Localisation of the Fanconi anaemia complementation group A gene to chromosome 16q24.3.

Fanconi anaemia (FA) is an autosomal recessive disorder associated with diverse developmental abnormalities, bone-marrow failure and predisposition to cancer. FA cells show increased chromosome breakage and hypersensitivity to DNA cross-linking agents such as diepoxybutane and mitomycin C. Somatic-cell hybridisation analysis of FA cell lines has demonstrated the existence of at least five complementation groups (FA-A to FA-E), the most common of which is FA-A. This genetic heterogeneity has been a major obstacle to the positional cloning of FA genes by classical linkage analysis. The FAC gene was cloned by functional complementation, and localised to chromosome 9q22.3 (ref. 2), but this approach has thus far failed to yield the genes for the other complementation groups. We have established a panel of families classified as FA-A by complementation analysis, and used them to search for the FAA gene by linkage analysis. We excluded the previous assignment by linkage of an FA gene to chromosome 20q, and obtained conclusive evidence for linkage of FAA to microsatellite markers on chromosome 16q24.3. Strong evidence of allelic association with the disease was detected with the marker D16S303 in the Afrikaner population of South Africa, indicating the presence of a founder effect.

Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.

Subunits of the translation initiation factor eIF2B are mutant in leukoencephalopathy with vanishing white matter.

Leukoencephalopathy with vanishing white matter (VWM) is an inherited brain disease that occurs mainly in children. The course is chronic-progressive with additional episodes of rapid deterioration following febrile infection or minor head trauma. We have identified mutations in EIF2B5 and EIF2B2, encoding the epsilon- and beta-subunits of the translation initiation factor eIF2B and located on chromosomes 3q27 and 14q24, respectively, as causing VWM. We found 16 different mutations in EIF2B5 in 29 patients from 23 families. We also found two distantly related individuals who were homozygous with respect to a missense mutation in EIF2B2, affecting a conserved amino acid. Three other patients also had mutations in EIF2B2. As eIF2B has an essential role in the regulation of translation under different conditions, including stress, this may explain the rapid deterioration of people with VWM under stress. Mutant translation initiation factors have not previously been implicated in disease.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Fanconi anemia A due to a novel frameshift mutation in hotspot motifs: lack of FANCA protein.

Homozygosity for a frameshift mutation at codon 1213 of FANCA gene was identified in a Turkish patient. Immunoprecipitation-western blot analysis showed the complete absence of the FANCA protein band. This novel mutation, a deletion of T at position 3639 in exon 37 (3639delT), is responsible for the disease and causes premature termination of translation 32 aa downstream. The deletion is (i) the T residue of 2 overlapping TGAGGC and CCTG hot spot motifs, (ii) flanked by several direct repeats, (iii) surrounded by the highly GC rich region that have frequently been identified at the site of human DNA deletions. The patient is the third living child of a first degree cousin marriage. The major abnormalities of the patient at the age of 6 months were growth retardation, microcephaly, hypoplastic right thumb, distal displacements of both thumbs and pelvic displacement of left kidney. Hematological presentation of the disease started before the age of 4 years.

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).

Cloning and sequencing of rhesus monkey pepsinogen A cDNA.

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Identification of a Glu greater than Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.

The influence of omeprazole on the synthesis and secretion of pepsinogen in isolated rabbit gastric glands.

Regulation mechanisms of pepsinogen (EC 3.4.23.) synthesis and secretion were studied by following newly synthesized [14C]-labeled pepsinogen during culture of isolated rabbit gastric glands. Omeprazole, a substituted benzimidazole, while almost completely abolishing acid production at 10(-4) M, strongly stimulated secretion of preformed and newly synthesized pepsinogen. Although the pepsinogen synthesis at this concentration of omeprazole was reduced to about 55% of the control rate, a two-fold absolute increase of total secreted pepsinogen was found. This increase was not due to a non specific leakage through disruption of chief cell membranes, as no increase of lactate dehydrogenase in the culture medium could be demonstrated. The stimulated secretion was influenced neither by 10(-3) M cimetidine, 10(-3) sodium thiocyanate nor 10(-4) M atropine. No additivity was found between the carbachol (10(-4) M) or dibutyryl cyclic AMP (10(-3) M) and the omeprazole induced pepsinogen secretion.

Linkage studies in autosomal dominant facioscapulohumeral muscular dystrophy.

Linkage studies were undertaken in 120 individuals from 10 kindreds with autosomal dominant facioscapulohumeral muscular dystrophy using 35 different marker genes. No linkage was found. The highest lod score was 1.438 for the immunoglobulin heavy chain gene cluster (IGH) at a recombination fraction of 0.2. IGH is located on the long arm of chromosome 14. Based on scores of other marker genes and on a recombination map of chromosome 14, the probability that the gene for facioscapulohumeral muscular dystrophy is located on chromosome 14 is estimated to be approximately 6%.

[From gene to disease; a defect in the regulation of protein production leading to vanishing white matter]. / Van gen naar ziekte; een stoornis in de regulatie van de eiwitproductie leidend tot 'vanishing white matter'.

Leukoencephalopathy with vanishing white matter (VWM) is a newly defined autosomal recessive disorder. The clinical course is chronically progressive with additional episodes of rapid deterioration, provoked by fever and minor head trauma. We recently identified the five genes associated with VWM: EIF2B1-5. They encode the five subunits of eIF2B, which is a eukaryotic translation initiation factor expressed in all human tissues and highly conserved during evolution. eIF2B has a key role in the regulation of protein synthesis. It is the most important factor to down-regulate protein synthesis during mild temperature stress, when a decrease in protein synthesis is necessary to prevent proteins, not protected by heat shock proteins, from coagulating. Most mutations found in the eIF2B genes are 'mild' and lead to the substitution of a single amino acid. Major rearrangements are only found in the heterozygous state with an amino acid substitution as second mutation. It is likely that the presence of two mutations, which lead to a total loss of one subunit, is not viable. Two founder effects were observed in the Dutch population. One concerned EIF2B5 and was observed in the region of Zwolle; the second was observed in the region of Weert and concerned EIF2B2. The diagnosis of VWM is based on typical MRI findings. DNA analysis is possible and will be limited to cases in which MRI findings are suggestive of VWM. Prenatal diagnosis is an option in the families in which the responsible mutations have been identified.

Vanishing white matter disease in a child presenting with ataxia.

Vanishing white matter disease is a recently described leukoencephalopathy that is characterized by chronic and episodic neurological deterioration. These episodes often follow periods of fever or minor head trauma. It frequently presents in childhood with problems of ataxia and tremor. Five genes have been identified for the disease, EIF2B1-5, which encode the five subunits of translation initiation factor eIF2B. Mutations in each of the genes may independently cause the disease. The defect in eIF2B results in abnormalities in translation and its regulation, leading to abnormalities in protein synthesis and its regulation. Magnetic resonance imaging of the brain reveals extensive cerebral white matter abnormalities with evidence of white matter rarefaction and cystic degeneration, which has been confirmed pathologically. We report the first confirmed Australasian patient.

Vacuolating megalencephalic leukoencephalopathy: 24 year follow-up of two siblings.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disorder with a chronic progressive course. The gene, MLC1, has been localized on chromosome 22qtell and 26 different mutations have been described. We report two siblings of non-consanguineous parents who presented with characteristic features of MLC. They showed macrocephaly from the first months of life. After a short time, motor clumsiness, ataxia, seizures and psychomotor retardation were observed. During childhood, both patients had a coma that lasted several days following a minor head trauma. The eldest sister experienced a permanent deterioration of the clinical picture after the coma. Epilepsy and electroencephalographic alterations were chronic, tending to improve during adulthood. Cerebral biopsy showed normal or minor changes in the cortical grey matter, and in the white matter gliosis, increased extracellular spaces and decreased numbers of fibres with thin myelin sheets. We have followed the patients during 24 years, from the ages of 4 and 8 years to the their present ages of 28 and 32 years. Clinical and neuro-imaging follow-up showed a chronic course with more prominent progression of the white matter abnormalities than of the neurological features. A homozygous mutation of the MLC1 gene was found in both siblings. The eldest patient, 32 years-old, needs a wheel-chair but has a good contact with the family and surrounding people. The youngest, 28-years-old, shows mild ataxia, spasticity and motor clumsiness, but she is able to participate in activities of daily life.