New multiplex PCR-based protocol allowing indirect diagnosis of FSHD on single cells: can PGD be offered despite high risk of recombination?

Abstract

Molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) involves the heterozygous contraction of the number of tandemly repeated D4Z4 units at chromosome 4q35.2. FSHD is associated with a range of 1-10 D4Z4 units instead of 11-150 in normal controls. Several factors complicate FSHD molecular diagnosis, especially the cis-segregation of D4Z4 contraction with a 4qA allele, whereas D4Z4 shortening is silent both on alleles 4qB and 10q. Discrimination of pathogenic 4q-D4Z4 alleles from highly homologous 10q-D4Z4 arrays requires the use of the conventional Southern blot, which is not suitable at the single-cell level. Preimplantation genetic diagnosis (PGD) is a frequent request from FSHD families with several affected relatives. We aimed to develop a rapid and sensitive PCR-based multiplex approach on single cells to perform an indirect familial segregation study of pathogenic alleles. Among several available polymorphic markers at 4q35.2, the four most proximal (D4S2390, D4S1652, D4S2930 and D4S1523, <1.23 Mb) showing the highest heterozygote frequencies (67-91%) were selected. Five recombination events in the D4S2390-D4S1523 interval were observed among 144 meioses. In the D4S2390-D4Z4 interval, no recombination event occurred among 28 FSHD meioses. Instead, a particular haplotype segregated with both clinical and molecular status, allowing the characterization of an at-risk allele in each tested FSHD family (maximal LOD score 2.98 for theta=0.0). This indirect protocol can easily complement conventional techniques in prenatal diagnosis. Although our multiplex PCR-based approach technically fulfils guidelines for single-cell analysis, the relatively high recombination risk hampers its application to PGD.

Figure 1

Schematic representation of the 4qter region. For each marker, we have reported (1) the physical distance to the D4Z4 locus in Mb (megabases) (according to http://genome.ucsc.edu/cgi-bin/hgGateway), (2) the positions in cM (according to the Marshfield genetic map), (3) the estimated heterozygote frequencies for the four selected markers (bold underlined) and (4) the heterozygote frequencies from the CEPH database (http://www.cephb.fr/fr/cephdb/). The estimated heterozygote frequencies were low for the D4S2283 and near zero for the newly described markers (BF992518, 17xTG, BG167386, BC087857, D4F104S1 and 22xTG). (^*) The D4F104S1 that overlaps the SSLP described by Lemmers et al.²⁸ displayed complex electrophoresis patterns (not shown) with more than two alleles. Het, heterozygote frequencies; CEN, centromer; TEL, telomer.

Figure 2

Segregation study of STS markers in a control family. Identification of five recombination events among 12 meioses in individuals II:5, II:6 and III:3. Recombinant alleles are surrounded.