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.

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).

Duplication and transposition of the NF1 pseudogene regions on chromosomes 2, 14, and 22.

Numerous NF1 pseudogenes have been identified in the human genome. Those in 2q21, 14q11, and 22q11 form a subset with a similar genomic organization and a high sequence homology. We have studied, by polymerase chain reaction and fluorescence in situ hybridization, the extent of homology of the regions surrounding these NF1 pseudogenes. Our analyses have demonstrated that a fragment of at least 640 kb is homologous between the three regions. Based on previous studies and these new findings, we propose a model for the spreading of the NF1 pseudogene-containing regions. A fragment of approximately 640 kb was first duplicated in chromosome region 2q21 and transposed to 14q11. Subsequently, this fragment was duplicated in 14q11 and transposed to 22q11. A part of the 640-kb fragment in 14q11, with a length of about 430 kb, was further duplicated to a variable extent in 14q11. In addition, we have identified sequences that may facilitate the duplication and transposition of the 640-kb and 430-kb fragments.

Type III collagen deficiency in a family with intracranial aneurysms.

We present a family with 2 female cousins with intracranial aneurysms and type III collagen deficiency. DNA analysis revealed no mutations in the COL3A1 gene, encoding type III collagen, and including the segment encoding the C-propeptide of type III collagen. The 2 patients with low type III collagen production and intracranial aneurysms had inherited different type III collagen alleles. The type III collagen deficiency in this family may results from defects during posttranslational modification or from an altered collagen metabolism.

Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth malformation syndrome that maps to human chromosome 11p15.5, a region that harbours a number of imprinted genes. We studied the methylation status of H19 and KCNQ1OT1 (LIT1/KvDMR1) in a large series of BWS patients. Different patient groups were identified: group I patients (20%) with uniparental disomy and hence aberrant methylation of H19 and KCNQ1OT1; group II patients (7%) with a BWS imprinting centre 1 (BWSIC1) defect causing aberrant methylation of H19 only; group III patients (55%) with a BWS imprinting centre 2 (BWSIC2) defect causing aberrant methylation of KCNQ1OT1 only; and group IV patients (18%) with normal methylation patterns for both H19 and KCNQ1OT1. BWS patients have an increased risk of developing childhood tumours. In our patient group, out of 31 patients (group III) with KCNQ1OT1 demethylation only, none developed a tumour. However, tumours were found in 33% of patients with H19 hypermethylation (group I and II) and in 20% of patients with no detectable genetic defect (group IV). All four familial cases of BWS showed reduced methylation of KCNQ1OT1, suggesting that in these cases the imprinting switch mechanism is disturbed.

Delineation and physical separation of novel translocation breakpoints on chromosome 1p in two genetically closely associated childhood tumors.

Sporadic childhood tumors associated with Beckwith-Wiedemann syndrome (BWS) all show abnormalities of the same region on chromosome 11. In addition to chromosome 11, other chromosome regions are affected in some of these tumor types. In this study we analyzed the region on chromosome 1p involved in the etiology of BWS-associated tumors, Wilms tumor, rhabdomyosarcoma, and hepatoblastoma. For this purpose we determined the location of two novel translocation breakpoints in this chromosome region in cells from a Wilms tumor and cells from a rhabdomyosarcoma. We constructed a map of the region and found that both breakpoints are separated by at least 875 kb. We identified a PAC clone which crosses the rhabdomyosarcoma breakpoint and found several exons within this clone. We established that this breakpoint is located proximal to the PAX7 gene and, therefore, identified a new region involved in the etiology of rhabdomyosarcomas.

Disruption of a novel imprinted zinc-finger gene, ZNF215, in Beckwith-Wiedemann syndrome.

The genetics of Beckwith-Wiedemann syndrome (BWS) is complex and is thought to involve multiple genes. It is known that three regions on chromosome 11p15 (BWSCR1, BWSCR2, and BWSCR3) may play a role in the development of BWS. BWSCR2 is defined by two BWS breakpoints. Here we describe the cloning and sequence analysis of 73 kb containing BWSCR2. Within this region, we detected a novel zinc-finger gene, ZNF215. We show that two of its five alternatively spliced transcripts are disrupted by both BWSCR2 breakpoints. Parts of the 3' end of these splice forms are transcribed from the antisense strand of a second zinc-finger gene, ZNF214. We show that ZNF215 is imprinted in a tissue-specific manner.

Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder that involves tissues derived from the embryonic neural crest. Besides the functional gene on chromosome arm 17q, NF1-related sequences (pseudogenes) are present on a number of chromosomes including 2, 12, 14, 15, 18, 21, and 22. We elucidated the complete nucleotide sequence of the NF1 pseudogene on chromosome 22. Only the middle part of the functional gene but not exons 21-27a, encoding the functionally important GAP-related domain of the NF1 protein, is presented in this pseudogene. In addition to the two known NF1 pseudogenes on chromosome 14 we identified two novel variants. A phylogenetic tree was constructed, from which we concluded that the NF1 pseudogenes on chromosomes 2, 14, and 22 are closely related to each other. Clones containing one of these pseudogenes cross-hybridised with the other pseudogenes in this subset, but did not reveal any in situ hybridisation with the functional NF1 gene or with NF1 pseudogenes on other chromosomes. This suggests that their hybridisation specificity is mainly determined by homologous sequences flanking the pseudogenes. Strong support for this concept was obtained by sequence analysis of the flanking regions, which revealed more than 95% homology. We hypothesise that during evolution this subset of NF1 pseudogenes initially arose by duplication and transposition of the middle part of the functional NF1 gene to chromosome 2. Subsequently, a much larger fragment, including flanking sequences, was duplicated and gave rise to the current NF1 pseudogene copies on chromosomes 14 and 22.

Genetics of Beckwith-Wiedemann syndrome-associated tumors: common genetic pathways.

A specific subset of solid childhood tumors-Wilms' tumor, adrenocortical carcinoma, rhabdomyosarcoma, and hepatoblastoma-is characterized by its association with Beckwith-Wiedemann syndrome. Genetic abnormalities found in these tumors affect the same chromosome region (11p15), which has been implicated in the etiology of Beckwith-Wiedemann syndrome. This suggests that the development of these tumors occurs along a common genetic pathway involving chromosome 11. To search for additional common genetic pathways, this article reviews the genetic data published for these tumors. It was found that, up until now, the only genetic abnormalities detected in all four tumors affect chromosome band 11p15 and the TP53 gene. In addition, there are several aberrations that occur in two or three of the neoplasms. It is concluded that, of the four tumors, the genetic relationship is most evident between Wilms' tumor and rhabdomyosarcoma.

Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. One of the characteristic features of this disease is the development of neurofibromas. Since the NF1 gene is supposed to be a tumour suppressor gene, these neurofibromas should develop upon inactivation of both NF1 alleles. So far, mutation and deletion have been found to be involved in NF1 gene inactivation. However, these inactivating mechanisms explain the development of only a limited fraction of analysed neurofibromas. In this study, we investigated microsatellite instability (MSI) and promoter methylation as potential contributors to NF1 gene inactivation. As site-specific methylation in the NF1 promoter inhibits binding of transcription factors Sp1 and CREB, we studied the methylation status of their binding sites in particular. We analysed 20 neurofibromas and three neurofibrosarcomas, but did not find evidence for microsatellite instability or NF1 promoter methylation in any of the tumours. Thus, our data suggest that both microsatellite instability and promoter methylation are unlikely to be the major causes of NF1 gene inactivation in these tumours.

Multiple MSP pseudogenes in a local repeat cluster on 1p36.2: An expanding genomic graveyard?

Chromosomal region 1p36.2 harbors an intriguing gene cluster of about 1 Mb. In addition to normal high-copy-number repeats, this cluster consists entirely of locally repeated sequences among which there are tRNA and small nuclear RNA (snRNA) genes. In 23 PACs and YACs from the 1p36.2 cluster, we identified eight different copies of a sequence with about 97% homology to the macrophage stimulating protein (MSP) gene located on chromosomal band 3p21. These MSP-like (MSPL) sequences on 1p36.2 are scattered over the repeat region. Nucleotide substitutions and single nucleotide deletions in exons of all identified MSPL genes on 1p36.2 mark them as pseudogenes. We constructed a phylogenetic tree of these sequences with their most likely order of origin in evolution. MSP from 3p21 could be identified as the ancestral sequence, a copy of which was captured into the cluster of tRNA and snRNA genes on 1p36.2 about 6 million years (MY) ago. MSP subsequently coamplified with the other sequences in the cluster. Analysis of the DNA of 18 individuals shows that the MSPL copy number is polymorphic, with a range of four to seven or more copies per haploid genome. Analysis of corresponding clusters in macaque chromosomes indicated an age for the tRNA/snRNA cluster of at least 30 MY. The MSPL sequence thus functions as a probe for the more recent primate evolution of this cluster and suggests a continuation of its unusual activity over the last 6 MY.

Evidence for an ependymoma tumour suppressor gene in chromosome region 22pter-22q11.2.

Ependymomas are glial tumours of the brain and spinal cord. The most frequent genetic change in sporadic ependymoma is monosomy 22, suggesting the presence of an ependymoma tumour suppressor gene on that chromosome. Clustering of ependymomas has been reported to occur in some families. From an earlier study in a family in which four cousins developed an ependymoma, we concluded that an ependymoma-susceptibility gene, which is not the NF2 gene in 22q12, might be located on chromosome 22. To localize that gene, we performed a segregation analysis with chromosome 22 markers in this family. This analysis revealed that the susceptibility gene may be located proximal to marker D22S941 in 22pter-22q11.2. Comparative genomic hybridization showed that monosomy 22 was the sole detectable genetic aberration in the tumour of one of the patients. Loss of heterozygosity studies in that tumour revealed that, in accordance to Knudson's two-hit theory of tumorigenesis, the lost chromosome 22 originated from the parent presumed to have contributed the wild-type allele of the susceptibility gene. Thus, our segregation and tumour studies collectively indicate that an ependymoma tumour suppressor gene may be present in region 22pter-22q11.2.

Comparative genomic hybridization analysis of hepatoblastomas: additional evidence for a genetic link with Wilms tumor and rhabdomyosarcoma.

We applied the technique of comparative genomic hybridization (CGH) to a series of 16 hepatoblastomas. Our goals were (1) to identify all quantitative chromosome abnormalities that appear in this type of tumor and (2) to compare the results with data from similar studies on other tumors associated with the Beckwith-Wiedemann syndrome (BWS). We found that the most commonly detected (> 30%) chromosome abnormalities were gains of chromosomes 1, 2, 7, 8, and 17. Losses of chromosomes were found in only a few cases. On comparing our results with those from studies on the BWS-associated tumors, Wilms tumor and rhabdomyosarcoma, it became clear that three chromosome regions, namely, 7q, 8q, and 17q, were the ones most commonly involved in all three types of tumors. These regions, therefore, may harbor genes that play a role in the etiology of BWS-associated tumors in general.

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

Type III collagen deficiency in saccular intracranial aneurysms. Defect in gene regulation?

BACKGROUND AND PURPOSE: We sought to determine whether there are mutations in the COL3A1 gene in patients with saccular intracranial aneurysms with a type III collagen deficiency and whether there is an association between a marker in the COL3A1 gene and saccular intracranial aneurysms. One of the heritable factors possibly involved in the pathogenesis of saccular intracranial aneurysms is a reduced production of type III collagen, demonstrated earlier by protein studies. METHODS: We analyzed the type III collagen gene in a group of 41 consecutive patients with an intracranial aneurysm, of whom 6 patients had shown a reduced production of type III collagen in cultured diploid fibroblasts from a skin biopsy. RESULTS: No mutations could be demonstrated in the COL3A1 gene, especially not in the globular N- and C-terminal regions. A null allele was excluded in 25 patients, including 1 patient with a decreased type III collagen production. No differences were found between 41 patients and 41 controls in allele frequencies of a DNA tandem repeat polymorphism located in the COL3A1 gene. CONCLUSIONS: It is concluded that the COL3A1 gene is not directly involved in the pathogenesis of most of intracranial aneurysms. The reduced type III collagen production in cultured fibroblasts found in some patients with an intracranial aneurysm is not explained by the present study and needs further exploration.

Integrated genetic and physical map of the 1q31-->q32.1 region, encompassing the RP12 locus, the F13B and HF1 genes, and the EEF1AL11 and RPL30 pseudogenes.

The gene for autosomal recessive retinitis pigmentosa (RP12) with preserved para-arteriolar retinal pigment epithelium was previously mapped close to the F13B gene in region 1q31-->q32.1. A 4-Mb yeast artificial chromosome contig spanning this interval was constructed to facilitate cloning of the RP12 gene. The contig comprises 25 sequence-tagged sites, polymorphic markers, and single-copy probes, including five newly obtained probes. The contig orders the F13B and HF1 genes, as well as five expressed sequence tags, with respect to the integrated genetic map of this region. Homozygosity mapping resulted in refinement of the candidate gene locus for RP12 to a 1. 3-cM region. Currently, approximately 1 Mb of the contig is represented in P1-derived artificial chromosome (PAC) clones. Direct screening of a cDNA library derived from neural retina with PACs resulted in identification of the human elongation factor 1alpha pseudogene (EEF1AL11) and a human ribosomal protein L30 pseudogene (RPL30). A physical and genetic map covering the entire RP12 candidate gene region was constructed.

Periconceptional use of folic acid amongst women of advanced maternal age.

Between February 1994 and February 1997 a group of 881 women completed a questionnaire on the use of folic acid. During the study period the percentage of women who had been informed about the benefits of folic acid rose from 41 per cent to 90 per cent and the percentage taking supplementation rose from 18 per cent to 60 per cent. Nevertheless, few women used folic acid at the right time and dose and although 90 per cent would have been willing to follow recommendations, correct use did not exceed 25 per cent. It is concluded that effective folic acid supplementation requires food fortification rather than information provided by healthcare workers.

Retinitis pigmentosa: defined from a molecular point of view.

Retinitis pigmentosa (RP) denotes a group of hereditary retinal dystrophies, characterized by the early onset of night blindness followed by a progressive loss of the visual field. The primary defect underlying RP affects the function of the rod photoreceptor cell, and, subsequently, mostly unknown molecular and cellular mechanisms trigger the apoptotic degeneration of these photoreceptor cells. Retinitis pigmentosa is very heterogeneous, both phenotypically and genetically. In this review we propose a tentative classification of RP based on the functional systems affected by the mutated proteins. This classification connects the variety of phenotypes to the mutations and segregation patterns observed in RP. Current progress in the identification of the molecular defects underlying RP reveals that at least three distinct functional mechanisms may be affected: 1) the daily renewal and shedding of the photoreceptor outer segments, 2) the visual transduction cascade, and 3) the retinol (vitamin A) metabolism. The first group includes the rhodopsin and peripherin/RDS genes, and mutations in these genes often result in a dominant phenotype. The second group is predominantly associated with a recessive phenotype that results, as we argue, from continuous inactivation of the transduction pathway. Disturbances in the retinal metabolism seem to be associated with equal rod and cone involvement and the presence of deposits in the retinal pigment epithelium.

The role of type III collagen in spontaneous cervical arterial dissections.

A case-control study was carried out to investigate whether type III collagen deficiency plays a role in the pathogenesis of spontaneous cervical arterial dissections. In 16 patients with spontaneous cervical arterial dissections and in 41 healthy controls, protein analysis of type III collagen (ratio of type III/type I collagen) was performed. Furthermore, single-stranded conformation polymorphism/heteroduplex analysis was used to investigate the type III collagen gene in the 16 patients with spontaneous cervical dissections to detect mutations. The ratios of type III/type I collagen in the controls ranged from 5.5 to 19.8% (median, 10%). The ratios of type III/type I collagen in the patients with spontaneous cervical arterial dissections ranged from 3.2 to 17.9% (median, 9.3%). Two patients had a low ratio of type III/type I collagen (<5.5%). No abnormalities suggesting a mutation in the gene of type III collagen were demonstrated in any of the 16 patients. Our findings are in keeping with the hypothesis that a reduced production of type III collagen may contribute to the formation of spontaneous cervical arterial dissections in some patients. The absence of a responsible mutation indicates that the coding sequence of the type III collagen gene is not involved.