未经证实:X连锁Alport综合征(XLAS)是由COL4A5中的致病变种引起的,其特征是进行性肾脏疾病,听力损失,和眼部异常。这项研究的目的是确定一个中国XLAS家族的基因突变,确认诊断,并提供准确的遗传咨询。
未经证实:先证者是一名5岁男性,有5名亲属的镜下血尿和肾病家族史。他的亲属有微血尿伴或不伴蛋白尿。他的叔叔在35岁时患上了肾衰竭。他通过肾活检进行了评估,全外显子组测序(WES)和全基因组测序(WGS)用于Alport综合征。对从先证者皮肤提取的RNA进行RT-PCR和cDNASanger测序。然后,剪接报告子小基因分析用于检查变异对转染细胞中初级转录物剪接的影响。
未经证实:先证者肾脏的病理检查显示肾小球基底膜弥漫性变薄,免疫荧光分析表明基底膜中α5链的正常表达。通过WES在先证者中未检测到表型相关的候选变体。一种新颖的深内含子COL4A5变体(c.385-716G>A),在这个家庭中与疾病隔离,使用WGS识别。体外小基因测定和体内RT-PCR分析表明,该变体可产生正常和异常转录本。异常转录本显示该变体激活了一个隐蔽的剪接位点,在mRNA序列中引入147bp的假外显子,从而产生过早的终止密码子(p。G129Afs*38),并导致α5(胶原蛋白IV)蛋白的移码和截短。
UNASSIGNED:这是COL4A5基因中新型c.385-716G>A剪接突变的首次报道,这说明了在高度怀疑的遗传性疾病的WES阴性患者中进行WGS以发现其他突变的重要性。从体外和体内剪接实验获得的相同结果证实了小基因测定和RT-PCR分析之间的一致性。此外,这项研究强调了功能分析在AS诊断和遗传咨询中的重要性.
UNASSIGNED: X-linked Alport syndrome (XLAS) is caused by pathogenic variants in COL4A5 and is characterized by progressive kidney disease, hearing loss, and ocular abnormalities.The aim of this study was to identify gene mutations in a Chinese family with XLAS, confirm a diagnosis, and provide an accurate genetic counseling.
UNASSIGNED: The proband was a 5-year-old male with microscopic hematuria and a family history of renal disease in 5 relatives.His relatives had microhematuria with or without proteinuria. His maternal uncle developed renal failure at the age of 35 years. He was evaluated by renal biopsy,whole-exome sequencing (WES) and whole-genome sequencing (WGS) for Alport syndrome. RT-PCR and cDNA Sanger sequencing were performed on RNA extracted from the skin of the proband. Then, a splicing reporter minigene assay was used to examine the effect of the variation on the splicing of the primary transcript in transfected cells.
UNASSIGNED: Pathological examination of the kidney of the proband revealed diffuse thinning of the glomerular basement membrane, and immunofluorescence analysis indicated normal expression of the α5 chain in the basement membrane. No phenotype-associated candidate variant was detected in the proband via WES. A novel deep intronic COL4A5 variant (c.385-716G > A), which is segregated with disease in this family, was identified using WGS. In-vitro minigene assay and in-vivo RT-PCR analysis demonstrated that the variant could produce both normal and abnormal transcripts. The abnormal transcripts showed that the variant activated a cryptic splice site, introducing a 147 bp pseudoexon into the mRNA sequence and consequently generating a premature termination codon (p.G129Afs*38) and leading to frameshifting and truncation of the α5 (collagen IV) protein.
UNASSIGNED: This is the first report of the novel c.385-716G > A splicing mutation in the COL4A5 gene, which illustrates the importance of performing WGS to find additional mutations in WES-negative patients with highly suspected forms of genetic diseases. The same results obtained from the in-vitro and in-vivo splicing experiments confirm the consistency between the minigene assay and RT-PCR analysis. In addition, this study highlights the importance of functional analysis in diagnosis and genetic counseling in AS.