A new genome scan for primary nonsyndromic vesicoureteric reflux emphasizes high genetic heterogeneity and shows linkage and association with various genes already implicated in urinary tract development.

Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder toward the kidneys, results from a developmental anomaly of the vesicoureteric valve mechanism, and is often associated with other urinary tract anomalies. It is the most common urological problem in children, with an estimated prevalence of 1-2%, and is a major cause of hypertension in childhood and of renal failure in childhood or adult life. We present the results of a genetic linkage and association scan using 900,000 markers. Our linkage results show a large number of suggestive linkage peaks, with different results in two groups of families, suggesting that VUR is even more genetically heterogeneous than previously imagined. The only marker achieving P < 0.02 for linkage in both groups of families is 270 kb from EMX2. In three sibships, we found recessive linkage to KHDRBS3, previously reported in a Somali family. In another family we discovered sex-reversal associated with VUR, implicating PRKX, for which there was weak support for dominant linkage in the overall data set. Several other candidate genes are suggested by our linkage or association results, and four of our linkage peaks are within copy-number variants recently found to be associated with renal hypodysplasia. Undoubtedly there are many genes related to VUR. Our study gives support to some loci suggested by earlier studies as well as suggesting new ones, and provides numerous indications for further investigations.

A genome-wide scan for genes involved in primary vesicoureteric reflux.

BACKGROUND: Vesicoureteric reflux (VUR) is the retrograde flow of urine from the bladder into the ureters. It is the most common urological anomaly in children, and a major cause of end-stage renal failure and hypertension in both children and adults. VUR is seen in approximately 1-2% of Caucasian newborns and is frequently familial. OBJECTIVE AND METHODS: In order to search for genetic loci involved in VUR, we performed a genome-wide linkage scan using 4710 single-nucleotide polymorphisms (SNPs) in 609 individuals from 129 Irish families with >1 affected member. RESULTS: Nonparametric linkage (NPL) analysis of the dataset yielded moderately suggestive linkage at chromosome 2q37 (NPL(max) = 2.67, p<0.001). Analysis of a subset without any additional features, such as duplex kidneys, yielded a maximum NPL score of 4.1 (p = 0.001), reaching levels of genome-wide statistical significance. Suggestive linkage was also seen at 10q26 and 6q27, and there were several smaller peaks. CONCLUSION: Our results confirm the previous conclusion that VUR is genetically heterogeneous, and support the identification of several disease-associated regions indicated by smaller studies, as well as indicating new regions of interest for investigation.

Secondary structures in d(CGG) and d(CCG) repeat tracts.

Several studies have been made to elucidate the nature of secondary structures in the single strands of d(CGG).d(CCG) repeat tracts but with conflicting conclusions. Here, we review this work and attempt to come towards consensus. Some investigators find that the G-rich strand forms hairpins. Of these, some conclude that pairing is in the alignment d(GGC).d(GGC) with two Watson-Crick bonds and one G.G bond per duplex repeat, others conclude that the alignment is d(GCG).d(GCG) with two G.G bonds and one C.C bond per duplex repeat. Others find quadruplex formation and conclude that this is in the latter alignment with two G4-quartets per quadruplex repeat and C.C bonds. We investigate why these different results were obtained and conclude that quadruplexes are likely to form under physiological conditions. We argue that they are probably bonded in the alignment d(GGC).d(GGC) with one G4-quartet and two C.G.C.G. quartets per quadruplex repeat. The C-rich strand does not appear to form quadruplexes under physiological conditions but forms hairpins. Apparently, short hairpins adopt the alignment d(CCG).d(CCG) with mismatched cytosine residues stacked into the helix but with 15 or more repeat units, the dominant form is a distorted hairpin aligned as d(GCC).d(GCC) with unpaired cytosine residues possibly turned outwards and stacked in the minor groove.

Evidence for two preferred hairpin folding patterns in d(CGG).d(CCG) repeat tracts in vivo.

Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that has been found to be responsible for a growing number of human inherited disorders and folate-sensitive fragile chromosome sites. By inserting trinucleotide repeat sequences into a palindromic clamp in lambda phage we are able to investigate their tendencies to form hairpins in vivo in any particular alignment and with odd or even numbers of repeat units in the hairpin. We previously showed that with d(CAG).d(CTG) repeat tracts there was a markedly greater tendency to form hairpins with even numbers of repeat units than with odd numbers, whereas d(GAC).d(GTC) repeats showed no such alternation despite having the same base composition. We expected that d(CGG).d(CCG) repeats, might show the same pattern as d(CAG).(CTG) repeats since they are also involved in trinucleotide repeat expansion disorders. The pattern was not so clear and we wondered whether this might be because d(CGG).d(CCG) repeats have more than one possible alignment in which they could self-anneal. We now present results for all three alignments, which suggest that while even-membered hairpins are preferred in the frame d(CGG).d(CCG), hairpins with odd numbers of trinucleotides are more stable in the frame d(GGC).d(GCC). In both cases the base-pair predicted to close the terminal loop of unpaired bases is 5'C.3'G which has previously been found to be a favoured loop-closing pair.

The effects of trinucleotide repeats found in human inherited disorders on palindrome inviability in Escherichia coli suggest hairpin folding preferences in vivo.

Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that are associated with several human inherited disorders. We present evidence consistent with the folding of these trinucleotide repeats into hairpin loops at the center of a long DNA palindrome in vivo. Our assay utilizes a palindrome in bacteriophage lambda, the center of which determines its ability to inhibit plaque formation in a manner that is consistent with folding into a hairpin or cruciform structure. We show that central inserts of even numbers of d(CAG).d(CTG) repeats inhibit plaque formation more than do odd numbers. Both d(CAG)2.d(CTG)2 and d(CGG)2.d(CCG)2 central sequences behave like DNA sequences known to form two-base loops in vitro, suggesting that they may also form compact and stable loops. By contrast, repeats of d(GAC).d(GTC) do not show any evidence consistent with unusual loop stability. These results agree with in vitro evidence that the unstable repeats can form hairpin secondary structures and suggest a favored position of folding. We discuss the potential roles of secondary structures, DNA replication and recombination in models of repeat tract expansion.