Complex Genetic Approaches to Monogenic Disease: Genomic and Transcriptomic Dissection of Normal Expression of CTNS, the Gene Involved in Nephropathic Cystinosis

Cystinosis Research Foundation Post-Doctoral Fellowship

Complex genetic approaches to monogenic disease: Genomic and transcriptomic dissection of normal expression of CTNS, the gene involved in nephropathic Cystinosis

Dr Katy Freed

Progress report

30 October 2007

Overview

While rare human genetic diseases like cystinosis are caused by mutations in a single gene there is a growing realization that genes rarely work alone but rather are posited within complex global regulatory networks in which they may potentially interact with many other genes. This realization underpinned the recently completed first phase of our cystinosis research program in which we set out to study the genetics of the CTNS gene in a large sample of unaffected families, employing normal human variation as a model for pathological human variation.

Using a genome-wide scanning strategy we identified the VPS13A gene on chromosome 9 to be a plausible positional and functional candidate for a trans-acting regulator of CTNS expression. In this current fellowship project our aim was to exhaustively enumerate all genetic variation in the VPS13A gene to confirm and ultimately identify those variants most likely to be functionally involved in the trans-regulation of CTNS expression.

Another gene of interest is STUB1. In many biological systems redundancy has been observed in gene function. This has been observed for the yeast homologue (ERS1) of human CTNS, with the recent identification of the functionally related MEH1 gene. Using a combinatorial strategy involving database sequence interrogation and genetically correlated expression data from our transcriptome dataset we have identified STUB1 to be a plausible candidate for the human homologue of yeast MEH1. This raises the possibility that Stub1 may work in parallel with Ctns having an overlapping or complementary function

Progress to date

One of the major aims of the proposal was to re-sequence the genes STUB1 and VPS13A to identify all known variants in our Mexican-American population. Initially PCR reactions were optimized then PCR amplicons, of approximately 700 bp, were generated then sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit. The products were analyzed on an Applied Biosystems 3730 DNA Analyzer. An overview of the experimental progress to date for each gene is presented in Table 1.

Table 1: An overview of the PCR and sequencing reactions for the genes STUB1 and VPS13A

The sequences generated from the re-sequencing were analyzed in the SeqScape program to identify single nucleotide polymorphisms (SNPs). The SNPs identified were then compared to the known SNPs in the NCBI dbSNP database. SNPs found in our population, which were not found in the public SNP database, were designated as novel SNPs. All of STUB1 was sequenced, including 1.5 kb of the 5’ promoter region. The results for STUB1 from the re-sequencing effort are presented in Table 2.

Table 2: The number and genomic location of the SNPs identified by re-sequencing STUB1

VPS13A is a large gene spanning 240 kb. In the first instance, we re-sequenced 1.5 kb of the 5’ promoter region. The data generated so far for VPS13A from the re-sequencing effort are presented in Table 3.

Table 3: The number and genomic location of the SNPs identified by re-sequencing VPS13A

The next phase of the project is to analyze SNPs in STUB1 and VPS13A in 1,240 individuals derived from the Mexican-American population. This will be done by genotyping DNA samples using the Illumina BeadExpress system. The results of this genotyping will be presented in the next progress report.