CHU de Montpellier, Laboratoire de Génétique Moléculaire, Montpellier, France.
Eur J Hum Genet. 2010 May;18(5):533-8. doi: 10.1038/ejhg.2009.207. Epub 2009 Nov 25.
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.
面肩肱型肌营养不良症（FSHD）的分子发病机制涉及 4q35.2 染色体上串联重复 D4Z4 单元数量的杂合收缩。FSHD 与 1-10 个 D4Z4 单元相关，而正常对照中则为 11-150 个。几个因素使 FSHD 的分子诊断变得复杂，特别是 D4Z4 收缩与 4qA 等位基因的顺式分离，而 D4Z4 缩短在 4qB 和 10q 等位基因上均不沉默。从高度同源的 10q-D4Z4 阵列中区分致病性 4q-D4Z4 等位基因需要使用不适合单细胞水平的常规 Southern 印迹。具有多个受累亲属的 FSHD 家族经常要求进行胚胎植入前遗传诊断（PGD）。我们旨在开发一种快速而敏感的基于 PCR 的多重方法，用于对单个细胞进行致病性等位基因的间接家族分离研究。在 4q35.2 上有几个可用的多态性标记中，选择了四个最接近的（D4S2390、D4S1652、D4S2930 和 D4S1523，<1.23Mb），它们显示出最高的杂合频率（67-91%）。在 144 个减数分裂中观察到 D4S2390-D4S1523 间隔中的 5 个重组事件。在 D4S2390-D4Z4 间隔中，在 28 个 FSHD 减数分裂中没有发生重组事件。相反，一个特定的单倍型与临床和分子状态一起分离，允许在每个测试的 FSHD 家族中鉴定出一个风险等位基因（最大对数似然比为 2.98，theta=0.0）。这种间接方案可以轻松地补充产前诊断中的常规技术。尽管我们基于多重 PCR 的方法在技术上符合单细胞分析的指南，但相对较高的重组风险阻碍了其在 PGD 中的应用。
