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

AbstractThe 22q11.2 deletion syndrome (22q11DS) encompasses DiGeorge syndrome, velo-cardio-facial syndrome and conotruncal anomaly face syndrome and is due to a microdeletion of chromosome 22q11.2. This is the most frequent known interstitial deletion found in human with an incidence of 1 in 4,000 live births. Approximately 90% of patients have a typical deleted region (TDR) and the typical deletion of 22q11.2 deletion is large, approximately 3 Mb.Genitourinary Malformations is one of most common malformations that does severe harm to newborn's health, with an incidence of about 1～8‰and 17th rankamong all birth defects and a uprising tendency with unknown reasons . Recent research has indicated that microdeletion in 22q11.2 is strongly associated with Genitourinary Malformations. Specific anomalies include renal malformations (such as single kidney, small kidneys, renal lithiasis, agenesis or dysplasia, polycystic kidney and horseshoe kidney) , metanephric duct/urinary bladder (blockage, backflow and irregularity urinary bladder) 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 in early embryonic stages. Such a gene may also be responsible for isolated renal malformations.Material and Methods1,To detect microdeletions of 22q11.2 in patients of congenital genitourinary system or cardiac malformation with Fluorescence in situ Hybridization (FISH).2,To analyze the expression of genes within the DGCR region in mouse embryonic kidneys with RT-PCR. To apply bioinformatics and technique of molecular biology for identify susceptibility gene(s) in urogenital tract anomalies located at chromosomal 22q11.2.3,To select genes with specific expression to mutation screen in 44 patients with congenital genitourinary system malformations and 50 normal controls with PCR-SSCP. Then sequencing the abnormal gene to check if mutations are existed or not.Results1,Microdeletions detectionFISH was used for screening of 22q11.2 deletion among 30 patients of congenital genitourinary system or cardiac malformation. None of whom, however, were found to have carried the deletion.2,Among 33 genes selected from the DGCR region, RT-PCR has found 9 genes have no expression in the whole embryonic development process. 18 genes have continue expression from the beginning of development or from some later periods on, the differences are only showed in quantity.6 genes (Snap29,Cldn5,Cdc45l,Dgcr8,Arvcf,Ufd1l) have their expressions only in a period of time. 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. Sequencing shows that two mutations in the SNAP29 gene were identified in collected genomic DNA from 3 patients: a G to A transition (GAG→AAG) at exon 2, two A to G transitions (AGC→GGC) at exon 3.Conclusion1,Our FISH has failed to detect any 22q11.2 deletion among the tested congenital genitourinary system or cardiac malformation cases. This is probably due to the small sample size. Recruiting more samples may improve the chance for identification.2,Expression analysis of DGCR genes during mouse embryonic development has suggested that 6 genes (Snap29,Cldn5,Cdc45l,Dgcr8,Arvcf,Ufd1l) have their expressions only in a period of time. They may play critical roles in the development of urogenital tract at early embryonic stages.3,Two mutations have been found in SNAP29 gene in three patients with genitourinary malformations. Further study about SNAP29 may eventually prove its role in kidney development.