Amsellem Sabine, Briffaut Dorothée, Carrié Alain, Rabès Jean Pierre, Girardet Jean Philippe, Fredenrich Alexandre, Moulin Philippe, Krempf Michel, Reznik Yves, Vialettes Bernard, de Gennes Jean Luc, Brukert Eric, Benlian Pascale
Department of Biochemistry and Molecular Biology, APHP Saint Antoine Hospital, Paris, France.
Hum Genet. 2002 Dec;111(6):501-10. doi: 10.1007/s00439-002-0813-4. Epub 2002 Sep 13.
Familial hypercholesterolemia (FH), a frequent monogenic condition complicated by premature cardiovascular disease, is characterized by high allelic heterogeneity at the low-density lipoprotein receptor ( LDLR) locus. Despite more than a decade of genetic testing, knowledge about intronic disease-causing mutations has remained limited because of lack of available genomic sequences. Based on the finding from bioinformatic analysis that Alu repeats represent 85% of LDLR intronic sequences outside exon-intron junctions, we designed a strategy to improve the exploration of genomic regions in the vicinity of exons in 110 FH subjects from an admixed population. In the first group of 42 patients of negative mutation carriers, as previously established by former screening strategies (denaturing gradient gel electrophoresis, DNA sequencing with former primers overlapping splice-sites, Southern Blotting), about half ( n=22) were found to be carriers of at least one heterozygous mutation. Among a second group of 68 newly recruited patients, 27% of mutation carriers ( n=37) had a splicing regulatory mutation. Overall, out of the 54 mutations identified, 13 were intronic, and 18 were novel, out of which nearly half were intronic. Two novel intronic mutations (IVS8-10G-->A within the polypyrimidine tract and IVS7+10G-->A downstream of donor site) might create potential aberrant splice sites according to neural-network computed estimation, contrary to 31 common single nucleotide variations also identified at exon-intron junctions. This new strategy of detecting the most likely disease-causing LDLR mutations outside of Alu-rich genomic regions reveals that intronic mutations may have a greater impact than previously reported on the molecular basis of FH.
家族性高胆固醇血症（FH）是一种常见的单基因疾病，常并发早发性心血管疾病，其特征是低密度脂蛋白受体（LDLR）基因座存在高度等位基因异质性。尽管进行了十多年的基因检测，但由于缺乏可用的基因组序列，关于内含子致病突变的了解仍然有限。基于生物信息学分析的结果，即Alu重复序列占外显子-内含子交界处以外LDLR内含子序列的85%，我们设计了一种策略，以改进对来自混合人群的110名FH受试者外显子附近基因组区域的探索。在第一组42名先前筛查策略（变性梯度凝胶电泳、使用与剪接位点重叠的先前引物进行DNA测序、Southern印迹）确定的阴性突变携带者患者中，约一半（n = 22）被发现是至少一种杂合突变的携带者。在第二组68名新招募的患者中，27%的突变携带者（n = 37）有剪接调节突变。总体而言，在鉴定出的54个突变中，13个是内含子突变，18个是新突变，其中近一半是内含子突变。根据神经网络计算估计，两个新的内含子突变（多嘧啶序列内的IVS8-10G→A和供体位点下游的IVS7+10G→A）可能会产生潜在的异常剪接位点，这与在外显子-内含子交界处也鉴定出的31个常见单核苷酸变异相反。这种在富含Alu的基因组区域之外检测最可能致病的LDLR突变的新策略表明，内含子突变对FH分子基础的影响可能比先前报道的更大。
