Niemann-Pick type C disease: a novel NPC1 mutation segregating in a Greek island.

Niemann-Pick type C (NPC) disease is a rare autosomal recessive lysosomal storage disease, exhibiting an extremely heterogeneous clinical phenotype. It is a cellular lipid trafficking disorder characterized by the accumulation in the lysosomal/late endosomal system of a variety of lipids, especially unesterified cholesterol. So far two genes, NPC1 or NPC2, have been linked to the disorder. It is a panethnic disease for which two isolates have been described. We present a novel NPC1 mutation (p.A1132P; c.3394G>C) identified in homozygosity in two patients originating from the same small town of an Aegean Sea island and the results of the broad screening of their extended families. Overall 153 individuals have so far been investigated and a total of 64 carriers were identified. Moreover a common descent of the individuals tested was revealed and all carriers could be traced back to a common surname, apparently originating from a common ancestor couple six generations back. The mutation was found associated with an uncommon haplotype in the island that is also present in other populations.

Molecular analysis of 30 Niemann-Pick type C patients from Spain.

Mutations in the NPC1 or NPC2 gene are responsible for Niemann-Pick type C (NPC) disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by an incorrect regulation of intracellular lipid trafficking. A molecular analysis carried out in 30 unrelated patients identified 43 distinct mutations in the NPC1 gene, 12 of which had not been previously described. The novel NPC1 alleles were four amino acid substitutions (p.F995L, p.F1079S, p.L1106P and p.G1209E), a nonsense mutation (p.E1089X), a 1-bp insertion (p.L1117PfsX4), an in-frame deletion (p.N916del), four intronic changes (c.58-3280C>G, c.882-28A>T, c.2604+5G>A and c.3591+5G>A) that affect the splicing mechanism, and the first deletion including the whole gene described in NPC disease. In all the splice site mutations, the formation of abnormal spliced transcripts was confirmed by cDNA analysis, and mRNA degradation by the nonsense-mediated mRNA decay process was also assessed. As it has been previously reported in this disease, genotype-phenotype correlations are limited due to the large number of private mutations. We describe for the first time one homozygous patient for p.I1061T mutation, who presented the severe infantile clinical onset, and another patient with the variant biochemical phenotype, whose clinical presentation was the neonatal form of the disease.

Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles.

The Sanfilippo syndrome type C [mucopolysaccharidosis IIIC (MPS IIIC)] is caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. We report the first molecular study on several Spanish Sanfilippo syndrome type C patients. Seven Spanish patients, one Argentinean and three Moroccan patients were analysed. All mutant alleles were identified and comprised nine distinct mutant alleles, seven of which were novel, including four missense mutations (p.A54V, p.L113P, p.G424V and p.L445P) and three splicing mutations due to two point mutations (c.633+1G>A and c.1378-1G>A) and an intronic deletion (c.821-31_821-13del). Furthermore, we found a new single nucleotide polymorphism (SNP) (c.564-98T>C). The two most frequent changes were the previously described c.372-2A>G and c.234+1G>A mutations. All five splicing mutations were experimentally confirmed by studies at the RNA level, and a minigene experiment was carried out in one case for which no fibroblasts were available. Expression assays allowed us to show the pathogenic effect of the four novel missense mutations and to confirm that the already known c.710C>A (p.P237Q) is a non-pathogenic SNP. Haplotype analyses suggested that the two mutations (c.234+1G>A and c.372-2A>G) that were present in more than one patient have a common origin, including one (c.234+1G>A) that was found in Spanish and Moroccan patients.

Spectrum of ABCA4 (ABCR) gene mutations in Spanish patients with autosomal recessive macular dystrophies.

The ABCA4 gene has been involved in several forms of inherited macular dystrophy. In order to further characterize the complex genotype-phenotype relationships involving this gene, we have performed a mutation analysis of ABCA4 in 14 Spanish patients comprising eight STGD (Stargardt), four FFM (fundus flavimaculatus), and two CRD (Cone-rod dystrophy) patients. SSCP (single-strand conformation polymorphism) analysis and DNA sequencing of the coding and 5' upstream regions of this gene allowed the identification of 16 putatively pathogenic alterations, nine of which are novel. Most of these were missense changes, and no patient was found to carry two null alleles. Overall, the new data agree with a working model relating the different pathogenic phenotypes to the severity of the mutations. When considering the information presented here together with that of previous reports, a picture of the geographic distribution of three particular mutations emerges. The R212C change has been found in French, Italian, Dutch, German, and Spanish but not in British patients. In the Spanish collection, R212C was found in a CRD patient, indicating that it may be a rather severe change. In contrast, c.2588G>C, a very common mild allele in the Dutch population, is rarely found in Southern Europe. Interestingly, the c.2588G>C mutation has been found in a double mutant allele together with the missense R1055W. Finally, the newly described L1940P was found in two unrelated Spanish patients, and may be a moderate to severe allele.

Two new mild homozygous mutations in Gaucher disease patients: clinical signs and biochemical analyses.

Gaucher disease (GD) is a lysosomal storage disorder resulting from impaired activity of lysosomal beta-glucocerebrosidase. More than 60 mutations have been described in the GBA gene. They have been classified as lethal, severe, and mild on the basis of the corresponding phenotype. The fact that most GD patients are compound heterozygous and that most type 1 patients bear the N370S allele, which by itself causes a mild phenotype, make it difficult to correlate the clinical signs with the mutations. Besides N370S, about 10 mild mutations have been described, but only one undoubtedly classified as mild was found at homozygosity. Here we report 2 novel mutations, I402T and V375L, at homozygosity in 2 adult Italian type 1 GD patients. Some properties of the I402T fibroblast enzyme have been compared to those of the enzyme from cells of several N370S/N370S patients. Analysis of the catalytic properties and heat stability as well as the response to phosphatidylserine and sphingolipid activator protein indicate a marked similarity between the 2 enzymes. The finding of another, unrelated patient bearing the I402T mutation (in this case as a compound heterozygote with mutation N370S) suggests that this allele might be quite frequent in the area of Sicily from where both patients originated. In conclusion, the phenotypic expression in the 2 homozygous patients presented here and the biochemical data for one of them allowed the classification of these mutations as mild thus extending the group of mild mutations found at homozygosity.

Mutation and haplotype analyses in 26 Spanish Sanfilippo syndrome type A patients: possible single origin for 1091delC mutation.

Mucopolysaccharidosis IIIA, also known as Sanfilippo syndrome type A, is an autosomal recessive storage disorder caused by deficiency of sulfamidase. The disease results in severe central nervous system degeneration often with mild somatic features that may delay the clinical diagnosis. Molecular analyses would allow early and unequivocal heterozygote detection, providing a useful tool for genetic counselling. About 40 mutations have been reported in the sulfamidase gene, with a very uneven distribution in different patient populations. We have previously described the high prevalence of mutation 1091delC in a small number of Spanish Sanfilippo A patients. The aim of the present work is to extend the mutational study to a total of 26 unrelated patients and perform haplotype analysis in order to study the origin of some mutations. The whole coding region of the gene was scanned by SSCP analysis and sequencing. This allowed the identification of 14 different mutations, corresponding to 90% of the mutant alleles. Seven of these mutations were only found in this Spanish group of patients, three of which, R150W, R433Q and R433W, are described here for the first time. We have also analyzed four internal polymorphisms and constructed the corresponding haplotypes. Chromosomes bearing mutation 1091delC show a conserved haplotype suggesting a common origin for this mutation. Moreover, all other mutations found twice or more also have conserved haplotypes for those polymorphic markers.

Mutation analysis of Gaucher disease patients from Argentina: high prevalence of the RecNciI mutation.

Gaucher disease (GD) is caused by a deficiency of beta-glucocerebrosidase activity mainly due to mutations in the gene coding for the enzyme. More than 100 mutations have been identified to date and their frequencies have been established in several populations, including Ashkenazi Jews, among whom the disease is particularly prevalent. In order to study the molecular pathology of the disease in patients from Argentina, we conducted a systematic search for mutations in the glucocerebrosidase gene. Genomic DNA from 31 unrelated GD patients was screened for seven previously described mutations: N370S (1226A-->G), L444P (1448T-->C), D409H (1342G-->C), R463C (1504C-->T), 1263de155, RecNciI, and RecTL. This allowed the identification of 77.4% of the GD alleles: N370S and RecNciI were the most prevalent mutations found (46.8% and 21% respectively). Southern analysis demonstrated three distinct patterns for the RecNciI alleles. In order to identify the remaining alleles, the full coding region of the gene, all the splice sites, and part of the promoter region were analyzed by single-strand conformational polymorphism analysis (SSCP) after polymerase chain reaction amplification. This extensive screening allowed the identification of 13 different mutations, accounting for 93% of the total number of GD alleles. Three novel missense mutations, I161S (599T-->G), G265D (911G-->A), and F411I (1348T-->A), were detected. Twelve polymorphic sites within the glucocerebrosidase gene are in complete linkage disequilibrium and define two major haplotypes, "-" and "+". Mutation N370S was always associated with the "-" haplotype, as described in other populations. Interestingly, the RecNciI alleles with the same Southern-blot pattern were always associated with the same haplotype.

Identification of a novel pseudodeficiency allele in the GLB1 gene in a carrier of GM1 gangliosidosis.

The term 'pseudodeficiency' is used in lysosomal storage diseases to denote the situation in which individuals show greatly reduced enzyme activity but remain clinically healthy. Pseudodeficiencies have been reported for several lysosomal hydrolases. GM1 gangliosidosis is a rare autosomal recessive lysosomal storage disorder caused by beta-galactosidase hydrolase deficiency as a result of mutations in the GLB1 gene. Until now, two variants altering the beta-galactosidase activity have been described, p.Arg521Cys and p.Ser532Gly. Here we report the new variant p.Arg595Trp in the GLB1 gene, which markedly reduces beta-galactosidase activity when expressed in COS-1 cells. The variant was identified in the healthy father of a girl with GM1 gangliosidosis. He was a heterozygous compound with p.Arg595Trp in trans with one of the disease-causing mutations identified in his daughter; in leukocytes and plasma he showed lower beta-galactosidase activity than that observed in GM1 gangliosidosis carriers. As this family originated from the Basque Country in the north of Spain, we decided to analyse individuals of Basque and non-Basque origin, finding the p.Arg595Trp allele in 3.2% of Basque and in 0.8% of non-Basque alleles. The detection of the presence of alterations resulting in pseudodeficient activity in leukocytes and plasma is important for the correct diagnosis of GM1 gangliosidosis.

Identification of 14 novel GLB1 mutations, including five deletions, in 19 patients with GM1 gangliosidosis from South America.

GM1 gangliosidosis is a lysosomal storage disorder caused by the absence or reduction of lysosomal beta-galactosidase activity because of mutations in the GLB1 gene. Three major clinical forms have been established: type I (infantile), type II (late infantile/juvenile) and type III (adult). A mutational analysis was performed in 19 patients with GM1 gangliosidosis from South America, mainly from Argentina. Two of them were of Gypsy origin. Main clinical findings of the patients are presented. All 38 mutant alleles were identified: of the 22 different mutations found, 14 mutations are described here for the first time. Among the novel mutations, five deletions were found. Four of them are relatively small (c.435_440delTCT, c.845_846delC, c.1131_1145del15 and c.1706_1707delC), while the other one is a deletion of 1529 nucleotides that includes exon 5 and is caused by an unequal crossover between intronic Alu sequences. All the described patients with GM1 gangliosidosis were affected by the infantile form, except for four unrelated patients classified as type II, III, and II/III (two cases). The two type II/III patients bore the previously described p.R201H mutation, while the adult patient bore the new p.L155R. The juvenile patient bore two novel mutations: p.S434L and p.G554E. The two Gypsy patients are homozygous for the p.R59H mutation as are all Gypsy patients previously genotyped.

Stabilisation of a yeast artificial chromosome containing plant DNA using a recombination-deficient host.

The large genomes of many plant species contain numerous dispersed repeat sequences. The problem of large-scale structural instability due to the presence of such repeats in yeast artificial chromosome (YAC) clones was assessed. The feasibility of stabilising plant sequences prone to rearrangement in YACs was demonstrated using a host yeast strain deficient in recombination.

Alcohol dehydrogenase from Drosophila funebris and Drosophila immigrans: molecular and evolutionary aspects.

Alcohol dehydrogenase from Drosophila funebris and D. immigrans is evident at all developmental stages. The highest activity level appears in third-instar larvae and declines to a lower level at all later stages of development. Both species are monomorphic. The enzyme is a dimer consisting of two identical subunits with molecular weight 27,600. The pI values are 8.6 for D. funebris and 9.02 for D. immigrans. The optimum pH is 8.6 and 8.7 for D. funebris and D. immigrans, respectively. The Km values for NAD+, propan-2-ol, and butan-2-ol are 0.15, 2.90, and 2.08 mM, respectively, for D. funebris and 0.16, 1.53, and 1.49 mM, respectively, for D. immigrans. The half-life for the purified enzyme is 45 days for D. funebris and 18 days for D. immigrans at 4 degrees C. Data on the amino acid composition of both enzymes and peptide maps of alcohol dehydrogenase of D. immigrans reveal that they have marked homologies between them and also with alcohol dehydrogenases of other species. D. funebris shows reduced levels of alcohol dehydrogenase synthesis but has the highest specific activity reported to date for a Drosophila species. D. immigrans synthesises six times more enzyme but the specific activity is comparable to that of other species of Drosophila. This evidence could explain their different alcohol tolerance. The molecular properties of these alcohol dehydrogenases together with other species of Drosophila suggest that the alcohol dehydrogenase of Drosophila has arisen by divergent evolution from a common ancestral gene.

Metabolic response to ethanol and isopropanol in D. funebris and D. immigrans.

Ethanol at low concentration increases larval viability in D. funebris and D. melanogaster and decreases it in D. immigrans. Isopropanol decreases viability in all three species. Isopropanol depresses alcohol dehydrogenase activity. This is accompanied by a change in the relative proportion of electrophoretic forms of the enzyme. Acetone appears in the medium when larvae of D. funebris and D. immigrans develop in the presence of isopropanol. It can be transformed again into isopropanol on reaching a threshold value. It seems that this transformation cannot be carried out by alcohol dehydrogenase alone.

Functional and biochemical features of alcohol dehydrogenase in four species of the obscura group of Drosophila.

The biochemical features of ADH of four Drosophila species of the obscura group have been studied. The relationship between ethanol tolerance and ADH activity has been investigated. Propan-2-ol and acetone concentrations have been determined in propan-2-ol treated flies and ADH activity has been followed during 96 h of propan-2-ol treatment. Data on the ADH system confirm constructed phylogenies based on electrophoretic variation and chromosome homologies.

A new gene-pseudogene fusion allele due to a recombination in intron 2 of the glucocerebrosidase gene causes Gaucher disease.

Gaucher disease is the most prevalent sphingolipid storage disorder in humans caused by a recessively inherited deficiency of the enzyme glucocerebrosidase. More than 100 mutations have been described in the glucocerebrosidase gene causing Gaucher disease. Some of them are complex alleles with several mutations due to recombination events between the gene and its highly homologous pseudogene. The generation of these recombinant alleles involves, in most cases, a crossover in the 3' end of the gene, beyond exon 8. However, in a few cases recombination took place in a more upstream location. Here we describe the analysis of a patient with type I Gaucher disease who bears a new complex allele. This allele was originated by a crossover between the gene and the pseudogene at intron 2, the most upstream recombination site described so far, which gave rise to a fusion gene. The patient was first diagnosed as homozygous for the c.1226 A --> G (N370S) mutation but the early onset of the disease prompted us to perform parental DNA analysis which showed that the mother was not a N370S carrier, suggesting deletion of at least part of the gene. Molecular analysis of the complex allele was carried out by Southern blot, PCR, and sequencing. We were able to close down the region of the recombination event to an interval of 18 nucleotides, corresponding to the last 15 nucleotides of intron 2 and the first 3 nucleotides of exon 3 of the gene. These 18 nucleotides are identical between the gene and pseudogene making any further refinement impossible. An exhaustive list of published glucocerebrosidase complex alleles, describing their recombination points, is included for comparison.

The Adh in Drosophila: chromosomal location and restriction analysis in species with different phylogenetic relationships.

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

Linkage disequilibrium detected between myotonic dystrophy and the anonymous marker D19S63 in the Spanish population.

We used the following polymorphic markers: APOC2 (BanI, BglI, TaqI), CKMM (NcoI, TaqI), and D19S63 (PstI) to haplotype 33 Spanish myotonic dystrophy (DM) families. We analysed the allele and haplotype frequencies of our sample, and the possible association of alleles or haplotypes with the disease. We found a slight linkage disequilibrium between APOC2 (BanI) and DM, but no disequilibrium when using all other APOC2 and CKMM RFLPs; this agrees with data previously reported. In addition, we found a very strong linkage disequilibrium when using D19S63 (PstI), the + allele being associated with the DM locus. This disequilibrium in the Spanish population indicates that D19S63 is very close to the DM locus.

Genetic fine localization of the beta-glucocerebrosidase (GBA) and prosaposin (PSAP) genes: implications for Gaucher disease.

Mutations in the glucocerebrosidase (GBA) and prosaposin (PSAP) genes are responsible for Gaucher disease, the most prevalent sphingolipidosis. Somatic cell hybrid analysis and in situ hybridization experiments have localized the GBA gene to 1q21 and the PSAP gene to 10q21-q22. We performed pairwise and multi-point linkage analyses between the two genes and several highly polymorphic markers from the Généthon human linkage map. Our results show that six markers cosegregate with the GBA gene (Zmax = 8.73 at theta = 0.00 for marker D1S2714) and define a 3.2-cM interval between D1S305 and D1S2624 as the most probable location for the gene. Three of these markers (D1S2777, D1S303, and D1S2140), as well as the gene encoding pyruvate kinase (PKLR), are contained in a single YAC clone together with the GBA gene. A new polymorphism was identified within the PSAP gene (C16045T) and used for linkage studies. The multi-point analysis places the gene in a 9.8-cM interval between D10S1688 and D10S607. The fine localization of these genes provides a useful tool for cosegregation analysis, indirect molecular diagnosis, and population genetic studies.

Molecular analysis and clinical findings in the Spanish Gaucher disease population: putative haplotype of the N370S ancestral chromosome.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the lysosomal beta-glucocerebrosidase (GBA) gene. As the disease is particularly prevalent among Ashkenazi Jews, most studies have been carried out on this ethnic group. In the current study, we present a mutation analysis of the GBA gene in Spanish patients together with the clinical findings. We conducted a systematic analysis in 53 unrelated GD patients. The GBA gene was initially scanned for nine previously described mutations by ASO hybridization or restriction analysis after PCR amplification. The remaining unidentified alleles were screened by nonisotopic PCR-SSCP analysis and sequenced. This approach allowed the identification of 101 of 106 GD alleles (95.3%) involving 24 different mutations, 11 of which are described for the first time: G113E (455G-->A), T134P (517A-->C), G389E (1283G-->A), P391L (1289C-->T), N392I (1292A-->T), Y412H (1351T-->G), W(-4)X (108G-->A), Q169X (662C-->T), R257X (886C-->T), 500insT, and IVS5+1G-->T. Most mutations are present in one or few GD chromosomes. However, two mutations, N370S (1226A-->G) and L444P (1448T-->C), are very frequent and account for 66.1% of the total number of alleles. Linkage disequilibrium was detected between these two mutations and an intragenic polymorphism, indicating that expansion of founder alleles occurred in both cases. Analysis of several microsatellite markers close to the GBA gene allowed us to establish the putative haplotype of the ancestral N370S chromosome.