两例无关先证者 FXIII 缺陷的表型和基因型：新型 F13A1 大片段缺失的鉴定，该缺失由复杂重排介导。
Phenotype and genotype of FXIII deficiency in two unrelated probands: identification of a novel F13A1 large deletion mediated by complex rearrangement.
机构信息
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Second Road, Shanghai, 200025, China.
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Second Road, Shanghai, 200025, China.
出版信息
Orphanet J Rare Dis. 2019 Jul 24;14(1):182. doi: 10.1186/s13023-019-1144-z.
BACKGROUND
Inherited Factor XIII deficiency (FXIIID) is one of the most severe and under-diagnosed rare bleeding disorders. Only 5 large deletions involving one or more exons in F13A1 have been reported, and lacking of multiplex ligation-dependent probe amplification (MLPA) assay might underestimate the copy number variations (CNVs) in F13A1 and F13B. We had characterized the clinical presentation of two unrelated severe FXIIID probands and explored the pathogenic mechanisms.
RESULTS
Both probands experienced several episodes of fatal bleeding and delayed wound healings prior to diagnosis. FXIII activity was measured by the ammonia release assay, and FXIII-A and FXIII-B antigens were determined by ELISA. All the exons including exon-intron boundaries and promoter regions of F13A1 and F13B were amplified and directly sequenced. Copy number variations (CNVs) of F13A1 and F13B were detected by the CNVplex® method. Breakpoints of the F13A1 large deletion were identified by quantitative primer walking combined long-range PCR (LR-PCR) strategies. Proband 1 was found to have compound heterozygous mutations of a novel small deletion (c.1147del) and a missense mutation p.Arg383Ser. Proband 2 was compound heterozygous for a novel large deletion (g.[77815_112815del;112837_116628del]) and a missense mutation p.Arg716Gly in F13A1. Bioinformatics analysis of the large deletion breakpoints predicted that two fork stalling and template switching and/or microhomology-mediated break-induced replication (FoSTeS/MMBIR) events with two homologies of TCT and C might be responsible for the complex rearrangement. Prophylactic replacement therapy was immediately administered for the two probands upon establishment of the diagnosis.
CONCLUSIONS
We detected two type I FXIIID pedigrees and adopted CNVplex® method to detect CNVs of F13A1 and F13B for the first time. A large heterozygous deletion of g.[77815_112815del;112837_116628del] in F13A1, mediated by two FoSTeS/MMBIR events, was identified.
背景
遗传性因子 XIII 缺乏症（FXIIID）是最严重和诊断不足的罕见出血性疾病之一。仅报道了涉及 F13A1 一个或多个外显子的 5 个大型缺失，并且缺乏多重连接依赖性探针扩增（MLPA）检测可能会低估 F13A1 和 F13B 的拷贝数变异（CNVs）。我们已经对两个不相关的严重 FXIIID 先证者的临床表现进行了描述，并探讨了其发病机制。
结果
两名先证者在确诊前均经历了数次致命性出血和伤口愈合延迟。通过氨释放测定法测量 FXIII 活性，通过 ELISA 测定 FXIII-A 和 FXIII-B 抗原。扩增并直接测序了包括 F13A1 和 F13B 的外显子-内含子边界和启动子区域在内的所有外显子。使用 CNVplex®方法检测 F13A1 和 F13B 的拷贝数变异（CNVs）。通过定量引物步行结合长距离 PCR（LR-PCR）策略确定 F13A1 大片段缺失的断点。发现先证者 1 存在新型小缺失（c.1147del）和错义突变 p.Arg383Ser 的复合杂合突变。先证者 2 为 F13A1 中的新型大片段缺失（g.[77815_112815del；112837_116628del]）和错义突变 p.Arg716Gly 的复合杂合子。大片段缺失断点的生物信息学分析预测，两个 TCT 和 C 的同源性可能导致两个 fork stalling and template switching and/or microhomology-mediated break-induced replication（FoSTeS/MMBIR）事件负责这种复杂的重排。在确诊后，立即对两名先证者进行了预防性替代治疗。
结论
我们检测了两个 I 型 FXIIID 家系，并首次采用 CNVplex®方法检测了 F13A1 和 F13B 的 CNVs。鉴定出 F13A1 中的大片段杂合缺失 g.[77815_112815del；112837_116628del]，由两个 FoSTeS/MMBIR 事件介导。
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