Sinha Sajan, Dutta Atanu Kumar, Bhattacharya Paramita, Bhattacharya Subham, Das Mrinal Kanti
Department of Paediatrics, IPGME&R and SSKM Hospital, Kolkata, India.
Department of Biochemistry, All India Institute of Medical Sciences, Second Floor, Medical College Block, NH - 34 Connector Basantapur, Saguna, 741245 Kalyani, West Bengal India.
Indian J Clin Biochem. 2024 Apr;39(2):207-213. doi: 10.1007/s12291-022-01098-w. Epub 2023 Jan 3.
There is limited data available regarding the clinical utility of routine molecular diagnosis of β Thalassaemia in addition to HPLC-based screening in low resource settings. The current study highlights the caveats of an HPLC-based screening compared to the inclusion of genetic confirmation as a second-tier test and its implications in terms of genotype-phenotype correlation. A prospective, institution-based, observational study was conducted at the Department of Paediatric Medicine, including 103 children aged up to 12 years. Five common mutations for β Thalassemia and the HbE mutation in the gene were tested by a two-tiered approach using multiplex ARMS PCR and PCR RFLP methods respectively. Sanger sequencing of all three exons of the gene was performed in all negative cases. Sequencing revealed many rare pathogenic mutations like c.316-106 C > G (dbSNP: 34,690,599); Hb Kairouan (c.92G > C); c.33 C > A (dbSNP rs35799536); c.47G > A (dbSNP rs63750783); c.51delC (HbVar ID 799); c.[93-2 A > C] and c.118 C > T (HbVar ID 845). We detected a novel Pathogenic M_000518.5():c.164_168delinsGGCATCA (p.Val55fs) mutation in a heterozygous state which was reported in the ClinVar database with accession ID VCV000590977.2. We also encountered several cases of silent carrier on HPLC and de novo occurrence of mutation. We conclude that the multiplex touchdown ARMS PCR methodology employed in the present study provides a low-cost solution for molecular diagnostics of Β Thalassaemia. The problem of silent carriers in HPLC is significant enough to rethink if we need supplemental genetic testing in the couple when one of the partners is a carrier.
The online version contains supplementary material available at 10.1007/s12291-022-01098-w.
在资源匮乏地区，除了基于高效液相色谱（HPLC）的筛查外，关于β地中海贫血常规分子诊断的临床效用的数据有限。当前研究强调了与将基因确认作为二级检测相比，基于HPLC筛查的注意事项及其在基因型-表型相关性方面的影响。在儿科学系进行了一项前瞻性、基于机构的观察性研究，纳入了103名12岁及以下儿童。分别使用多重扩增阻滞突变系统聚合酶链反应（ARMS PCR）和聚合酶链反应-限制性片段长度多态性（PCR RFLP）方法，通过两级方法检测β地中海贫血的5种常见突变和基因中的HbE突变。对所有阴性病例进行基因的所有三个外显子的桑格测序。测序发现了许多罕见的致病突变，如c.316-106 C＞G（dbSNP：34,690,599）；开罗血红蛋白（c.92G＞C）；c.33 C＞A（dbSNP rs35799536）；c.47G＞A（dbSNP rs63750783）；c.51delC（HbVar ID 799）；c.[93-2 A＞C]和c.118 C＞T（HbVar ID 845）。我们在一名杂合状态个体中检测到一种新的致病突变M_000518.5():c.164_168delinsGGCATCA（p.Val55fs），该突变已在ClinVar数据库中报告，登录号为VCV000590977.2。我们还遇到了几例在HPLC检测中为沉默携带者以及新发突变的病例。我们得出结论，本研究中采用的多重降落ARMS PCR方法为β地中海贫血的分子诊断提供了一种低成本解决方案。当夫妻一方为携带者时，HPLC检测中沉默携带者的问题严重到足以让我们重新思考是否需要进行补充基因检测。
在线版本包含可在10.1007/s12291-022-01098-w获取的补充材料。
