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

Abstract

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.

Figure 1

Schematic presentation of the telomeric regions of chromosome 10q26 and the two variants of chromosome 4q35, 4qA and 4qB. The region distal to D4S2463 shows 98% homology between chromosomes 10q and 4q, except for the region distal to D4Z4 on the 4qB variant. Both 4q alleles are almost equally present in the population, but FSHD has been associated with only the 4qA variant. Chromosome 4–type D4Z4 repeats are depicted as blackened triangles, and the chromosome 10 D4Z4 repeats are shown as unblackened triangles. The locations of the EcoRI (E) and HindIII (H) restriction sites adjacent to D4Z4 are shown, as well as probes p13E-11 (blackened box), 4qB (blackened box), and 4qA (unblackened box). Chromosomal assignment depicted in figure 2G–2I makes use of the NotI restriction site (N) proximal to the chromosome 4–specific probe B31 (unblackened box).

Figure 2

A–F, Southern blot analysis of samples from three families with FSHD (Rf215, Rf202, and Rf207). DNA was double-digested with EcoRI-HindIII (E) and EcoRI-BlnI (B), separated by PFGE, and hybridized with probe p13E-11. The FSHD-sized 4qB alleles (unblackened circles) of 26 kb, 30 kb, and 34 kb, respectively, are underlined (A–C). FSHD-causing 4qA alleles are indicated with blackened circles. For allelotyping (4qA/4qB), DNA was digested with HindIII, separated by PFGE, and subsequently hybridized with probes 4qA and 4qB (D–F). Marker sizes (in kb) are indicated on the right. A and D, In family Rf215, the affected proband (III-1) carried two chromosome 4–type FSHD-sized D4Z4 repeats. Allelotyping shows that the 20-kb D4Z4 repeat is of 4qA origin and was inherited from the affected father. The 26-kb D4Z4 repeat was inherited from the healthy mother and is of 4qB origin (D). Furthermore, the affected child carried chromosome 10 repeats of 70 kb and 145 kb. Segregation analysis showed that the FSHD-causing allele was inherited from the affected grandfather (I-1) and that the nonpathogenic short 4qB allele was inherited from the healthy maternal grandmother (I-4) and is also observed in a healthy uncle (II-3) of the affected proband (III-1) (A). B and E, In family Rf202, three affected sisters (II-1, II-2, and II-3) were identified as carriers of two FSHD-sized D4Z4 repeats. Allele-sizing and allelotyping identified a 25-kb 4qA allele and a 30-kb 4qB allele (B and E). The 30-kb 4qB allele was inherited from the healthy father (I-1), and the FSHD-causing 25-kb 4qA allele was inherited from the affected mother (I-2). Comigrating chromosome 10 alleles of 75 kb in the father are marked with “*1.” The mother carried a 45-kb chromosome 4–type allele (marked with “*2”), which originated from chromosome 10 (translocated chromosome 4–type repeat) and was passed to her daughters II-1 and II-3. C and F, In family Rf207, the oldest affected daughter (II-1) carried two FSHD-sized repeats. Allele-sizing and allelotyping identified a 27-kb 4qA allele and a 34-kb 4qB allele (C and F). The 34-kb 4qB allele was inherited from the healthy father (I-1), and the FSHD-causing 27-kb 4qA allele was inherited from the affected mother (I-2). Further analyses show that the FSHD allele in the mother comigrates with a chromosome 10 allele of 27 kb (marked with “*3”). G–I, Chromosome assignment of the D4Z4 repeats in three families with FSHD (Rf215, Rf202, and Rf207). NotI-digested DNA plugs and the chromosome 4–specific probe B31 were used. The NotI fragment is 185 kb larger than the EcoRI-HindIII fragment on chromosome 4. Marker sizes (in kb) are indicated on the right. G, The two chromosome 4–derived repeats of 20 kb and 26 kb in the compound heterozygous affected proband (III-1) in family Rf215 are visualized at 205 kb (20 kb + 185 kb) and 211 kb (26 kb + 185 kb). These results show that both FSHD-sized repeats do indeed reside on chromosome 4. Furthermore, the chromosomal assignment reveals that the chromosome 4–type 75-kb repeat in the father resides on chromosome 4 (75 kb + 185 kb = 260 kb), just like the 200-kb repeat in the mother (200 kb + 185 kb = 385 kb). H, Chromosomal assignment in family Rf202 shows that the two FSHD-sized alleles inherited by the three affected sisters (II-1, II-2, and II-3) are both located on chromosome 4. They are visualized at 210 kb (25 kb + 185 kb) and 215 kb (30 kb + 185 kb), corresponding to the paternal 25-kb repeat and the maternal 30-kb repeat, respectively. The other chromosome 4 alleles from the father and the mother are visualized at 281 kb (96 kb + 185 kb) and 240 kb (55 kb + 185 kb), respectively. I, The two FSHD-sized alleles in the affected daughter (II-1) of family Rf207 are located on chromosome 4 and are visible at 212 kb (27 kb + 185 kb) and 219 kb (34 kb + 185 kb).