Use of patient derived urine renal epithelial cells to confirm pathogenicity of PKHD1 alleles.
Use of patient derived urine renal epithelial cells to confirm pathogenicity of PKHD1 alleles.
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DOI：
10.1186/s12882-020-02094-z
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发表时间：
2020-10-15
期刊：
影响因子：
2.3
通讯作者：
Sayer JA
中科院分区：
文献类型：
--
作者：
Molinari E;Srivastava S;Dewhurst RM;Sayer JA
PKHD 1是常染色体隐性遗传性多囊肾病（ARPKD）的主要遗传原因，ARPKD是一种遗传性肝肾纤维囊性疾病，是儿童早期终末期肾病的最重要原因。ARPKD也可以在成年期出现较温和的表型。在这项研究中，我们描述了一名患有非典型多囊肾的24岁女性，没有肾脏疾病家族史，也没有明显的肾外表现，她被转诊进行基因调查。我们使用下一代测序、桑格测序和RNA以及对尿源性肾上皮细胞（UREC）进行的显微镜研究的组合来提供ARPKD的遗传诊断。下一代囊性纤毛病变基因测序组允许鉴定PKHD 1中的两个杂合序列变化（c.6900C > T; p.（Asn2300=）和c.7964A > C; p.（His2655Pro））。同义PKHD 1变体的致病性尚不清楚，需要RNA研究，由于PKHD 1在淋巴细胞中的低表达水平，不能有效地对从先证者血液中提取的RNA进行研究。使用URECs作为肾脏特异性RNA的来源，我们发现PKHD 1在对照组和先证者URECs中围绕外显子43选择性剪接。变种P（Asn 2300 =）改变表达比率，有利于更短的框外转录物。为了进一步研究这些变异的表型结果，我们研究了患者UREC的纤毛表型，其异常伸长并沿轴丝沿着呈现多个泡。我们证实了URECs作为遗传性肾病候选变体功能研究工具的作用，特别是当感兴趣的基因的表达仅限于肾脏时，我们首次描述了ARPKD患者细胞中的纤毛异常。
PKHD1 is the main genetic cause of autosomal recessive polycystic kidney disease (ARPKD), a hereditary hepato-renal fibrocystic disorder which is the most important cause of end-stage renal disease during early childhood. ARPKD can also present in adulthood with milder phenotypes. In this study, we describe a 24-year-old woman with atypical polycystic kidney, no family history of renal disease and no obvious extra-renal manifestations who was referred for genetic investigation. We used a combination of next generation sequencing, Sanger sequencing and RNA and microscopy studies performed on urine-derived renal epithelial cells (URECs) to provide a genetic diagnosis of ARPKD. A next generation sequencing panel of cystic ciliopathy genes allowed the identification of two heterozygous sequence changes in PKHD1 (c.6900C > T; p.(Asn2300=) and c.7964A > C; p.(His2655Pro)). The pathogenicity of the synonymous PKHD1 variant is not clear and requires RNA studies, which cannot be carried out efficiently on RNA extracted from proband blood, due to the low expression levels of PKHD1 in lymphocytes. Using URECs as a source of kidney-specific RNA, we show that PKHD1 is alternatively spliced around exon 43, both in control and proband URECs. The variant p.(Asn2300=) shifts the expression ratio in favour of a shorter, out-of-frame transcript. To further study the phenotypic consequence of these variants, we investigated the ciliary phenotype of patient URECs, which were abnormally elongated and presented multiple blebs along the axoneme. We confirm the power of URECs as a tool for functional studies on candidate variants in inherited renal disease, especially when the expression of the gene of interest is restricted to the kidney and we describe, for the first time, ciliary abnormalities in ARPKD patient cells.
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发表时间：
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