Identification of mutations in the F8 and F9 gene in families with haemophilia using targeted high-throughput sequencing
Identification of mutations in the F8 and F9 gene in families with haemophilia using targeted high-throughput sequencing
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使用靶向高通量测序鉴定血友病家族中 F8 和 F9 基因的突变
DOI：
10.1111/hae.12924
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发表时间：
2016-09-01
期刊：
影响因子：
3.9
通讯作者：
Zhang, L.
中科院分区：
文献类型：
--
作者：
Lyu, C.;Xue, F.;Zhang, L.
目前血友病的遗传诊断方法很多，包括间接连锁分析、直接测序等。然而，这些方法耗时，费力，并且在应用中受到限制。因此，开发新的、更有效的技术是必要的。目的：检测29个血友病A （HA）家族和11个血友病B （HB）家族血友病患者及其女性亲属中F8和F9基因的突变。方法：采用一期法分析FVIII:C和FIX: C，采用Bethesda法检测因子VIII和因子IX抑制剂。采用长距离聚合酶链式反应（PCR）和标准PCR鉴定内含子22和1个反转。F8和F9基因的非反转突变通过靶向高通量测序鉴定。所有突变均通过Sanger测序进行验证。结果：8个HA家族中检测到内含子22反转，1个HA家族中检测到内含子1反转。除倒置突变外，在HA家族中鉴定出20个突变，包括17个先前报道的突变和3个新突变：C. 5724g >A (p.Trp1908*)， C. 6116- 1_6120delgagtgtinstcc （p.Lys2039Ilefs*13）和C. 5220- 2a >C。我们在HA中发现了一个复杂的重排：内含子1反转伴随着外显子1缺失。在HB中，检测到8个复发突变，包括6个错义突变和2个无义突变。结论：靶向高通量测序是检测F8和F9基因突变的有效技术，尤其对发现新突变具有重要意义。该方法准确度高，省时，在发现大缺失突变和区分野生型和杂合型大缺失方面具有很大的优势。
Introduction: At present, many methods are available for the genetic diagnosis of haemophilia, including indirect linkage analysis, direct sequencing. However, these methods are time-consuming, labourious, and limited in their application. Therefore, the development of new, more effective techniques is necessary. Aim: To detect the F8 and F9 gene mutations in patients with haemophilia and their female relatives in 29 haemophilia A (HA) and 11 haemophilia B (HB) families. Methods: FVIII:C and FIX: C were analyzed using one-stage method, and factor VIII and factor IX inhibitors were tested using the Bethesda method. Intron 22 and one inversions were identified using long-distance polymerase chain reaction (PCR) and standard PCR. Non-inversion mutations of the F8 and F9 gene were identified by targeted high-throughput sequencing. All mutations were verified by Sanger sequencing. Results: Intron 22 inversion was detected in eight HA families and intron one inversion was detected in one HA family. Apart from the inversion mutations, 20 mutations were identified in HA families, including 17 previously reported and three novel mutations: c.5724G>A (p.Trp1908*), c.6116-1_6120delGAGTGTinsTCC (p.Lys2039Ilefs*13), and c.5220-2A>C. We found a complex rearrangement in HA: intron one inversion concomitant with exon one deletion. In HB, eight recurrent mutations were detected, including six missense mutations and two nonsense mutations. Conclusion: Targeted high-throughput sequencing is an effective technique to detect the F8 and F9 gene mutations, especially for the discovery of novel mutations. The method is highly accurate, time-saving and shows great advantage in uncovering large deletion mutations and also in distinguishing the wild-type genotype and heterozygous large deletions.