Background & Aims Zebrafish mutants generated by ethylnitrosourea (ENU)-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, “flotte lotte” (flo), displays striking defects in intestinal, liver, pancreas and eye formation at 78hpf. In this study we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. Methods Positional cloning was employed to map the flo mutation. Sub-cellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, confocal and electron microscopy. Results The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, ELYS has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Though elys mRNA is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally-expressed elys mRNA until 24hpf. From 72hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately intestinal epithelial cells undergo apoptosis. Conclusion Our results demonstrate that Elys regulates digestive organ formation.

Thin-basement-membrane nephropathy (TBMN) is characterized by persistent dysmorphic hematuria, and the presence of proteinuria is a risk factor for renal impairment. TBMN is often due to mutations in the COL4A3 and COL4A4 genes, and this study determined whether additional mutations in genes encoding other structures in the glomerular filtration barrier contributed to the development of proteinuria. Fifty-six unrelated individuals with TBMN including 18 (32%) with proteinuria > or = 300 mg/L and ten (18%) with proteinuria > or = 500 mg/L were studied. Deoxyribonucleic acid (DNA) was screened for NPHS2 mutations and variants (R138Q and P375L) using single-stranded conformational analysis (SSCA) and for the R229Q mutation by sequencing. DNA was also screened for ACTN4 mutations. R229Q was more common in patients with TBMN and proteinuria > or = 500 mg/L (p < 0.05), and a possible NPHS2 mutation (671G>A, R224H) was identified in one patient with proteinuria 700 mg/L. No other NPHS2 variants correlated with proteinuria, and no ACTN4 mutations were found. Individuals with TBMN and R229Q are carriers of the autosomal recessive forms of both Alport syndrome and familial focal segmental glomerulosclerosis (FSGS). The early demonstration of R229Q in individuals with TBMN may indicate those at increased risk of proteinuria and renal impairment.

BackgroundMany research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays.ResultsWe demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method.ConclusionTheoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.