Chen Xu, Luo Mingyue, Pan Lei, Huang Yuting, Yan Zehao, Shen Kai, Mai Guangxing, Liang Hui, Li Jiaqi, Chen Yiwei, Xiong Likuan
Central Laboratory, Baoan Women's and Children's Hospital, Shenzhen, China; Shenzhen Key Laboratory of Birth Defects Research, Shenzhen, China.
Department of Medical Genetics and Prenatal Diagnosis, Baoan Women's and Children's Hospital, Shenzhen, China.
Clin Chim Acta. 2022 Apr 1;529:10-16. doi: 10.1016/j.cca.2022.01.024. Epub 2022 Feb 9.
Thalassemia is a common inherited haemoglobin disorder worldwide, several methods have been utilized in the step-wise screening. Even though hundreds of mutations in globin genes have been reported, novel mutations are continuously emerging as the development of DNA sequencing.
The case is a 27-year-old female with abnormal values of routine hematological indices, who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays cover 26 mutations in HBA and HBB genes: gap-PCR and agarose gel electrophoresis were performed to detect deletions, while PCR-reverse dot blot was used to detect point mutations. The next- and third- generation sequencing were used to identify the known and potential novel genotypes of thalassemia, and multiplex ligation-dependent probe amplification (MLPA) was used for genotype validation.
Hematological results indicate microcytic hypochromic anemia, high HbA2 (7.2%) and high HbF (6.2%). None of the known genotypes of thalassemia were matched for this case, but a novel 4.9 Kb deletion at HBB gene (hg38, Chr11: 5226187-5231089) was discovered by the third-generation sequencing, the novel deletion was also validated by MLPA (8 probes, 11p15.4: 203314-207652).
This study suggests the third-generation sequencing has promising potentiality to discover novel genotypes (especially deletions) of thalassemia.
地中海贫血是全球常见的遗传性血红蛋白疾病，在逐步筛查中已采用多种方法。尽管已报道了数百种珠蛋白基因突变，但随着DNA测序技术的发展，新的突变仍在不断出现。
该病例为一名27岁女性，其常规血液学指标异常，因地中海贫血基因筛查入院。提取基因组DNA并用于基因检测，检测HBA和HBB基因中的26种突变：采用缺口聚合酶链反应（gap-PCR）和琼脂糖凝胶电泳检测缺失，采用聚合酶链反应-反向点杂交（PCR-反向点杂交）检测点突变。使用二代和三代测序来鉴定已知和潜在的地中海贫血新基因型，并采用多重连接依赖探针扩增技术（MLPA）进行基因型验证。
血液学检查结果显示为小细胞低色素性贫血，HbA2升高（7.2%），HbF升高（6.2%）。该病例未匹配任何已知的地中海贫血基因型，但通过三代测序发现了HBB基因上一个新的4.9 kb缺失（hg38，Chr11: 5226187-5231089），该新缺失也通过MLPA（8个探针，11p15.4: 203314-207652）得到验证。
本研究表明，三代测序在发现地中海贫血新基因型（尤其是缺失）方面具有广阔的潜力。
