Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Hubrecht Institute-KNAW and University Medical Center, Utrecht, The Netherlands.
J Med Genet. 2019 Dec;56(12):828-837. doi: 10.1136/jmedgenet-2019-106402. Epub 2019 Nov 1.
Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in .
We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic variant from patient myoblasts.
We identified intronic variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of .
The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic variants identified here, emphasises the importance of screening for intronic variants in . Furthermore, the efficient suppression of after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.
面肩肱型肌营养不良症（FSHD）与包含在 4 号染色体上的 D4Z4 重复基因的部分染色质松弛以及骨骼肌中转录去抑制有关。FSHD 的常见形式 FSHD1 是由 D4Z4 重复阵列收缩引起的。不太常见的形式 FSHD2 通常是由 中的杂合变体引起的。
我们采用外显子组测序结合 Sanger 测序，对未明确诊断的 FSHD2 患者进行外显子突变筛查。我们还使用 CRISPR-Cas9 基因组编辑修复来自患者成肌细胞的致病性内含子 中的变体。
我们在两个 FSHD 家族中发现了内含子 中的变体。在第一个家族中，内含子变体导致部分内含子保留，并将内含子 13 的远端 14 个核苷酸包含在转录本中。在第二个家族中，内含子 34 中的深内含子变体导致内含子 34 中的 53 个核苷酸外显子化。在两个家族中，异常转录本被预测为无功能的。通过 CRISPR-Cas9 介导的基因组编辑从第二个家族的索引病例的原代和永生化成肌细胞中删除假外显子，将野生型 SMCHD1 表达水平恢复到有效抑制 的水平。
FSHD2 中的内含子突变频率估计接近 2%，正如这里鉴定的两个新的内含子 变体所例示的那样，强调了在 中筛查内含子变体的重要性。此外，通过突变等位基因的基因组编辑恢复 SMCHD1 水平后对 的有效抑制为治疗策略提供了进一步的指导。
