Service de génétique, Groupe Hospitalier Est, Hospices Civils de Lyon, Bron, France.
CRNL, équipe GENDEV INSERM U1028, CNRS UMR5292, Université Claude Bernard, Lyon, France.
J Thromb Haemost. 2019 Jul;17(7):1097-1103. doi: 10.1111/jth.14460. Epub 2019 May 20.
Essentials No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients. Detection of F8 structural variants remains a challenge. We identified a new F8 rearrangement in a severe hemophilia A patient using nanopore sequencing. We highlight the value of single-molecule long-read sequencing technologies in a genomics laboratory.
No F8 genetic abnormality is detected in about 2% of severe hemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal.
To characterize, in a genetically unresolved severe hemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing.
Long-range polymerase chain reaction (PCR) performed throughout F8 identified a nonamplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8 messanger ribonucleic acid (mRNA) analysis including 3'rapid amplification of complementary deoxyribonucleic acid (cDNA) ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742-bp pseudoexon corresponding to a noncoding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66 310 352-66 311 093, GRCh37/hg19). Cytogenetic microarray analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. The PCR amplification of junction fragments and fluorescent in situ hybridization (FISH) analysis found that the Xq11q12 duplicated region was inserted in the F8 intron 25 genomic region.
We characterized a novel genomic rearrangement in which a 3.8-Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe hemophilia A (HA), using comprehensive molecular techniques. This study highlights the value of single-molecule long-read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.
在大约 2%的重度血友病 A 患者中，未检测到 F8 基因异常。F8 结构变异的检测仍然是一个挑战。我们使用纳米孔测序在一名重度血友病 A 患者中发现了一种新的 F8 重排。我们强调了单分子长读测序技术在基因组学实验室中的价值。
使用传统的遗传方法，大约 2%的重度血友病 A 患者未检测到 F8 遗传异常。在这些患者中，深内含子变异或 F8 破坏基因组重排可能是致病原因。
使用包括纳米孔测序在内的综合分子技术，对一名遗传上未解决的重度血友病 A 患者中一种新的 Xq28 重排进行分析，该重排破坏了 F8。
在整个 F8 中进行长距离聚合酶链反应（PCR），在 25 号内含子中发现了一个无法扩增的区域，表明存在基因组重排。F8 信使核糖核酸（mRNA）分析包括 3'快速扩增互补脱氧核糖核酸（cDNA）末端和纳米孔测序，发现存在一种 F8 融合转录本，其中 F8 外显子 26 被一个 742 碱基对的假外显子取代，该假外显子对应于 X 染色体长臂（Xq12；chrX:66310352-6631109，GRCh37/hg19）起始处的非编码区。染色体微阵列分析发现存在 Xq11.1q12 增益 3.8 Mb。连接片段的 PCR 扩增和荧光原位杂交（FISH）分析发现，Xq11q12 重复区域插入 F8 内含子 25 基因组区域。
我们使用综合分子技术，在一名重度血友病 A（HA）患者中，鉴定了一种新型基因组重排，其中 Xq11.1q12 增益 3.8 Mb 插入 F8 内含子 25 导致异常融合转录本。本研究强调了单分子长读测序技术在 HA 分子诊断中的价值，特别是在传统遗传方法失败时。
