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.

Predictive testing for BRCA1 and 2 mutations: a male contribution.

BACKGROUND: Management strategies for women carrying BRCA1 and 2 mutations are becoming clearer and predictive testing for a known family mutation is commonly undertaken. Implications for men are not as clear and they participate less frequently. PATIENTS AND METHODS: Twenty-six men from 10 extended families underwent predictive testing. Their motivation, reaction and outcome were studied. Subjects had appropriate pre- and post-test counselling. Informed consent was obtained before predictive testing for known deleterious mutations. DNA analysis followed standard procedures. RESULTS: Eighteen tested positive and eight negative. Four had adverse psychological reactions and three reneged on their commitments to impart results. The spouse of another man had an adverse psychological reaction to the disclosure of his positive result. Two, already suffering from prostate cancer, were phenocopies and paternal lineage transmission was unexpectedly determined in another. Risk was removed from 33 offspring and confirmed for 56. CONCLUSIONS: Complex themes associated with genetic testing are confirmed and the spectrum extended. Men appear to understand the importance of participating in this process. Methods of avoiding adverse reactions merit further study along with other aspects of the process.

The detection of large deletions or duplications in genomic DNA.

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Quality control in molecular genetic testing.

DNA-based testing for genetic diseases has developed from nothing into a principal part of laboratory medicine over the past 15 years. In the rush to bring these powerful new technologies into medical use, issues of quality have not always been given sufficient attention. Efforts are now being made to assess the quality of the output of genetic testing laboratories, and the results show that there is room for improvement.

A comparison of methods for gene dosage analysis in HMSN type 1.

A number of different approaches are used in diagnostic laboratories to detect the 1.5 Mb duplication at 17p11.2 seen in approximately 70% of patients with hereditary motor and sensory neuropathy type 1 (HMSN1). Here we compare the methods used in UK diagnostic laboratories to detect the duplication. Samples referred to participating centres for HMSN testing were collected, randomised, and distributed for testing. One hundred samples were examined using five different methods; each method was tested by two independent laboratories. Identical results were obtained from all laboratories for 44 samples. The remaining samples were classified as duplication positive or duplication negative on the basis of the same result by two or more methods. A total of 95 samples were classified by more than one method, two were withdrawn from the study as the same result was not obtained by two methods, and three are thought to have a duplication smaller than 1.5 Mb. Seven of 49 duplications were not detected by methods used to detect the common junction fragment and the use of microsatellites failed to yield a result in four of 95 samples. Sequence tagged site (STS) dosage analysis was found to be the most sensitive of the methods tested, although this method was found to be the most likely to require repeat analysis. Eight samples gave discordant results between the two laboratories testing by the same method. Upon retesting, reasons for the initial incorrect result included processing and typographical errors.

The relationship between Y chromosome DNA haplotypes and Y chromosome deletions leading to male infertility.

Microdeletions on the short arm of the Y chromosome have defined three non-overlapping regions (AZFa, b, c) recurrently deleted among infertile males. These regions contain several genes or gene families involved in male germ-cell development and maintenance. Even though a meiotic origin for these microdeletions is assumed, the mechanisms and causes leading to microdeletion formation are largely unknown. In order to assess whether some Y chromosome groups (or haplogroups) are predisposed to, or protected against, deletion formation during male meiosis, we have defined and compared Y chromosome haplogroup distribution in a group of infertile/subfertile males harbouring Yq deletions and in a relevant Northwestern European control population. Our analyses suggest that Y chromosome deletion formation is, at least in the study populations, a stochastic event independent of the Y chromosome background on which they arise and may be caused by other genetic and/or environmental factors.

Rapid-onset dystonia-parkinsonism: a clinical and genetic analysis of a new kindred.

BACKGROUND: Rapid-onset dystonia-parkinsonism (RDP) is an autosomal dominant disorder linked to chromosome 19q13 that is characterized by sudden onset of primarily bulbar and upper limb dystonia with parkinsonism. METHODS: The authors evaluated 12 individuals from three generations of an Irish family and obtained detailed medical records on a deceased member. The authors describe the clinical, psychiatric, and genetic features of the affected individuals. RESULTS: Five of eight affected members developed sudden-onset (several hours to days) dystonia with postural instability. Four of the five also had bulbar symptoms. Two have stable focal or segmental limb dystonia. One has intermittent hemidystonia with dysarthria that comes on abruptly in times of stress or anxiety. Three had a history of profound difficulty socializing, and at presentation two developed depression. Three patients had a trial of dopamine agonists without benefit. Genetic analysis suggests linkage to chromosome 19 with lod score of 2.1 at zero recombination. CONCLUSION: This is the third reported family with chromosome 19q13 rapid-onset dystonia-parkinsonism. Psychiatric morbidity appeared common in affected members of this family and may be part of the RDP phenotype.

Typical Friedreich's ataxia without GAA expansions and GAA expansion without typical Friedreich's ataxia.

We clinically assessed and performed polymerase chain reaction analysis for the GAA trinucleotide repeat expansion in 103 patients from 73 families in Ireland, with a prior clinical diagnosis of Friedreich's ataxia (FA) or an unclassified progressive ataxic syndrome. The patients were classified as "typical" or "atypical" FA according to Harding's mandatory clinical diagnostic criteria. All patients underwent blood glucose analysis, and electrocardiography and echocardiography was performed in 99 and 101 patients, respectively. Mutation screening for expanded CAG trinucleotide repeats, associated with spinocerebellar ataxia (SCA) 1, 2, 3 and 6 was performed in 86 patients overall, including all GAA negative patients. Forty-nine of 56 typical patients and 13 of 47 atypical patients were either homozygous or heterozygous for the GAA expansion. Seven patients with a typical FA phenotype were negative for the GAA expansion. Although one of these patients had vitamin E deficiency, and two had raised alpha-fetoprotein levels, three other GAA negative patients with a typical FA phenotype had no other identifiable cause for their ataxia, once again raising the possibility of locus heterogeneity in FA. It is also possible that these patients have two point mutations in the X25 gene, or that they have another ataxic syndrome mimicking the FA phenotype. Two families who were homozygous for the GAA expansion exhibited intrafamilial phenotypic variability. Only one GAA negative patient had the SCA 3 mutation, and this was the only patient in the study with a possible autosomal dominant inheritance pattern. In the homozygous GAA population typical patients had significantly more repeats on the smaller allele than atypical patients, and there was an inverse relationship between the number of repeats on the smaller allele and the age at presentation. There was also an inverse relationship between the repeat size on both the larger and the smaller of the two alleles and the age at becoming wheelchair bound. There was no significant relationship between repeat size and the other indices of disease severity, including the presence or absence of diabetes or cardiomyopathy. This is the first large study of an Irish population with progressive ataxia that has shown a similar phenotype/genotype relationship to studies of FA in other European and non-European populations. The relatively low sensitivity and specificity of Harding's clinical diagnostic criteria must be appreciated when clinically assessing patients with a progressive ataxic syndrome. Although molecular genetic analysis now plays an essential role in diagnosis and classification, patients with a typical FA phenotype without any identifiable cause for their ataxia exist.

Hyperinsulinism caused by paternal-specific inheritance of a recessive mutation in the sulfonylurea-receptor gene.

Neonatal hyperinsulinism (HI) is a genetic disorder of pancreatic beta-cells characterized by failure to suppress insulin secretion in the presence of hypoglycemia, resulting in brain damage or death if not adequately treated. Germline mutations in four genes have been associated with HI. Some patients have focal regions of beta-cell proliferation (focal HI). Seventy HI probands in whom at least one SUR-1 mutation was identified were studied. Clinical data from patients with two SUR-1 mutant alleles were compared with those from patients with single paternally inherited mutations. Thirty-seven probands were homozygous or compound heterozygous for SUR-1 mutations. In 33 probands, only a single mutation was identified, and in 31, the parental origin of the proband could be determined; in 29, the mutation was on the paternal allele (P < 0.0002). For three of these, pancreatic tissue was available and showed focal beta-cell hyperplasia. DNA extracted from the focal lesion and adjacent normal pancreas revealed loss of the maternal chromosome 11p15, resulting in reduction to homozygosity for the SUR-1 mutation within the focal lesion only. Using the Tdt-mediated dUTP nick end labeling (TUNEL) reaction, apoptotic beta-cells were identified exclusively within the focal region. At diagnosis, disease severity was similar in patients with paternally inherited mutations and those with two mutations. For patients who did not undergo surgery, those with only paternal mutations entered clinical remission within 16 +/- 6.2 months, compared with 48 +/- 23 months for those with two SUR-1 mutations (P = 0.001). In conclusion, we identified a novel mechanism to explain the pathophysiology of focal HI and provide evidence to suggest that this entity may be self-limiting, since affected beta-cells undergo apoptosis.

Fine-resolution mapping by haplotype evaluation: the examples of PFIC1 and BRIC.

Loci for two inherited liver diseases, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), have previously been mapped to 18q21 by a search for shared haplotypes in patients in two isolated populations. This paper describes the use of further haplotype evaluation with a larger sample of patients for both disorders, drawn from several different populations. Our assessment places both loci in the same interval of less than 1 cM and has led to the discovery of the PFIC1/BRIC gene, FIC1; this discovery permits retrospective examination of the general utility of haplotype evaluation and highlights possible caveats regarding this method of genetic mapping.

Hyperinsulinism: molecular aetiology of focal disease.

Persistent hypoglycaemia in infancy is most commonly caused by hyperinsulinism. A case is reported of the somatic loss of the maternal 11p in an insulin secreting focal adenoma in association with a germline SUR-1 mutation on the paternal allele in a baby boy with hyperinsulinism diagnosed at 49 days old. A reduction to homozygosity of an SUR-1 mutation is proposed as a critical part of the cause of focal hyperinsulinism.

Instability of normal (CTG)n alleles in the DM kinase gene.

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.

A molecular, clinical, and immunohistochemical study of vestibular schwannoma.

The molecular pathogenesis of vestibular schwannoma has been investigated by determining the extent of chromosome 22 loss of heterozygosity in 77 tumors and relating these findings to clinical and immunohistochemical indexes of tumor behavior. Loss of heterozygosity was looked for at eight chromosome 22q loci. Clinical details were obtained in all 77 cases, and a clinical growth index was calculated for each tumor. The proliferative index was estimated in all tumors by using a monoclonal antibody to the proliferating cell nuclear antigen and by calculating the labeling index. Forty percent (31 of 77) of the tumors showed allele loss, and in each case this loss involved the region of the neurofibromatosis type 2 gene. No evidence was found that the presence of chromosome 22 allele loss was associated with the clinical growth index. On the log scale, however, an association was seen between the clinical growth index and the proliferating cell nuclear antigen labeling index p = 0.001). These results suggest that chromsome 22 allele loss is a frequent event in vestibular schwannoma. Tumor behavior, however, appears to be independent of the chromosome 22 mutation. It is proposed that chromosome 22 allele loss and neurofibromatosis type 2 gene inactivation is an early event, possibly involved in the initiation of tumorigenesis in vestibular schwannoma. Tumor growth appears to be independent of this mutation and is likely to be determined by other as yet undefined factors.

Improved genetic mapping of X linked retinoschisis.

X linked retinoschisis (RS) causes poor vision in affected males owing to radial cystic changes at the macula. Genetic linkage analysis was carried out in 16 British families with X linked retinoschisis using markers from the Xp22 region. Linkage was confirmed between the RS locus and the markers DXS207 (lod score, Zmax = 17.9 at recombination fraction theta = 0.03; confidence interval for theta = 0.007-0.09), DXS1053 (Zmax = 18.0 at theta = 0.01, CI = 0.001-0.06), DXS43 (Zmax = 12.9 at theta = 0.03, CI = 0.004-0.09), DXS1195 (Zmax = 6.4 at theta = 0.00), DXS418 (Zmax = 8.2 at theta = 0.00), DXS999 (Zmax = 21.2 at theta = 0.01, CI = 0.001-0.05), DXS443 (Zmax = 14.2 at theta = 0.03, CI = 0.004-0.09), DXS365 (Zmax = 24.5 at theta = 0.008, CI = 0.001-0.04). Key recombinants placed RS between DXS43 distally and DXS999 proximally. Multipoint linkage analysis gave odds of 344:1 in favour of this location for RS and supported the map Xpter-(DXS207, DXS1053)-DXS43-1 cM-RS-1 cM-DXS999-DXS443-DXS365-DXS1052-Xcen.