Cogan Joy D, Vnencak-Jones Cindy L, Phillips John A, Lane Kirk B, Wheeler Lisa A, Robbins Ivan M, Garrison Gladys, Hedges Lora K, Loyd James E
Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2578, USA.
Genet Med. 2005 Mar;7(3):169-74. doi: 10.1097/01.gim.0000156525.09595.e9.
Approximately 50% of patients with familial primary pulmonary hypertension (FPPH) have been reported to have mutations within the bone morphogenic protein receptor type 2 (BMPR2) gene. The vast majority of these mutations were identified by PCR amplification and sequencing of individual exons. The aim of our study was to determine if additional BMPR2 mutations not found by exon sequencing alone could account for a significant portion of these negative cases.
We examined DNA samples from 12 families, previously found to be negative for BMPR2 mutations, to identify any large BMPR2 gene rearrangements.
Southern blot analysis found large gene rearrangements in four (33%) unrelated kindreds. Further analysis by reverse transcriptase PCR (RT-PCR) of BMPR2 transcripts from two of these kindreds found one to be heterozygous for a exon 10 duplication and the second to be heterozygous for a deletion of exons 4 to 5. Nonhomologous recombination is believed to be the cause of these large insertions/deletions.
Our results demonstrate the inherent problems associated with exon-by-exon sequencing and the importance of other screening methods such as Southern blot and RT-PCR in the identification of BMPR2 mutations.
据报道，约50%的家族性原发性肺动脉高压（FPPH）患者骨形态发生蛋白受体2型（BMPR2）基因存在突变。这些突变绝大多数是通过对单个外显子进行PCR扩增和测序鉴定出来的。我们研究的目的是确定仅通过外显子测序未发现的其他BMPR2突变是否能解释这些阴性病例中的很大一部分。
我们检测了来自12个家族的DNA样本，这些家族先前被发现BMPR2突变呈阴性，以确定是否存在任何BMPR2基因大片段重排。
Southern印迹分析在4个（33%）无关家族中发现了大片段基因重排。对其中两个家族的BMPR2转录本进行逆转录PCR（RT-PCR）进一步分析发现，一个家族外显子10重复呈杂合状态，另一个家族外显子4至5缺失呈杂合状态。非同源重组被认为是这些大片段插入/缺失的原因。
我们的结果证明了逐外显子测序存在的固有问题，以及Southern印迹和RT-PCR等其他筛查方法在鉴定BMPR2突变中的重要性。
