Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (mucopolysaccharidosis Type II) patients. Mutations in brief no. 202. Online

Hunter syndrome is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS deficiency can be caused by several different types of mutations in the IDS gene. We have performed a molecular and mutation analysis of a total 19 unrelated MPS II patients of different ethnic origin and identified 19 different IDS mutations, 9 of which were novel and unique. SSCP analysis followed by DNA sequencing revealed four novel missense mutations: S143F, associated with the 562C-->T polymorphism, C184W, D269V and Y348H. Two novel nonsense mutations were found: Y103X (433C-->A) and Y234X (826C-->G). In two patients two novel minor insertions (42linsA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466).     

Mutation analysis of 19 North American mucopolysaccharidosis type I patients: Identification of two additional frequent mutations

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive genetic disorder caused by deficiency of the lysosomal glycosidase α-L-iduronidase. Patients with this disorder present with varied clinical phenotypes ranging from early severe onset of disease and death in early childhood to mild manifestations compatible with adult life. An understanding of the molecular basis of iduronidase deficiency and its correlation to clinical phenotype will improve prognostic prediction at diagnosis, aid in genetic counselling of families, and provide a framework to more accurately assess experimental treatment protocols. We have used the approach of single-strand conformational polymorphism analysis and direct sequencing of the α-L-iduronidase gene in an attempt to define the molecular basis of iduronidase deficiency in affected individuals. An initial series of 19 patients representing 35 independently segregating mutant alleles were studied. In addition to five previously identified mutations (W402X, Q70X, E274X, H82P, and P533R) two novel mutations (A75T and 474-2a→g) were found. These seven mutations account for 71% of the mutant alleles and 53% of the genotypes in this group of patients. Analysis of a larger independently ascertained group of 103 MPS I patients, mainly of Northern European origin, revealed that together the two novel mutations account for 7% of mutant alleles and are associated with severe clinical phenotypes. These mutations are the most frequent MPS I mutations detected so far after W402X and Q70X. With the definition of these two mutations, a clear picture of the molecular heterogeneity of MPS I is emerging. © 1994 Wiley-Liss, Inc.     

Identification of 6 new mutations in the iduronate sulfatase gene

Mucopolysaccharidosis type II (Hunter syndrome) is an X‐linked lysosomal storage disorder caused by a deficiency of the enzyme iduronate‐2‐sulfatase. We sequenced genomic DNA and RT‐PCR products in the iduronate sulfatase (IDS) gene in 6 unrelated patients with Hunter syndrome to assess genotype / phenotype relationships and offer carrier testing where required. Six novel mutations were identified: four missense mutations, one four‐base pair deletion (596‐599delAACA) and a cryptic splice site mutation. Three of the missense mutations were significant amino acid substitutions (S143F, S491F, E341K) of which the latter two involve amino acids conserved amongst sulfatase enzymes. The patients identified with these mutations all had a severe clinical phenotype. One missense mutation with a minimal amino acid substitution (H342Y), in a non‐conserved region of the gene, was associated with a mild clinical phenotype. We identified a novel cryptic splice site (IVS5+934G>A) with some normal (wild type) mRNA processing. We predict that the normal mRNA product confered some residual functional enzyme, resulting in a mild phenotype associated with the absence of overt central nervous system disease. © 1999 Wiley‐Liss, Inc.     

Molecular analysis of Turkish mucopolysaccharidosis IVA (Morquio A) patients: identification of novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4). The deficiency of N-acetylgalactosamine-6-sulfate sulfatase leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate. Mutation screening of the GALNS gene was performed by SSCP and direct sequence analyses using genomic DNA samples from 10 Morquio A patients. By nonradioactive SSCP screening, 6 different gene mutations and 2 polymorphisms were identified in 10 severely affected MPS IVA patients. Five of the mutations and one of the polymorphisms are novel. The vast majority of the gene alterations were found to be single nucleotide deletions (389delG, 929delG, and 763delT) or insertions (1232-1233insT). The other two mutations were one previously identified missense mutation (Q473X) and one novel nonsense (P179S) mutation. Together they account for 95% of the disease alleles of the patients investigated. Beside mutations, one previously identified E477 polymorphism and one novel W520 polymorphism were found among Turkish MPS IVA patients.     

Two cases of steroid sulfatase deficiency with complex phenotype due to contiguous gene deletions

We report contiguous gene deletions in the distal short arm of the X chromosome in two patients with ichthyosis, due to steroid sulfatase deficiency, and other complex phenotypes. One patient had chondrodysplasia punctata (CDP) and ichthyosis with a normal chromosome constitution. Another patient had a CDP-like phenotype, ichthyosis, and hypogonadism. His karyotype was 46, -X,Y, +der(X)t(X;Y)(p22;q11). DNA from the two patients was analyzed by Southern blotting using cloned fragments mapped in the Xp21-Xpter region to investigate gene deletions. DNA from the patient with CDP showed a gene deletion of the STS, DXS31, and DXS89 loci, and DNA from the patient with X-Y translocation lacked fragments of the STS, DXS31, DXS89, and DXS143 loci. These findings suggest that the common deleted region involving the STS locus might have caused the similar phenotypes in both patients.     

Metachromatic leukodystrophy: Identification of the first deletion in exon 1 and nine novel point mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459+1G→A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459+1G→a, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene. Hum Mutat 9:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Identification of 6 new mutations in the iduronate sulfatase gene. Mutation in brief no. 233. Online

Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase. We sequenced genomic DNA and RT-PCR products in the iduronate sulfatase (IDS) gene in 6 unrelated patients with Hunter syndrome to assess genotype/phenotype relationships and offer carrier testing where required. Six novel mutations were identified: four missense mutations, one four-base pair deletion (596-599delAACA) and a cryptic splice site mutation. Three of the missense mutations were significant amino acid substitutions (S143F, S491F, E341K) of which the latter two involve amino acids conserved amongst sulfatase enzymes. The patients identified with these mutations all had a severe clinical phenotype. One missense mutation with a minimal amino acid substitution (H342Y), in a non-conserved region of the gene, was associated with a mild clinical phenotype. We identified a novel cryptic splice site (IVS5+934G>A) with some normal (wild type) mRNA processing. We predict that the normal mRNA product confered some residual functional enzyme, resulting in a mild phenotype associated with the absence of overt central nervous system disease.     

Identification of 12 novel mutations and two new polymorphisms in the arylsulfatase A gene: Haplotype and genotype–phenotype correlation studies in spanish metachromatic leukodystrophy patients

Arylsulfatase A (ARSA) deficiency is the main cause of metachromatic leukodystrophy (MLD), a lysosomal disorder with no specific treatment. In view of the importance of genetic counseling, analyses of mutations and polymorphisms, including the ARSA pseudodeficiency allele, were carried out in 18 unrelated Spanish MLD patients. A systematic search allowed us to identify 100% of the alleles involving 17 different mutations, 12 of which are novel: G32S, L68P, R84W, P94A, G99V, P136S, W193X, H227Y, R288H, G308D, T327I, and IVS6‐12C→G. Two new polymorphisms, 2033C>T and 2059C>T, were identified in intron 6 which, in combination with two polymorphisms previously described (2161C>G and 2213C>G), gave rise to four different haplotypes in the control population. In addition, we also studied polymorphism 842G>T. Linkage disequilibrium was detected between mutations IVS2+1G→A, D255H, and T327I and specific haplotypes, suggesting a unique origin for these mutations. Moreover, mutation T327I was always associated with the T allele of the new rare variant A210A (893C>T). The distribution of mutation D255H (frequency 19.4%) among patients with different MLD clinical presentation revealed a clear genotype–phenotype correlation paralleling that reported for mutation IVS2+1G→A (frequency 25%). Among the novel mutations, only P136S and R288H occurred on a background of the ARSA pseudodeficiency allele. Screening 182 normal chromosomes identified a frequency of 8.8% of this allele; moreover, we identified two unrelated subjects with the polyA‐ mutation in the absence of the N350S mutation, and this infrequent haplotype reinforced the heterogeneity of conditions with ARSA deficiency. Hum Mutat 14:240–248, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.     

Identification of 12 novel mutations in the alpha-N-acetylglucosaminidase gene in 14 patients with Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB).

Sanfilippo syndrome type B or mucopolysaccharidosis type IIIB (MPS IIIB) is one of a group of lysosomal storage disorders that are characterised by the inability to breakdown heparan sulphate. In MPS IIIB, there is a deficiency in the enzyme alpha-N-acetylglucosaminidase (NAGLU) and early clinical symptoms include aggressive behaviour and hyperactivity followed by progressive mental retardation. The disease is autosomal recessive and the gene for NAGLU, which is situated on chromosome 17q21, is approximately 8.5 kb in length and contains six exons. Primers were designed to amplify the entire coding region and intron/exon boundaries of the NAGLU gene in 10 fragments. The PCR products were analysed for sequence changes using SSCP analysis and fluorescent DNA sequencing technology. Sixteen different putative mutations were detected in DNA from 14 MPS IIIB patients, 12 of which have not been found previously. The mutations include four deletions (219-237del19, 334-358del25, 1335delC, 2099delA), two insertions (1447-1448insT, 1932-1933insGCTAC), two nonsense mutations (R297X, R626X), and eight missense mutations (F48C, Y140C, R234C, W268R, P521L, R565W, L591P, E705K). In this study, the Y140C, R297X, and R626X mutations were all found in more than one patient and together accounted for 25% of mutant alleles.     

Multipoint linkage of 9 anonymous probes to HPRT, factor 9, and fragile X

We have analyzed the segregation of restriction fragment length polymorphisms (RFLPs) associated with 9 anonymous probes detecting loci DXS10, DXS15, DXS19, DXS37, DXS51, DXS52, DXS98, DXS99, and DXS100 and probes for HPRT and F9 in a set of 40 families segregating fragile X (fra(X]. Using two-point and multipoint analysis, we have established their relative genetic locations. The results indicate that DXS99 and DXS10, unlike previous reports, are not tightly linked to F9. A new locus was found to map within the F9 - fra(X) region. DXS98 showed 6% recombination with fra(X) and appeared to be the closest locus to fra(X). These results will be useful for mapping the relative position of newly defined X probes in this region and for future genetic studies of families with fra(X), hemophilia B, or Lesch-Nyhan mutations.     

Seven novel mutations in the ORNT1 gene (SLC25A15) in patients with hyperornithinemia,hyperammonemia, and homocitrullinuria syndrome

Eight unrelated Italian patients with the hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome were analyzed for mutations in the ORNT1 gene. Seven novel mutations were identified (Q89X, G27R, G190D, R275Q, c.861insG, c.164insA, and IVS5+1G→A). Other previously described variants were a heterozygous deletion of a phenylalanine residue (F188del) in one allele and the R179X in two. The G27R mutation was carried by two patients. Analyses of ORNT1 mRNA in four patients showed that mutant alleles were stable and of the predicted size. The current study expands the spectrum of mutations in ORNT1 gene. © Wiley‐Liss, Inc.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.     

Identification of the molecular defects in Spanish and Argentinian mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) patients, including 9 novel mutations

Maroteaux-Lamy syndrome, or mucopolysaccharidosis VI (MPS VI), is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase or arylsulfatase B (ARSB). We aimed to analyze the spectrum of mutations responsible for the disorder in Spanish and Argentinian patients, not previously studied. We identified all the ARSB mutant alleles, nine of them novel, in 12 Spanish and 4 Argentinian patients. The new changes were as follows: six missense mutations: c.245T>G [p.L82R], c.413A>G [p.Y138C], c.719C>T [p.S240F], c.922G>A [p.G308R], c.1340G>T [p.C447F] and c.1415T>C [p.L472P]; one nonsense mutation: c.966G>A [p.W322X]; and two intronic changes involving splice sites: c.1142+2T>A, in the donor splice site of intron 5, which promotes skipping of exon 5, and c.1143-1G>C, which disrupts the acceptor site of intron 5, resulting in skipping of exon 6. We also report 10 previously described mutations as well as several non-pathogenic polymorphisms. Haplotype analysis indicated a common origin for most of the mutations found more than once. Most of the patients were compound heterozygotes, whereas only four of them were homozygous. These observations confirm the broad allelic heterogeneity of the disease, with 19 different mutations in 16 patients. However, the two most frequent mutations, c.1143-1G>C and c.1143-8T>G, present in both populations, accounted for one-third of the mutant alleles in this group of patients.     

Mutation 1091delC is highly prevalent in Spanish Sanfilippo syndrome type A patients

The gene resposible for Sanfilippo syndrome type A, a lysosomal disorder caused by deficiency of sulfamidase, was recently cloned and more than 40 mutations were identified. This paper presents the mutation analysis and clinical findings in 11 Spanish patients in whom 19 of the 22 mutant alleles have been identified. This is the first report on mutations in Spanish Sanfilippo A patients. Seven different mutations were found, four of which (Q85R, R206P, A354P, and L386R) were not previously described. Mutation 1091del C was the most prevalent, accounting for nearly one-half of the mutated alleles, while mutations R245H and R74C were not found. Haplotype analysis suggests a founder effect as the cause of the high frequency of 1091del C in this population. Hum Mutat 12:274–279, 1998. © 1998 Wiley-Liss, Inc.     

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 β-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. Hum Mutat 11:295–305, 1998. © 1998 Wiley-Liss, Inc.