FSH dystrophy and a subtelomeric 4q haplotype: a new assay and associations with disease

Abstract

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease associated with contraction of arrays of tandem 3.3-kb units (D4Z4) on subtelomeric 4q. Disease-linked arrays usually have fewer than 11 repeat units. Equally short D4Z4 arrays at subtelomeric 10q are not linked to FSHD. The newly described 4qA161 haplotype, which is more prevalent in pathogenic 4q alleles, involves sequences in and near D4Z4.

Methods: We developed two new assays for 4qA161, which are based upon direct sequencing of PCR products or detecting restriction fragment length polymorphisms. They were used to analyse single nucleotide polymorphisms (SNPs) indicative of 4q161 alleles.

Results: All (35/35) FSHD patients had one or two 4qA161 alleles (60% or 40%, respectively). In contrast, 46% (21/46) of control individuals had no 4qA161 allele (p<10(-4)), and 26% had homozygous 4qB163 alleles.

Conclusions: Our results from a heterogeneous population are consistent with the previously described association of the 4qA161 haplotype with FSHD, but a causal association with pathogenesis is uncertain. In addition, we found that haplotype analysis is complicated by the presence of minor 10q alleles. Nonetheless, our sequencing assay for the 4qA161allele can enhance molecular diagnosis of FSHD, including prenatal diagnosis, and is simpler to perform than the previously described assay.

Figure 1

Cartoon of the major 4q haplotypes. The D4Z4 array consists of variable numbers of 3.3 kb units (green triangles) ~25–33 kb from the telomeric (TTAGGG)_n (distal end sequence derived from van Geel et al and Genome Reference Consortium h37 10qA and 4qB sequences). Proximal to D4Z4 is a SSLP subregion (thin red or grey line) and SNP-rich 0.4-kb subregion (thick red or grey line); the 0.8-kb p13E-11 subregion (purple double-headed arrow) is indicated. Not included are less frequent haplotypes with unreported sequences. 4qA and 4qB alleles have almost equal prevalence at 4q. The predominant D4Z4-distal allele at 10q is very similar to 4qA and called 10qA. 4qA and 10qA, but not 4qB, contain an ~3-kb subregion of 68-bp tandem repeats (red bar) (A–D). Dense SNPs in the 0.4-kb region adjacent to D4Z4. SNP3 and SNP6 are highlighted. The BtsI site that distinguishes the main 10q allele (BtsI^S) and 4q alleles (BtsI^R) is shown (E).

Figure 2

Testing for the 4qA161 haplotype by differential digestion with Hpy188I and Hpy188III. Genomic DNAs and human-rodent somatic cell hybrids (GM14193, GM11687, and GM10926 containing chr4, chr4, and chr10, respectively; Coriell Institute) were analysed for overlapping Hpy188I (purple) or Hpy188III (blue) sites. Whether one or two 4qA161 alleles were present was subsequently determined by the sequencing assay. GM11448 and GM10115 containing chr4 were negative for 4qA161 (not shown) (A). The Hpy188I or Hpy188III sites are indicated for the forward strand in the SNP-rich region of 4qA161, the other main 4qA alleles, and the predominant 10qA allele (B).

Figure 3

Testing for the 4qA161 haplotype by sequencing through SNP3 and SNP6 on the reverse strand. Sequence alignment of the 4qA alleles and 10qA166 allele illustrates the 4q-specific nature of PCR primer Bts-R. The BtsI site overlapping the primer in 10qA166 is highlighted (A). PCR with Bts-R as one of the primers followed by sequencing the reverse strand allows identification of the 4qA161haplotype. As described in the text, digestion with BtsI to eliminate 10qA166 amplicons before PCR is not necessary (B).

Figure 4

Results from the DNA sequencing test for 4qA161. Sequencing data from representative samples containing two 4qA161 alleles, none or one (A). Genotype frequencies from our assays of 35 FSHD patients and 46 controls (B).

Figure 5

A possible scheme for use of the sequencing SNP assay in clinical testing for FSHD1, which involves short 4q D4Z4 arrays and constitutes ~95% of FSHD cases. Analysis paths are shown by arrows in series. Thick arrows indicate most likely outcomes. ^aCanonical 10q D4Z4 arrays in EcoRI/BlnI digests and canonical 4q arrays in XapI digests are reduced to small fragments that electrophorese off the gel. PFGE is highly preferable to LGE, for example, PFGE can reveal somatic mosaics having five hybridising EcoRI fragments. If LGE is used, the so-called dosage test should be done to determine if the most proximal D4Z4 repeat unit on both 4q alleles is 4q-type (BlnI^R) and on both 10q alleles is 10q-type (BlnI^S), and the blot should be re-probed with D4Z4. EcoRI/HindIII digests can be used instead of EcoRI digests to get cleaner bands, but fragments are ~2 kb smaller. The maximum size of pathogenic D4Z4 alleles is only an approximate threshold due to incomplete penetrance, especially when EcoRI fragments are 35–41 kb; also, some patients may have very slightly larger D4Z4 arrays. ^bMatching EcoRI/BlnI fragments are ~3 kb shorter and XapI fragments are ~5 kb shorter than the corresponding EcoRI fragment. ^cThe sequencing test is the recommended assay for 4qA161, the predominant haplotype of pathogenic D4Z4 alleles. It can serve as a surrogate for 4qA/4qB testing by PFGE on HindIII digests with 4qA- and 4qB-specific oligonucleotide probes. There can be pathogenic non-4qA161 alleles with 10q-type BlnI^S D4Z4 repeat units on 4q. However, categories I, III and VI in figure 6 do not involve such arrays; therefore, 4qA161 testing would be informative for samples giving these types of PFGE patterns, especially because ~40–50% of controls have no 4qA161 allele. ^dThe D4Z4 subfragment probe under stringent hybridisation conditions detects fragments with p13E-11 deletions and gives stronger signals than the p13E-11 probe from XapI digests. ^eCategories IV, Va and Vb are non-standard D4Z4 arrays with canonical 10q-type repeat units on 4q or vice versa, as evidenced by a missing EcoRI/BlnI fragment (only one EcoRI/BlnI fragment instead of two, figure 6, Vb) or an EcoRI/BlnI fragment that is less than 3 kb shorter than the corresponding EcoRI fragment (figure 6, IV and Va). ^fSometimes, the chromosomal origin of a short EcoRI fragment has to be determined by PFGE on NotI digests blot-hybridised to a 4q35-specific probe (B31), for example, figure 6, category VI versus category VII, and unusual cases with all four arrays beginning with 10q-type D4Z4 units. The NotI fragment length will be the sum of 187 kb and the length of the corresponding EcoRI fragment.

Figure 6

A cartoon illustrating various categories of subtelomeric 4q and 10q genotypes (I–VII; large white rectangles) analysed by Southern blotting after digestion with EcoRI, EcoRI plus BlnI and XapI (ApoI) and probing with p13E-11 (large yellow rectangles) or D4Z4 (single large light-green rectangle). Non-standard D4Z4 arrays on 4q that contain BlnI^S units (black-filled boxes) and those on 10q that contain BlnI^R units (white boxes) complicate the determination of whether a short array is present on 4q. These types of D4Z4 arrays with repeat units from chr10 on 4q are almost always mixed with canonical chr4-type units in various patterns, and those from chr4 on 10q are usually homogeneously chr4-type, as illustrated for categories IV–VII. Other combinations of translocated arrays are possible.