Centre for Genetics and Genomics, School of Biology, Queen's Medical Centre, The University of Nottingham, Nottingham, UK.
Eur J Hum Genet. 2012 Sep;20(9):999-1003. doi: 10.1038/ejhg.2012.42. Epub 2012 Feb 29.
We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.
我们研究并验证了 FACIOSCAPULOHUMERAL 肌营养不良症（FSHD）患者无 D4Z4 收缩（FSHD2 或“表型 FSHD”）的样本。为此，我们开发了非放射性方案来测试 D4Z4 等位基因组成和 DNA 甲基化，并将其应用于科里尔研究所细胞库的样本。D4Z4 定序显示两个相关的个体具有经典的染色体 4 收缩依赖性 FSHD1。第三个样本（GM17726）没有短的染色体 4 片段，并且被分配为非 4q FSHD（FSHD2）。我们对该个体的 D4Z4 单倍型和甲基化进行了测试，但发现两者均与该诊断不一致。使用外显子组测序，我们鉴定了 CAPN3 中的两个已知致病性突变（Arg490Gln 和 Thr184Argfs(*)36），表明这是 LGMD2A 而不是 FSHD 的病例。我们的研究表明，通过全外显子组测序，通过在生成数据后将变异分析限制在候选基因中，很容易纠正错误的诊断。这种新的“测序诊断”方法可能会在遗传诊断实验室中变得很常见。我们还发布了一种 DIGOXIGENIN 标记的 Southern 方案来测试 D4Z4 甲基化。我们的数据支持低甲基化作为 FSHD2 的良好表观遗传预测因子。非放射性方案将有助于使该检测更易于临床诊断实验室和更广泛的 FSHD 研究社区使用。
