Amr Khalda, Fahmy Nagia, El-Kamah Ghada
Medical Molecular Genetics, Human Genetics and Genome Research Institute, National Research Centre, Egypt.
Neurology Ain Shams University, Egypt.
J Genet Eng Biotechnol. 2024 Dec;22(4):100436. doi: 10.1016/j.jgeb.2024.100436. Epub 2024 Nov 11.
Duchenne muscular dystrophy (DMD/BMD) is the most common type of muscular dystrophy, together with Becker muscular dystrophy represent more than half of all cases. DMD is a single-gene, X-linked recessive disorder that predominantly affects boys, causing progressive muscle deterioration and eventually leading to fatal cardiopulmonary complications. This study aimed to implement a cost-effective molecular diagnostic method using the SALSA MLPA Kit (probe mixes 034 and 035) to screen a large group of Egyptian DMD patients. The study included 1250 clinically diagnosed DMD males, following complete family history, pedigree analyses and an accurate clinical examination and laboratory investigations mainly considering high levels of creatine phosphokinase (>2000 U/L). We also analysed the carrier status of 100 mothers of 100 probands to gauge the inherited mutation through their patients with familial disease. The negative results of MLPA were further analysed with NGS for ten patients and the results were validated for novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. Results SALSA MLPA analysis confirmed the diagnoses in 733/1250 (58.6 %) DMD patients and the remaining of 517/1250 (41.4%) were negative. DMD patients having large deletions were 632/1250 (50.6%) while duplications occurred in 101/1250 (8%). The most common single exon deletion was 45 (50/632, 7.9%). In addition, 163 different deletion and duplication patterns were characterized among positive MLPA analyses. 30% of our studied cohort exhibited new patterns of rearragements in addition to seven cases of double deletion and duplication rearrangements identified, within nine patients. Using NGS, for small mutations detection, revealed six novel and three previously reported mutations among screened ten patients. In conclusion, our findings expand the spectrum of known DMD mutations by offering an effective diagnostic method and identifying novel point mutations through NGS analysis. We recommend using NGS to uncover uncharacterized mutations in patients who test negative with MLPA, which could contribute to the treatment of DMD.
杜氏肌营养不良症（DMD/BMD）是最常见的肌营养不良症类型，与贝克型肌营养不良症一起占所有病例的一半以上。DMD是一种单基因X连锁隐性疾病，主要影响男孩，导致进行性肌肉退化，最终导致致命的心肺并发症。本研究旨在使用SALSA MLPA试剂盒（探针混合物034和035）实施一种具有成本效益的分子诊断方法，以筛查一大群埃及DMD患者。该研究纳入了1250名临床诊断为DMD的男性，在完善家族史、系谱分析以及准确的临床检查和实验室检查后，主要考虑肌酸磷酸激酶水平较高（>2000 U/L）。我们还分析了100名先证者的100名母亲的携带者状态，以评估通过其家族性疾病患者遗传的突变。对10名患者的MLPA阴性结果进一步进行NGS分析，并对新的错义突变结果进行验证，使用PolyPhen2和突变预测软件分析表型-基因型相关性。结果SALSA MLPA分析在1250名DMD患者中的733名（58.6%）中确诊，其余517名（41.4%）为阴性。有大片段缺失的DMD患者为632名（50.6%），而重复发生在101名（8%）。最常见的单个外显子缺失是45（50/632，7.9%）。此外，在阳性MLPA分析中鉴定出163种不同的缺失和重复模式。在我们研究的队列中，30%表现出新的重排模式，此外在9名患者中还发现了7例双缺失和重复重排。使用NGS检测小突变，在筛查的10名患者中发现了6个新突变和3个先前报道的突变。总之，我们的研究结果通过提供一种有效的诊断方法并通过NGS分析鉴定新的点突变，扩展了已知DMD突变的谱。我们建议使用NGS来发现MLPA检测为阴性的患者中未表征的突变，这可能有助于DMD的治疗。
