Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood
Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood
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DOI：
10.1160/th06-10-0592
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发表时间：
2007-01
影响因子：
6.7
通讯作者：
Ting-Chang Hsu;S. Nakaya;A. Thompson
中科院分区：
文献类型：
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作者：
Ting-Chang Hsu;S. Nakaya;A. Thompson
在对重度血友病B受试者进行基因分型时，外显子1-3和5-8正常。外显子4未扩增，提示部分基因缺失。以前，一个外显子4缺失的法国家族患有重度血友病B，其循环功能失调的因子IX蛋白缺失其第一个生长因子样结构域;未分析断点。使用外显子3的5'引物和外显子5片段的3'引物，受试者的因子IX基因扩增了5 kb片段，而预测为11 kb，表明6 kb缺失。限制性内切酶将3'内含子4缺失断裂点定位于外显子5'的1.2kb处。通过断裂点进行测序，发现5，969 bp缺失，包括外显子4，并伴随着在3'断裂点附近插入的13 bp重复。血友病为家族性;经检测，他的母亲被证实为杂合子携带者，而他的妹妹则是正常的、较大片段的纯合子。由于因子IX基因的外显子4和5在读码框内，该缺失应产生缩短的转录物，缺失114 bp（第一生长因子样结构域的38个密码子）。从全血制备的mRNA的逆转录和PCR鉴定了较短的cDNA片段。Western blotting显示较小的因子IX蛋白。
Summary In genotyping a severe hemophilia B subject, exons 1–3 and 5–8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5’ primer for exon 3 and a 3’ primer for exon 5 fragments, the subject’s factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3’ intron 4 deletion breakpoint to 1.2 kb 5’ to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3’ breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.