Severe digital abnormalities in a patient heterozygous for both a novel missense mutation in HOXD13 and a polyalanine tract expansion in HOXA13
Severe digital abnormalities in a patient heterozygous for both a novel missense mutation in HOXD13 and a polyalanine tract expansion in HOXA13
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DOI：
10.1136/jmg.39.11.852
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发表时间：
2002-11-01
影响因子：
4
通讯作者：
Goodman, FR
中科院分区：
文献类型：
--
作者：
Debeer, P;Bacchelli, C;Goodman, FR
方法 在征得先证者、父母双方、家庭 1 的其他 13 名成员和家庭 2 的其他 2 名成员的知情同意和当地研究伦理委员会的批准的情况下，采集用于 DNA 提取的静脉血样本。为了寻找 HOXD13（GenBank 登录号 AF005219 和 AF005220）和 HOXA13（GenBank 登录号 U82827）中的突变，如前所述，通过 PCR 将每个基因的整个编码区分为四个片段进行扩增。 5 9 扩增片段直接进行循环测序（Applied Biosystems Prism Dye Terminator Kit）或亚克隆到 pCRScript (Stratagene)，然后在 ABI 377 自动测序仪（Applied Biosystems）上进行循环测序和分析。 结果 先证者 HOXD13 的直接测序显示编码序列第 892 位存在杂合性 C 到 T 转换，该转换将氨基酸 298 转换（同源结构域的残基 31）从精氨酸到色氨酸。在先证者的母亲和家庭 1 的其他 12 名受影响成员中也发现了相同的碱基变化（图 1），但在先证者的父亲或 50 名不相关的未受影响的对照中没有发现。先证者 HOXA13 外显子 1 5' 部分的 PCR 扩增不仅产生了预期大小的产物，而且还产生了第二个更大的产物，表明她携带了一个小插入。这些产物的克隆和测序表明，她在编码序列第 387 位碱基后插入了 27 bp 的读码框，位于编码聚丙氨酸束的三个不完美三核苷酸重复序列的三分之一内，是杂合的。该插入似乎是由于重复
METHODS Venous blood samples for DNA extraction were obtained from the proband, both parents, 13 other members of family 1, and two other members of family 2, with their informed consent and the approval of the local research ethics committee. To search for mutations in HOXD13 (GenBank accession numbers AF005219 and AF005220) and HOXA13 (GenBank accession number U82827), the entire coding region of each gene was amplified by PCR in four segments, as described previously. 5 9 Amplified fragments were either cycle sequenced directly (Applied Biosystems Prism Dye Terminator Kit) or subcloned into pCRScript (Stratagene) before being cycle sequenced and analysed on an ABI 377 automated sequencer (Applied Biosystems).RESULTS Direct sequencing of HOXD13 in the proband showed a heterozygous C to T transition at position 892 of the coding sequence, which converts amino acid 298 (residue 31 of the homeodomain) from arginine to tryptophan. The same base change was identified in the proband’s mother and in 12 other affected members of family 1 (fig 1) but not in the proband’s father or in 50 unrelated unaffected controls. PCR amplification of the 5′ portion of exon 1 of HOXA13 in the proband yielded not only a product of the expected size, but also a second larger product, suggesting that she carried a small insertion. Cloning and sequencing of these products showed that she was heterozygous for a 27 bp in frame insertion after base 387 of the coding sequence, within the third of three imperfect trinucleotide repeats encoding polyalanine tracts. This insertion appears to have arisen by duplication of