Preliminary Screening For Susceptibility Genes For Urinary Malformations At Chromosome Region 22q11.2

Congenital anomalies of the kidney and urinary tract (CAKUT) are one of most common malformations that does severe harm to newborn's health, with an incidence of about 1～8‰and 17th rank among all birth defects and a uprising tendency with unknown reasons. Genetic factors play crucial roles in urinary tract development, albeit that the mechanisms are not completely known. In recent years, genes that express in the early development of kidney such as PAX2,KAL,EYA1,AGTR2,HNF-1beta,SIX1,SIX2,SALL1,FOXC1,WT1 and HOX11 have been confirmed to be closely associated with anomalies of the kidney and urinary tract. To study the molecule mechanisms during the development of urinary system may therefore help to reveal the pathogenetic mechanisms of the CAKUT and provide evidence for gene diagnosis and genetic counseling.Recent research has indicated that microdeletion in 22q11.2 is strongly associated with CAKUT. Specific anomalies may include renal malformations (such as single kidney, small kidneys, renal lithiasis, agenesis or dysplastic, polycystic kidney and horseshoe kidney, etc.), metanephric duct/urinary bladder (blockage, backflow and irregularity urinary bladder, etc.) and hypospadia. According to a recent study by Annegret et al., five out of six 22q11.2 deletion carriers (80%) have been found to have renal dysplasia, hydronephrosis and/or single kidney. The authors postulated that a single gene defect within the DiGeorge anomaly critical region (DGCR) region plays an important role during the development of urogenital tract at early embryonic stages. Such a gene may also be responsible for isolated renal malformations.The objectives in the study were:1) To screen the microdeletions of 22q11.2 in patients of CAKUT with real-time quantitative PCR (RT-Q PCR).2) To identify susceptible genes from the DGCR region through analyzing their expression during mouse embryonic development.3) To conduct mutation screening for candidate genes identified through steps 1 and 2.Methods1. To detect microdeletions of 22q11.2 in patients of CAKUT DNA of CAKUT patients and normal individuals were extracted with saturated sodium chloride method and diluted to 10, 100 and 1000 times for obtaining of standard curve with RT-Q PCR. Subsequently, microdeletions of 22q11.2 were screened among the samples with RT-Q PCR.2. To analyze the expression of genes within the DGCR region in mouse embryonic kidneysWhole RNA was extracted from kidney tissues obtained from mouse embryos at different developmental stages as well as adulthood expression strength of DGCR genes was measured with RT-PCR.3. To apply bioinformatics and technique of molecular biology for identify susceptibility gene(s) in urinary tract anomalies located at chromosomal 22q11.2.Results1. Microdeletions' detectionRT-Q PCR was used for screening of 22q11.2 deletion among 30 patients of CAKUT. None of whom, however, were found to have carried the deletion.2. Among 29 genes selected from the DGCR region, RT-PCR has found 9 have no expression at all developmental stages including adulthood of mouse kidney development. For the remainders, most of them have low expression at the selected time points, except that Pnutll, Ranbpl and Mapkl have high expression at all 4 stages, and Cdc451, Hira, Snap29, Ube213 have an upraise at the critical stages. With K-means methods, the expression curves of above genes were clustered into 6 groups. 3. Based on literature study, SNAP29 was selected for mutation screening. Specific primers were designed for all its 5 exons. PCR was performed on DNA samples from 10 patients. Sequencing of the amplified fragment has identified frequent mutations (six in all) within exon 2 in one of the samples, among which a nonsense mutation was discoverd.Conclusion1. Our RT-Q PCR has failed to detect any 22q11.2 deletion among the tested CAKUT cases. This is probably due to the small sample as well as the technical features of RT-Q PCR. Utilization of FISH and recruiting more samples may improve the chance for identification.2. Expression analysis of DGCR genes during mouse embryonic development has suggested that 9 genes have no expression in the developing kidneys at various stages of mouse life, whilst most of the remainders have low expression at these stages. Notably, Pnutll, Ranbpl and Mapkl have high expression at all stages, and Cdc45l,Hira,Snap29,Ube213 had particular upraise at the critical stages. It may be postulated that, while their continuous strong expression may suggest that Pnutll, Ranbpl and Mapkl are of housekeeping gene type, Cdc45l, Hira, Snap29 and Ube213 may play critical roles in the development of urogenital tract at early embryonic stages.3. Mutations of SNAP29 have already been associated with malformation of brain and cutaneous tissue. In this study, multide mutations within the second exon of SNAP29 have been found in one subject featuring urogenital malformations, among which one was of nonsense type. Further study about SNAP29 may be eventually prove its role in kidney development.