Erdos Melinda, Alapi Krisztina, Balogh István, Oroszlán György, Rákóczi Eva, Sümegi János, Maródi László
Department of Infectious and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Exp Hematol. 2006 Nov;34(11):1517-21. doi: 10.1016/j.exphem.2006.06.009.
A 5-month-old male infant presenting with recurrent respiratory tract infections, chronic diarrhea, and failure to thrive was found to be pancytopenic. Bone marrow and x-ray examinations were consistent with Shwachman-Diamond syndrome (SDS). Genomic DNA sequencing, restriction fragment analysis, and studies of the mutant proteins were performed to gain further knowledge on the molecular pathology of SDS.
Exons 1 to 5 of the SBDS gene were amplified and sequenced. COS-7 cells were transfected with expression vectors containing wild-type cDNA or mutant cDNAs generated by site-directed mutagenesis. Protein expression of SBDS variants were examined by Western blotting. Pulse-chase assay and densitometry were used to study protein stability.
Two novel missense mutations (c.362A > C in exon 3, and c.523C > T in exon 4) of the SBDS gene were identified in the patient. These mutations result in p.N121T and p.R175W amino acid replacements and correspond to amino acid residues that are highly conserved in SBDS proteins. In vitro expression studies revealed a markedly decreased half-life of the p.R175W protein, whereas stability of the p.N121T mutant was not significantly reduced compared to that of the wild type.
This is the first report of compound heterozygous missense mutations occurring in patients with SDS. These mutations may not eliminate SBDS expression but may result in impaired protein stability and protein function leading to severe disease.
一名5个月大的男婴反复出现呼吸道感染、慢性腹泻且生长发育迟缓，检查发现全血细胞减少。骨髓和X线检查结果符合施瓦赫曼-戴蒙德综合征（SDS）。进行了基因组DNA测序、限制性片段分析以及突变蛋白研究，以进一步了解SDS的分子病理学。
扩增并测序SBDS基因的外显子1至5。用含有野生型cDNA或通过定点诱变产生的突变cDNA的表达载体转染COS-7细胞。通过蛋白质免疫印迹法检测SBDS变体的蛋白质表达。采用脉冲追踪分析和光密度测定法研究蛋白质稳定性。
在该患者中鉴定出SBDS基因的两个新的错义突变（外显子3中c.362A>C，外显子4中c.523C>T）。这些突变导致p.N121T和p.R175W氨基酸替换，且对应于SBDS蛋白中高度保守的氨基酸残基。体外表达研究显示p.R175W蛋白的半衰期显著缩短，而与野生型相比，p.N121T突变体的稳定性未显著降低。
这是首次报道SDS患者中发生复合杂合错义突变。这些突变可能不会消除SBDS表达，但可能导致蛋白质稳定性受损和蛋白质功能障碍，从而引发严重疾病。
