Specific sequence variations within the 4q35 region are associated with facioscapulohumeral muscular dystrophy

Abstract

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is mainly characterized by progressive wasting and weakness of the facial, shoulder, and upper-arm muscles. FSHD is caused by contraction of the macrosatellite repeat D4Z4 on chromosome 4q35. The D4Z4 repeat is very polymorphic in length, and D4Z4 rearrangements occur almost exclusively via intrachromosomal gene conversions. Several disease mechanisms have been proposed, but none of these models can comprehensively explain FSHD, because repeat contraction alone is not sufficient to cause disease. Almost-identical D4Z4-repeat arrays have been identified on chromosome 10q26 and on two equally common chromosome 4 variants, 4qA and 4qB. Yet only repeat contractions of D4Z4 on chromosome 4qA cause FSHD; contractions on the other chromosomes are nonpathogenic. We hypothesized that allele-specific sequence differences among 4qA, 4qB, and 10q alleles underlie the 4qA specificity of FSHD. Sequence variations between these alleles have been described before, but the extent and significance of these variations proximal to, within, and distal to D4Z4 have not been studied in detail. We examined additional sequence variations in the FSHD locus, including a relatively stable simple sequence-length polymorphism proximal to D4Z4, a single-nucleotide polymorphism (SNP) within D4Z4, and the A/B variation distal to D4Z4. On the basis of these polymorphisms, we demonstrate that the subtelomeric domain of chromosome 4q can be subdivided into nine distinct haplotypes, of which three carry the distal 4qA variation. Interestingly, we show that repeat contractions in two of the nine haplotypes, one of which is a 4qA haplotype, are not associated with FSHD. We also show that each of these haplotypes has its unique sequence signature, and we propose that specific SNPs in the disease haplotype are essential for the development of FSHD.

Figure 1.

A, Schematic representation of the D4Z4 repeat on chromosomes 4q35 and 10q26 with the localization of the four polymorphic markers used in this study. The SSLP marker is localized 3.5 kb proximal to D4Z4. The D4Z4 SNP, the D4Z4-repeat size variation, and the distal A/B variation have been described elsewhere.^, The 475-bp D4F104S1 sequence is localized immediately proximal to D4Z4. B, Overview of the total number of control and patient alleles that were tested for these polymorphisms.

Figure 2.

Examples of D4Z4 genotyping, including the three polymorphic markers in control individual (1) and FSHD-affected patient (2). A, D4Z4 sizing and A/B typing after PFGE and Southern blotting. For D4Z4 sizing, DNA was digested with restriction enzymes EcoRI and HindIII (lane E), EcoRI and BlnI (lane B), or XapI (lane X), and the blotted DNA was hybridized with probe p13E-11 located immediately proximal to D4Z4 (left panel). The four fragments present in lane E represent two 4q- and two 10q-derived repeat arrays with different repeat sizes. The 4q or 10q origin of the D4Z4 repeats can be determined by digestions with BlnI or XapI, which fragmentize 10q- or 4q-derived repeat arrays, respectively. Consequently, individual 1 carries 4q D4Z4 repeats of 40 kb and 55 kb and 10q repeats of 95 kb and 130 kb, whereas individual 2 carries 4q alleles of 35 kb and 165 kb and 10q repeats of 50 kb and 65 kb. For A/B typing, DNA was digested with HindIII (lane H) and was hybridized serially with probes 4qA and 4qB, located immediately distal to D4Z4 (right panel). Both 4q alleles (40 kb and 55 kb) in individual 1 carry the B variant, and both 4q alleles (35 kb and 165 kb) in individual 2 carry the A variant. As was shown elsewhere, 10q alleles almost always carry the A variant. The molecular weight marker (M) in kilobases is shown in the middle. Cross-hybridizing Y-chromosome–derived fragments with probe p13E-11 in individual 2 (male) are indicated with “Y.” Non-4q and -10q cross-hybridizing fragments with probes 4qA and 4qB are marked with an asterisk (*). B, Analysis of the G/C SNP within D4Z4 (for description, see the work of Lemmers et al.4). Double digestion of genomic DNA with PvuII and BlnI (PB) reveals 10q fragments of 2,464 bp upon hybridization with p13E-11. 4q alleles reveal fragments of either 4,559 bp or 2,849 bp, depending on the presence (C variant) or absence (G variant) of the PvuII restriction site in D4Z4, respectively. One 4qB allele in individual 1 contains the C variant, whereas all other 4q alleles in both individuals carry the G variant. C, Example of the fragment run of the SSLP sequence. Altogether, this analysis demonstrates that individual 1 carries a 4qB161 and a 4qB163 allele next to the two 10qA166 alleles and that individual 2 carries two 4qA161 alleles and two 10qA166 alleles.

Figure 3.

Haplotype analyses of individual 4q and 10q DNA sources, of which the last six 10q sequences are obtained from GenBank. The identity of the DNA source is shown in the first column. The haplotype and the SSLP sequence are listed in columns 2 and 3, whereas the D4Z4 SNP is shown in column 4. The last two columns display the D4Z4-repeat length (in kb) and the distal variation.

Figure 4.

Overview of the different haplotypes that were defined after complete genotyping of 4qA, 4qB, and 10q alleles. A, All D4Z4-repeat units on 10q alleles encompass the BlnI restriction site (B), which is absent in D4Z4 in all 4q alleles. Likewise, all D4Z4-repeat units on 4q alleles encompass the XapI restriction site (X) that is absent in D4Z4-repeat units on 10q alleles, except for D4Z4 repeats in the 4qA166 haplotype, which lack the XapI restriction site in their most proximal D4Z4 unit (indicated with an asterisk [*]). B, Genotyping of 4q and 10q alleles in control individuals. The first column lists the different haplotypes that have been identified, and the second shows the sequence of the SSLP in each haplotype (M = A or C). Those haplotypes for which we could not obtain SSLP sequence information are indicated by “NA” (not analyzed). The third and fifth columns represent, respectively, the number of alleles found in each haplotype (SSLP n) and the prevalence (%) of each haplotype among 4qA, 4qB, and 10q alleles. The distribution of the G and C variants in the D4Z4 SNP is shown in the fourth column (D4Z4 SNP (G/C)). In total, 200 4qA alleles were genotyped, and, on the basis of SSLP length, they were subdivided into three haplotypes: 4qA161, 4qA163, and 4qA166. 4qB alleles (n=244) were subdivided into six haplotypes, designated as 4qB161, 4qB162, 4qB163, 4qB164, 4qB166, and 4qB168, and 10q alleles into haplotypes 10qA166 and 10qA164. There is a significant difference in D4Z4 SNP distribution between 4qA and 4qB haplotypes (P<.0001) and between 4qB163 and 4qB162, 4qB166, and 4qB168 haplotypes (P<.0001). C, Genotyping of alleles with an FSHD-sized D4Z4 repeat. Without exception, all FSHD alleles belong to the 4qA161 haplotype. All alleles with an FSHD-sized repeat that belong to the 4qA166 or 4qB163 haplotypes are nonpathogenic.

Figure 5.

Pedigrees of FSHD-affected families Rf10 and Rf204. The patients with FSHD in family Rf10 carry a 33-kb 4qA161 (33A¹⁶¹) allele and are moderately affected. Individuals II-9 and II-11 carry a 33-kb 4qA166 (33A¹⁶⁶) allele and do not display any clinical signs of muscular dystrophy. In family Rf204, all patients with FSHD carry a 17-kb 4qA161 (17A¹⁶¹) allele and are moderately to severely affected. Six individuals carry an FSHD-sized allele of 24 kb (24A¹⁶⁶), which belongs to the 4qA166 haplotype, and they are not affected (II-11, III-2, III-3, III-4, III-5, and III-6). Index cases are indicated with an arrow.

Figure 6.

Alignment of a 475-bp consensus sequence within D4F104S1 of the most common haplotypes (4qA161, 4qB163, 4qA166, 4qB168, and 10qA166). The consensus is based on the sequence of at least three independent alleles for each specific haplotype. The highlighted nucleotides indicate the presence of haplotype-specific SNPs. Similar to the SSLP, the 4qB163 sequence is very homologous to the pathogenic 4qA161 sequence, whereas the 4qA166 sequence is more homologous to the 10qA166 and 4qB168 sequences.

Figure 7.

A, Overview of D4Z4-repeat length distribution in the most common haplotypes, 4qA161, 4qB163, and 10qA166. Statistical analysis revealed that the D4Z4-repeat size distributions differ significantly between these haplotypes (P<.0001). B, Mean and median of the D4Z4-repeat length and number of alleles in the major haplotypes.