Frequency of Von Hippel-Lindau germline mutations in classic and non-classic Von Hippel-Lindau disease identified by DNA sequencing, Southern blot analysis and multiplex ligation-dependent probe amplification.

The current clinical diagnosis of Von Hippel-Lindau (VHL) disease demands at least one specific [corrected] VHL manifestation in a patient with familial VHL disease, or, in a [corrected] sporadic patient, at least two or more hemangioblastomas or a single hemangioblastoma in combination with a typical visceral lesion. To evaluate this definition, we studied the frequency of germline VHL mutation in three patients groups: (i) multi-organ involvement (classic VHL), (ii) limited VHL manifestations meeting criteria (non-classic VHL) and (iii) patients with VHL-associated tumors not meeting current diagnostic VHL criteria. In addition, we validated multiplex ligation-dependent probe amplification (MLPA) as a rapid and reliable quantitative method for the identification of germline VHL deletions. The frequency of germline VHL mutations was very high in classic VHL cases with multi-organ involvement (95%), lower in non-classic cases that meet current diagnostic criteria but have limited VHL manifestations or single-organ involvement (24%) and low (3.3%), but tangible in cases not meeting current diagnostic VHL criteria. The detection of germline VHL mutations in patients or families with limited VHL manifestations, or single-organ involvement is relevant for follow-up of probands and early identification of at-risk relatives.

The PIP5K2A and RGS4 genes are differentially associated with deficit and non-deficit schizophrenia.

Several putative schizophrenia susceptibility genes have recently been reported, but it is not clear whether these genes are associated with schizophrenia in general or with specific disease subtypes. In a previous study, we found an association of the neuregulin 1 (NRG1) gene with non-deficit schizophrenia only. We now report an association study of four schizophrenia candidate genes in patients with and without deficit schizophrenia, which is characterized by severe and enduring negative symptoms. Single-nucleotide polymorphisms (SNPs) were genotyped in the DTNBP1 (dysbindin), G72/G30 and RGS4 genes, and the relatively unknown PIP5K2A gene, which is located in a region of linkage with both schizophrenia and bipolar disorder. The sample consisted of 273 Dutch schizophrenia patients, 146 of whom were diagnosed with deficit schizophrenia and 580 controls. The strongest evidence for association was found for the A-allele of SNP rs10828317 in the PIP5K2A gene, which was associated with both clinical subtypes (P = 0.0004 in the entire group; non-deficit P = 0.016, deficit P = 0.002). Interestingly, this SNP leads to a change in protein composition. In RGS4, the G-allele of the previously reported SNP RGS4-1 (single and as part of haplotypes with SNP RGS4-18) was associated with non-deficit schizophrenia (P = 0.03) but not with deficit schizophrenia (P = 0.79). SNPs in the DTNBP1 and G72/G30 genes were not significantly associated in any group. In conclusion, our data provide further evidence that specific genes may be involved in different schizophrenia subtypes and suggest that the PIP5K2A gene deserves further study as a general susceptibility gene for schizophrenia.

Historical development of analysing large-scale changes in the human genome.

A widely held belief today is that genomics really only started with the DNA sequence information emanating from the genome programs for various organisms, with the human genome playing the leading role. In fact there is a discernable trail stretching for more than a 100 years from the observations of Boveri on tissue instability involving polyploidy in sea urchin embryos and human tumours to the present day. This historical review follows that trail and shows that many theoretical and technical advantages taken for granted in today's genomics era rely heavily on earlier cytogenetic and gene mapping discoveries. Three specific examples of technical developmental paths involving in situ hybridisation, flow-sorting and DNA reassociation kinetics will be explored. In the mid-1980s the two former approaches merged to give rise to several applications of which chromosome painting and chromosome CGH are arguably the most important. The latter developed into array CGH which has now become the pre-eminent method for detecting micro-imbalances in a large number of targets. A competing emerging technology is that of genome-wide SNP typing, which itself is a product of the much earlier RFLP approach linked to DNA sequence information. Do such approaches spell the final demise of the microscope? Perhaps for narrowly defined activities this may occur, but for addressing general questions, microscopic examination will remain pre-eminent.

Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations.

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1-->q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.

Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.

Studying the biological and technical sources of variation in telomere length of individual chromosomes.

BACKGROUND: Consistent average length differences between species and chromosome arm differences within species indicate that telomere length is genetically determined. This seems to contradict an observed large variation in lengths of the same human telomere between metaphases of the same individual. We examined the extent to which the variation in the telomeres of the human X and Y chromosomes is heritable, induced, or technical in origin. METHODS: Metaphase chromosomes were stained by fluorescence in situ hybridization with a telomere repeat-specific probe, and fluorescence intensities of the X and Y chromosomes were measured. If telomere length variation is predominantly genetically determined and a 50% probability of meiotic recombination between the pseudo-autosomal regions of Yp and Xp in the father is taken into account, one expects an equal chance that the Yp telomere of a son is derived from his father's Xp or Yp telomere. This implies that the Yp/Yq telomere ratios in fathers and sons will be identical in the absence of paternal meiotic recombination and different when recombination occurs. RESULTS: Among five father-son pairs, four showed similar Yp/Yq ratios (P > 0.05), whereas one pair exhibited a large difference in the Yp/Yq ratio that was attributable to a significantly longer Xp than Yp telomere in the father and a presumptive meiotic exchange between X and Y during paternal meiosis. Further, the Xq telomere exhibited a generally shorter telomere length than the others. CONCLUSIONS: The high variation in telomere length appeared to be intracellular (between sister chromatids) and, hence, technical in nature. We found no measurable induced variation in the cells studied, implying that, if induced variation exists, it is small compared with the technical variation.

Genome-wide linkage in a large Dutch consanguineous family maps a locus for intracranial aneurysms to chromosome 2p13.

BACKGROUND AND PURPOSE: Familial occurrence of intracranial aneurysms suggests a genetic factor in the development of these aneurysms. In this study, we present the identification of a susceptibility locus for the development of intracranial aneurysms detected by a genome-wide linkage approach in a large consanguineous pedigree. METHODS: Patients with clinical signs and symptoms of intracranial aneurysms, confirmed by radiological, surgical, or postmortem investigations, were included in the study. Magnetic resonance angiography was used to detect asymptomatic aneurysms in relatives. RESULTS: Seven out of 20 siblings had an intracranial aneurysm. Genome-wide multipoint linkage analysis showed a significant logarithm of the odds score of 3.55. CONCLUSIONS: In a large consanguineous pedigree intracranial aneurysms are linked to chromosome 2p13 in a region between markers D2S2206 and D2S2977.

Mapping of the SCA23 locus involved in autosomal dominant cerebellar ataxia to chromosome region 20p13-12.3.

We report upon a Dutch autosomal dominant cerebellar ataxia (ADCA) family, clinically characterized by a late-onset (>40 years), slowly progressive, isolated spinocerebellar ataxia (SCA). Neuropathological examination in one affected subject showed neuronal loss in the Purkinje cell layer, dentate nuclei and inferior olives, thinning of cerebellopontine tracts, demyelination of posterior and lateral columns in the spinal cord, as well as ubiquitin-positive intranuclear inclusions in nigral neurons that were considered to be Marinesco bodies. Data obtained from the genome-wide linkage analysis revealed a maximal lod score of 3.46 at = 0.00 for marker D20S199. This new SCA locus, on chromosome region 20p13-p12.3, was designated SCA23 after approval by the HUGO Nomenclature Committee. Currently, candidate genes are being screened for mutations within the SCA23 interval. In addition to the recently identified SCA14, SCA19 and FGF14 families, SCA23 is yet another novel SCA locus in the Dutch ADCA population, which further defines the genetic heterogeneity of ADCA families in the Netherlands.

Defining the contribution of the HLA region to cis DQ2-positive coeliac disease patients.

The major genetic susceptibility to coeliac disease is contributed by the human leukocyte antigen (HLA) region. The primary association is with the HLA-DQ2 molecule, encoded by the DQA1*05 and DQB1*02 alleles, which is expressed by over 90% of patients. The aim of our study was to perform an extensive scan of the entire HLA region to determine whether there is evidence for the presence of additional HLA susceptibility genes for coeliac disease in the Dutch population, acting independently of DQ2. In all, 16 microsatellite markers and the DQA1 and DQB1 genes were genotyped in simplex cis DQ2-positive coeliac disease families and cis DQ2-positive control families. Allele frequencies of markers on phase-known DQ2-positive haplotypes transmitted to patients were compared to a combined group of DQ2-positive nontransmitted and control haplotypes, thereby controlling for the DQ2 contribution. No significant differences at any of the marker loci were detected, suggesting that DQ2 is the major HLA risk factor for coeliac disease. Individuals homozygous for DQ2 or heterozygous for DQA1*05-DQB1*02/DQA1*0201-DQB1*02 were found to be at five-fold increased risk for development of coeliac disease (P<10(-8)). This risk seems to be conferred by the presence of a second DQB1*02 allele next to one DQA1*05-DQB1*02 haplotype, independently of the second DQA1 allele.

Identification of a novel SCA14 mutation in a Dutch autosomal dominant cerebellar ataxia family.

OBJECTIVE: To report a Dutch family with autosomal dominant cerebellar ataxia (ADCA) based on a novel mutation in the PRKCG gene. METHODS: The authors studied 13 affected members of the six-generation family. After excluding the known spinocerebellar ataxia (SCA) genes, a combination of the shared haplotype approach, linkage analysis, and genealogic investigations was used. Exons 4 and 5 of the candidate gene, PRKCG, were sequenced. RESULTS: Affected subjects displayed a relatively uncomplicated, slowly progressive cerebellar syndrome, with a mean age at onset of 40.8 years. A focal dystonia in two subjects with an onset of disease in their early 20s suggests extrapyramidal features in early onset disease. Significant linkage to a locus on chromosome 19q was found, overlapping the SCA-14 region. Based on the recent description of three missense mutations in the PRKCG gene, located within the boundaries of the SCA-14 locus, we sequenced exons 4 and 5 of this gene and detected a novel missense mutation in exon 4, which involves a G-->A transition in nucleotide 353 and results in a glycine-to-aspartic acid substitution at residue 118. CONCLUSION: A SCA-14-linked Dutch ADCA family with a novel missense mutation in the PRKCG gene was identified.

Genome-wide screen in obese pedigrees with type 2 diabetes mellitus from a defined Dutch population.

A genome scan was performed in obese type 2 diabetes mellitus pedigrees to identify susceptibility loci involved in obesity-driven type 2 diabetes mellitus. We studied the 20% most obese diabetes pedigrees from a confined Dutch population from around the town of Breda. Previously we, and others, have already shown that a susceptibility locus influencing obesity in diabetes may reside on chromosome 18p11. We now report evidence to also suggest linkage for type 2 diabetes in these obese pedigrees on chromosome regions 11p (genome-wide P-value

The effect of breed and intrauterine crowding on fetal erythropoiesis on day 35 of gestation in swine.

In a previous report, it was suggested that intrauterine crowding impaired fetal erythropoiesis and that fetal erythropoiesis was accelerated in Meishan pigs during early pregnancy. Because these conclusions were based on limited numbers of observations, the present experiment was undertaken to provide a more extensive investigation of these phenomena. Intact white crossbred gilts, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts, and intact Meishan gilts (n = 13 to 16 per group) were mated after at least one estrous cycle of normal duration (17 to 23 d). Gilts were laparotomized at d 35 of pregnancy, the uterine horns were exteriorized and opened near each fetus, and a blood sample was collected from each fetus. The uterine horn was then surgically removed, and each fetus and placenta was weighed. All fetal blood samples were measured for hematocrit, red blood cell number, and hemoglobin. Erythropoietin and the percentages of nucleated cells and reticulocytes were also measured in blood samples from the largest and smallest living fetus in each litter. Fetal hematocrits were not affected by treatment. Blood cell counts were greater (P < 0.01) in fetuses of Meishan gilts than in White crossbred intact or UHO gilts. Hemoglobin was less (P < 0.01) in fetuses of Meishan gilts than in fetuses of White crossbred intact or UHO gilts. The percentages of nucleated (immature) cells and reticulocytes were both less (P < 0.01) in fetuses of Meishan intact gilts. Erythropoietin was also lower (P < 0.01) in fetuses of Meishan gilts. As observed previously, fetal weight was correlated (r = 0.38; P < 0.01) with blood hemoglobin concentration. These results confirm that fetal erythropoiesis in Meishan gilts is accelerated compared with White crossbred gilts. These results are consistent with the hypothesis that faster blood cell development could be beneficial to fetal survival in swine.

Anticipation in familial intracranial aneurysms in consecutive generations.

Intracranial aneurysms (IA) are the major cause of subarachnoid haemorrhages (SAH). A positive family history for SAH is reported in 5-10% of the patients. The mode of inheritance is not unambiguously established; both autosomal dominant and recessive modes have been reported. In sporadic as well as in familial SAH, approximately 60% of the SAH patients are female. Recently, anticipation has been described in familial SAH. Since up to 15% of the SAHs are not caused by an IA, we have analysed anticipation, sex ratio and mode of inheritance only in families with patients with a proven IA in two consecutive generations. A total of 10 families were studied in which at least two persons in consecutive generations were affected by SAH, a symptomatic IA (SIA) or a presymptomatic IA (PIA). We also analysed published data from families with a proven IA in two consecutive generations on age of SIA onset and sex ratios among affected family members (both SIA and PIA). The age of SIA onset in the parental generation (mean 55.5 years) differed significantly from the age of onset in their children (mean 32.4 years). In the parental generation 11 men and 37 women were affected (both SIA and PIA), in the consecutive generation these numbers were 28 men and 32 women. There is a significant difference in sex ratio of affected family members when the generations are compared (P<0.02). No family could be found in which three consecutive generations were affected by an IA (SIA or PIA).

A genome-wide scan in type 2 diabetes mellitus provides independent replication of a susceptibility locus on 18p11 and suggests the existence of novel Loci on 2q12 and 19q13.

A genome-wide scan was performed, using nonparametric linkage analyses, to find susceptibility loci for type 2 diabetes mellitus in the Dutch population. We studied 178 families from The Netherlands, who constituted 312 affected sibling pairs. The first stage of the genome scan consisted of 270 DNA markers, with an average intermarker spacing of 13 cM. Because obesity and type 2 diabetes mellitus are interrelated, the data set was stratified for the subphenotype body mass index, corrected for age and gender. This resulted in a suggestive maximum multipoint LOD score of 2.3 (single-point P value, 9.7 x 10(-4); genome-wide P value, 0.028) for the most obese 20% pedigrees of the data set, between marker loci D18S471 and D18S843. In the lowest 80% obese pedigrees, two interesting loci on chromosome 2 and 19 were found, with LOD scores of 1.5 and 1.3. We provide independent evidence that the chromosome 18p11 locus, reported earlier from a Finnish/Swedish population, is of definite interest for type 2 diabetes mellitus in connection with obesity. Subsequently, our results indicate that two novel loci may reside on chromosomes 2 and 19, with minor effects involved in the development of type 2 diabetes mellitus in the Dutch population.

A whole-genome scan in 164 Dutch sib pairs with attention-deficit/hyperactivity disorder: suggestive evidence for linkage on chromosomes 7p and 15q.

A genome scan was performed on 164 Dutch affected sib pairs (ASPs) with attention-deficit/hyperactivity disorder (ADHD). All subjects were white and of Dutch descent and were phenotyped according to criteria set out in the Diagnostic and Statistical Manual Of Mental Disorders, 4th edition. Initially, a narrow phenotype was defined, in which all the sib pairs met the full ADHD criteria (117 ASPs). In a broad phenotype, additional sib pairs were included, in which one child had an autistic-spectrum disorder but also met the full ADHD criteria (164 ASPs). A set of 402 polymorphic microsatellite markers with an average intermarker distance of 10 cM was genotyped and analyzed using the Mapmaker/sibs program. Regions with multipoint maximum likelihood scores (MLSs) >1.5 in both phenotypes were fine mapped with additional markers. This genome scan indicated several regions of interest, two of which showed suggestive evidence for linkage. The most promising chromosome region was located at 15q, with an MLS of 3.54 under the broad phenotype definition. This region was previously implicated in reading disability and autism. In addition, MLSs of 3.04 and 2.05 were found for chromosome regions 7p and 9q in the narrow phenotype. Except for a region on chromosome 5, no overlap was found with regions mentioned in the only other independent genome scan in ADHD reported to date.

The role of genetic factors in age at natural menopause.

BACKGROUND: Environmental factors explain only a small part of the age variance at which menopause commences. The variation in natural menopause is a trait predominantly determined by interaction of multiple genes, whose identity and causative genetic variation remains to be determined. Menopause is a retrospective marker for the reproductive capacity of preceding years, since subfertility and infertility precede menopause at distinct time-intervals. In the present study we have investigated the contribution of genetic factors to menopausal age. METHODS: Data were collected from a random population sample of singleton and twin sisters participating in a prospective breast cancer screening project, who had subsequently experienced natural menopause. Heritability of menopausal age was estimated with analysis of variance, Mx modelling and Gibbs sampling. RESULTS: All produced almost identical heritability estimates of 0.85-0.87 for singleton sisters, suggesting a strong genetic contribution to menopausal age. Twin data were used to distinguish additive genetic from common environmental effects; a heritability of 0.71-0.72 was determined, which does not deviate significantly from the estimate for singleton sisters. CONCLUSIONS: According to our findings, a woman with a family history of early menopause risks early menopause and consequently early reproductive failure herself.

Characterization and chromosomal localization of five canine ATOX1 pseudogenes.

We have isolated six ATOX1 loci from the canine genome in BAC clones. Sequence analysis showed that five of these clones correspond to processed pseudogenes. Fluorescent in situ hybridization allowed us to map the genuine ATOX1 gene to CFA4q24-->q31 and the ATOX1 pseudogenes to CFA19q13.1, CFA4q24-->q31, CFA18q24-->q25, CFA9q22.1 -->q22.2 and CFA20q11-->q12.