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

Abstract

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.

Figure 1

A polymorphic PvuII site at position 6044 of GenBank accession number AF117653 was used to study recombination between 4qA and 4qB alleles. A, Double digestion with PvuII/BlnI normally gives rise to fragments of 4,559 bp (chromosome 4 alleles) and 2,464 bp (chromosome 10 alleles). However, the presence of an extra PvuII site within the proximal D4Z4 unit yields a fragment of 2,849 bp. B, Example of the modified PvuII/BlnI dosage test after hybridization with probe p13E-11. Samples in lanes 1–3 carry two 4qA alleles and two 10q alleles, all of which are PvuII resistant (PvuII⁻). Samples in lanes 4–6 carry two 4qB alleles and two 10q alleles; in lane 4 and lane 6, a PvuII-sensitive (PvuII⁺) fragment is visible. “Y” indicates the cross-hybridizing Y chromosome. The table on the right shows dosage experiment results of the PvuII RFLP in the proximal D4Z4 unit of 78 different 4qA and 62 different 4qB alleles. Twenty-nine percent of the 4qB alleles are PvuII⁺, whereas 4qA alleles are almost exclusively PvuII⁻ (P<10^-5). C, Distribution of the PvuII RFLP on 4qA- and 4qB-type alleles. The PvuII⁻ proximal D4Z4 unit on 4qA-type alleles is indicated by “−,” whereas D4Z4 units that were heterozygous for PvuII RFLP are indicated by “+/−.”

Figure 2

Example of p13E-11 hybridization after PFGE and Southern blot analysis: analysis of mosaic family 26 with FSHD (GC-CO = gene conversion without crossover). DNA was double digested with EcoRI/HindIII (“E”) and EcoRI/BlnI (“B”), separated by PFGE, and hybridized with p13E-11. The patient inherited the paternal (“p”) 60-kb chromosome 10 allele and 90-kb 4qB allele and, from his mother (“m”), the 100-kb chromosome 10 allele and the 140-kb 4qA allele (unblackened arrowhead), which is present in only 50% of his PBL. The 14-kb 4qA allele (blackened arrowhead) causing FSHD is derived from the 140-kb maternal allele and is detected in 50% of his PBL. Estimation of the proportion of mosaic alleles is based on the signal intensities of the D4Z4 fragments, with a CI of 5%. Allele types are not shown. Marker sizes (“M”) in kilobases are indicated on the left.

Figure 3

Two examples of p13E-11 hybridization after PFGE and Southern blot. Analyses, as in figure 2, of mitotic interchromatid gene conversion with crossover (GC+CO) in families 36 and 1 with FSHD. Estimation of the proportion of mosaic alleles is based on the signal intensities of the D4Z4 fragments, with a CI of 5%. Allele types are not shown. A, Male patient with de novo FSHD in family 36, who inherited 10q alleles of 50 kb and 130 kb and a 140-kb 4qA-type allele. He also has mosaic alleles of 384 kb (40%; 4A^exp) and 16 kb (60%; 4A^FSH), which are expanded and contracted alleles, respectively, most probably produced by an interchromatid gene conversion with crossover in the maternal 100-kb or 240-kb allele. Maternally and paternally inherited alleles are indicated by “m” and “p,” respectively. Comigrating chromosome 4 and 10 alleles of 130 kb in the father are marked with an asterisk. Marker sizes (“M”) in kilobases are indicated on the left. B, The left panel shows EcoRI/HindIII (“E”) and EcoRI/BlnI (“B”) digestion of DNA from family 1 with de novo FSHD. The patient from this family has three mosaic alleles—of 14kb, 70 kb, and 96 kb—that originated from the paternal 70-kb 4qA allele. The 96-kb 4qA mosaic allele is comigrating with a maternal 96-kb 4qB allele. Furthermore, he inherited a paternal 60-kb 10q allele and a maternal 135 kb 4qB-type allele (marked with an asterisk), which originated from chromosome 10 (translocated 4-type repeat (t10;4) (van Deutekom et al. 1996). Quantification of all alleles revealed 100% intensity for the 135-kb (t10;4) 4qB allele, 137% for the comigrating 96-kb 4qA and 4qB alleles, 37% for the 70-kb 4qA allele, 100% for the 60-kb 10q allele, and 26% for the 14-kb 4qA allele. An EcoRV (“EV”) digestion allows separation of the comigrating 4qA and 4qB alleles at 96 kb (137%). On the basis of EcoRV digestion (right panel) the patient clearly has six alleles; of these, the contracted 14-kb 4qA allele (26%; FSHD allele, 4A^FSH) and the expanded 96-kb 4qA allele (37%; 4A^exp) originate from the mitotic rearranged parental 70-kb allele (37%; 4A⁰). Two arrows indicate D4Z4 expansion and contraction. Allele quantifications after EcoRV digestion are indicated in the right example panel. Marker sizes (“M”) in kilobases are indicated at the right. In the box, the constitution of the three different cell populations of this patient is schematically depicted.