Besides the large variation of clinical severity and age of onset, FSHD manifests an almost complete penetrance after the age of 20 years and prognosis in terms of mobility loss, pain and fatigue might be pejorative. Moreover, there is no specific therapeutic and some FSHD patients experience a very poor quality of life, which often worsens with age. With a 50% chance of having affected offspring, D4Z4 contraction carriers and affected parents may request PGD as an alternative to termination of pregnancy after PND. Standard molecular testing for FSHD (Southern blot and PFGE) is not applicable to PGD, which is usually performed on single blastomere DNA sampled from a 6- to 8-cell embryo, and in a limited time frame of 24
h at day 3 after intracytoplasmic sperm injection. Thus, PCR-based methods are mandatory for single-cell genetic analysis.
Although counting D4Z4 repeats by long PCR seems promising in terms of cost, specificity and rapidity (14
h), it suffers from a number of weaknesses. In particular, it is unsuitable for PGD because it requires large amounts of genomic DNA (>500
Moreover, the absence of PCR amplicons might be observed in various situations: amplification failure, large FSHD-sized alleles (>18.4
kb, ie, >5 repeats) frequent in Caucasian patients, non-carrier individuals (>11 repeats), or p13E-11 deletion including the upstream primer (3% of FSHD cases).11, 30, 35
In addition, false-positive amplification of the short allele occurs in asymptomatic mosaic carriers.36
Thus, this approach constitutes a first-step screening method and Southern blotting is still required in at least 10% of FSHD cases.34
In addition, the UCSC in Silico
PCR gave no match with the long-range primers and The Human BLAT Search mapped the reverse primer on 10q but not on 4qter (http://genome.ucsc.edu/
We designed new primers more proximal to the D4Z4 locus and outside the p13E-11 sequence. Nonetheless, a complex array of PCR products (all below 10
kb) was observed instead of specific products of expected size, which is not surprising according to the GC richness (73%) and the complexity of this region (data not shown).
As the direct D4Z4 array sizing by PCR is not reliable in all situations, we turned towards an indirect molecular strategy by segregation analysis of microsatellite markers. We first evaluated a nested PCR protocol on single lymphocytes.31
After an exhaustive in silico
exploration of genomic 4q35.2 sequences, 14 putative polymorphic loci were combined into several multiplex PCRs and evaluated on 600 lymphocytes, followed by about 1500 specific PCRs. The electrophoretic patterns and the estimated amplification rates did not satisfy the recommended criteria for the single-cell level.37
Moreover, although highly sensitive, the nested PCR is costly and time-consuming, requiring a very tedious handling of a large number of PCR microtubes, increasing the risk of contamination and labelling errors.
Among the 14 markers, the D4F104S1
overlaps the recently described SSLP. On the basis of the electrophoresis sizes, it displays four frequent alleles 161, 163, 166 and 168 on 4qA, 4qB and/or 10qA haplotypes.28
As this marker is mapped within the highly homologous 4qter and 10qter regions, all our family members displayed complex electrophoretic patterns with 2–4 alleles. Moreover, the exhaustive 4qA/B haplotyping is unusable for single cell, as it requires Southern blotting for A/B variation and D4Z4 SNP genotyping, and fragment analysis of the SSLP
proved to be unsuitable for accurate genotyping.
Finally, 10 markers, including D4F104S1
, were dismissed and we selected 4 markers (D4S2390, D4S1652, D4S2930
) according to their estimated informativity, amplification efficacies and distance to the D4Z4
To analyse several microsatellite markers, we evaluated whole genome amplification using multiple displacement amplification (MDA) rapid protocols (1.5
Locus-specific PCRs on amplified single-cell genomic DNA showed poor efficacies and very high ADOs (data not shown). Similar observations were reported for single-cell applications, and surprisingly, despite these major drawbacks, MDA protocols have been successfully applied in PGD.39, 40, 41, 42, 43, 44, 45
Then, we developed a new one-step multiplex PCR with the four selected markers, which proved to be more rapid (<8
h for 16–32 samples), less expensive and requiring fewer sample handling steps than other protocols. The observed heterozygote rates at the four loci revealed full informativity in our control cohort and corroborated the CEPH data (http://www.cephb.fr/fr/index.php
). Haplotype analysis in the D4S2390-D4S1523 interval revealed five crossing-overs, all occurring in the same control family (). A double recombination (scored in individual II.5) is a very rare event considering the genetic and physical distances (). Therefore, we proposed that the D4S1652
bp allele be changed into a 139
bp allele by the addition of a GATA repeat during the replication (). Under this hypothesis, recombination events would be 3 out of 144 meioses (2.1%) instead of 5 out of 144 meioses (3.5%), corroborating the recombination fraction θ
=0.021, estimated by linkage analysis throughout the genetic interval (D4S2390-D4S1523
) in the whole sample of 22 pedigrees. Nonetheless, segregation analysis allowed the identification, in every FSHD family, of a specific haplotype that perfectly segregates with the disease phenotype and with the D4Z4 repeat sizing determined by the Southern blot reference diagnosis. In addition, the D4S1523
showed tight linkage to the D4Z4
locus (maximum LOD score=2.98 at θ
=0.0) and the recombination beyond D4S1523
remains low in our FSHD families.
Thus, the rapid, simple and sensitive protocol described in this paper allowed a robust indirect diagnosis that could easily solve or complement standard diagnosis in complex and urgent situations, particularly in the context of PND.
Finally, knowing the hope of young couples suffering from FSHD to benefit from a PGD, we evaluated the quadruplex PCR protocol on a large number of lymphocytes. The specificity and sensitivity of our strategy largely fulfils the recommended criteria for PGD (97.6% amplification rates, ADO and contamination rates both <1%).37
In conclusion, we have designed a new one-step multiplex PCR protocol that is technically efficient and suitable for the indirect diagnosis of FSHD at the single-cell level. This protocol could be theoretically offered to a very limited number of couples persistently requesting a PGD, who are fully informed regarding the selected markers and after careful exclusion of frequent sporadic cases and mosaicism in de novo FSHD families. Owing to the risk of recombination and the absence of flanking distal polymorphic markers, PGD for FSHD should not be recommended. Nevertheless, our improved protocol can be useful to some centres in the world that offer this diagnosis.