Hosseini S Mohsen, Herd Sarah, Vincent Andrea L, Héon Elise
Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
Mol Vis. 2008 Jan 16;14:71-80.
Posterior polymorphous corneal dystrophy (PPCD) is a genetically heterogeneous autosomal dominant condition which maps to the pericentromeric region of chromosome 20. Mutations in the VSX1 transcription factor have been reported in patients affected with PPCD, keratoconus, or a combination of both phenotypes. However, no mutation was identified in the coding region of VSX1 in the family used for the original mapping. To clarify the genetic basis of PPCD1, a thorough analysis was performed on the original PPCD1 family and two other PPCD1-linked families. As part of the analysis, the expression profile, transcript variants, and evolutionary conserved regions of VSX1, a key candidate gene within the linkage interval, were characterized.
Haplotype analysis was performed using highly informative markers on the pericentromeric region of chromosome 20. VSX1 transcript variants were identified using RT-PCR and characterized by 3'RACE assay. Temporal expression profile of VSX1 was evaluated using semi-quantitative real-time RT-PCR on human tissues. Evolutionary conserved regions (ECRs) were identified in the vicinity of VSX1 using publicly available sequence alignments (UCSC and rVista) and sequenced for mutation analysis.
Recombination events were identified that narrow the PPCD1-disease interval from 20 to 16.44 cM. This smaller interval includes the CHED1 locus and a recently described PPCD locus in Czech families. The three strongest candidate genes of the PPCD1-CHED1 overlap region (RBBP9, ZNF133, SLC24A3) did not show any mutations in our PPCD1-linked families. Semi-quantitative real-time RT-PCR detected VSX1 expression in neonatal human cornea. Six transcript variants of VSX1 were characterized. Four of the transcript variants spliced to two novel exons downstream of the gene. Mutation analysis of the PPCD1-linked families did not reveal any mutations in the full genomic sequence of VSX1 (considering all splice variants) or in the six cis- regulatory modules predicted in the vicinity of VSX1 (100 kb).
This is the first documentation of VSX1 expression in human neonatal cornea. We provide evidence for genetic heterogeneity of chromosome 20-related PPCD and refinement of the original PPCD1 interval. The full genomic sequence of VSX1 and coding exons of three other candidate genes were excluded from being pathogenic in the original PPCD1 family.
后极性多形性角膜营养不良（PPCD）是一种具有遗传异质性的常染色体显性疾病，定位于20号染色体的着丝粒周围区域。据报道，VSX1转录因子的突变存在于患有PPCD、圆锥角膜或两种表型组合的患者中。然而，在最初用于定位的家族中，未在VSX1的编码区发现突变。为了阐明PPCD1的遗传基础，对最初的PPCD1家族和另外两个与PPCD1连锁的家族进行了全面分析。作为分析的一部分，对连锁区间内的关键候选基因VSX1的表达谱、转录变体和进化保守区域进行了表征。
使用20号染色体着丝粒周围区域的高信息量标记进行单倍型分析。使用RT-PCR鉴定VSX1转录变体，并通过3'RACE分析进行表征。使用半定量实时RT-PCR评估人组织中VSX1的时间表达谱。使用公开可用的序列比对（UCSC和rVista）在VSX1附近鉴定进化保守区域（ECR），并进行测序以进行突变分析。
鉴定出重组事件，将PPCD1疾病区间从20 cM缩小到16.44 cM。这个较小的区间包括CHED1基因座和捷克家族中最近描述的PPCD基因座。PPCD1-CHED1重叠区域的三个最强候选基因（RBBP9、ZNF133、SLC24A3）在我们与PPCD1连锁的家族中未显示任何突变。半定量实时RT-PCR检测到新生儿人角膜中VSX1的表达。对VSX1的六个转录变体进行了表征。其中四个转录变体剪接到该基因下游的两个新外显子。对与PPCD1连锁的家族进行的突变分析未在VSX1的全基因组序列（考虑所有剪接变体）或VSX1附近预测的六个顺式调节模块（100 kb）中发现任何突变。
这是VSX1在人新生儿角膜中表达的首次记录。我们为20号染色体相关PPCD的遗传异质性和原始PPCD1区间的细化提供了证据。VSX1的全基因组序列和其他三个候选基因的编码外显子在最初的PPCD1家族中被排除为致病因素。
