Abstract：
BACKGROUND: No F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal.
OBJECTIVE: The study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed.
METHODS: We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction.
RESULTS: A structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5.
CONCLUSIONS: All the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.
OBJECTIVE: The study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed.
METHODS: We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction.
RESULTS: A structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5.
CONCLUSIONS: All the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.
摘要：
背景：使用常规遗传方法在大约1％至2％的重度血友病A（HA）患者中未检测到F8遗传异常。在这些患者中,深层内含子变异或F8破坏基因组重排可能是因果关系。
目的：该研究旨在确定遗传调查失败的有严重HA病史的家庭的因果变异。
方法：我们对8名患者进行了全F8基因测序。通过断点连接的Sanger测序和/或定量聚合酶链反应确认基因组重排。
结果：在每个命题中都发现了破坏F8的结构变体，因此，在我们实验室注册的所有815个有严重HA病史的家庭都获得了决定性的基因诊断。这些结构变体由3个平衡倒置组成，获得区域的3个大插入，和1个移动元素的反向移位。3次倒转是105Mb，1.97Mb,和0.362Mb的大小。在获得区域的插入中，一个对应于F8内含子6中6q27号染色体的34kb获得区的插入，另一个对应于F8内含子14中9p22.1号染色体的447kb重复区的插入，最后一个对应于F8内含子5中获得的Xq28349kb的插入。
结论：该队列中所有严重HA的遗传未解决病例都是由于结构变异破坏了F8。这项研究强调了当常规方法失败时，全F8测序对改善HA分子诊断的有效性。
目的：该研究旨在确定遗传调查失败的有严重HA病史的家庭的因果变异。
方法：我们对8名患者进行了全F8基因测序。通过断点连接的Sanger测序和/或定量聚合酶链反应确认基因组重排。
结果：在每个命题中都发现了破坏F8的结构变体，因此，在我们实验室注册的所有815个有严重HA病史的家庭都获得了决定性的基因诊断。这些结构变体由3个平衡倒置组成，获得区域的3个大插入，和1个移动元素的反向移位。3次倒转是105Mb，1.97Mb,和0.362Mb的大小。在获得区域的插入中，一个对应于F8内含子6中6q27号染色体的34kb获得区的插入，另一个对应于F8内含子14中9p22.1号染色体的447kb重复区的插入，最后一个对应于F8内含子5中获得的Xq28349kb的插入。
结论：该队列中所有严重HA的遗传未解决病例都是由于结构变异破坏了F8。这项研究强调了当常规方法失败时，全F8测序对改善HA分子诊断的有效性。
