 cases, which is suggestive of a mitotic gene conversion without crossover. However, in 3 cases, the D4Z4 rearrangement resulted in two different-sized D4Z4 repeats, indicative of a gene conversion with crossover. In all cases, DNA markers proximal and distal to D4Z4 showed no allelic exchanges, suggesting that all rearrangements were intrachromosomal. We propose that D4Z4 rearrangements occur via a synthesis-dependent strand annealing model that relatively frequently allows for crossovers. Furthermore, the distribution of different cell populations in mosaic patients with FSHD suggests that mosaicism here results from D4Z4 rearrangements occurring during the first few zygotic cell divisions after fertilization.