Mutations of the iduronate-2-sulfatase (IDS) gene in patients with hunter syndrome (mucopolysaccharidosis II)

Genomic DNA and cDNA from fibroblasts from nine unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency showing variable clinical manifestation were screened for point mutations and small structural aberrations. Direct sequencing revealed a splice mutation skipping exon A, one nonsense mutation, and five missense mutations concerning the exons B, F and I of the IDS gene. Several novel missense mutations were found: A68E, S426X, I485R, Q293H, and D478G. One of the point mutations eliminating a recognition site for the restriction enzyme MspI was used as a direct marker for a prenatal diagnosis. A relationship between type of mutation and clinical picture could not be recognized. © 1994 Wiley-Liss, Inc.     

Molecular diagnostics of 15 hemophilia A patients: Characterization of eight novel mutations in the factor VIII gene,two of which result in exon skipping

The X-linked bleeding disorder hemophilia A is caused by mutations in the coagulation factor VIII gene. A high frequency of de novo mutations and the large size of this gene complicate the molecular diagnostic of hemophilia A. Characterization of mutations, however, may help identify amino acids or regions with essential functional or structural properties and thereby clarify the mechanism of pathogenesis. In the present study, we describe the identification of 15 mutations in the factor VIII gene, of which eight are novel. Among the patients with severe hemophilia A, two splice mutations (IVS5-3 and IVS19-2), a 4-bp deletion (TACA) at codon 1215, and a missense mutation G1850V have been characterized. The missense mutations G479R, R531C, V537D, N2129S and I2190N were found for five patients with a moderate course of hemophilia A disease. A silent mutation resulting in activation of a cryptic acceptor splice site within exon 11 and four other missense mutations Y114C, R1689H, R2150H (2x), M2164V have been identified for six patients with mild hemophilia A. Hum Mutat 12:301–303, 1998.© 1998 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 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).     

Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1.

Subcortical band heterotopia (SBH) are bilateral and symmetric ribbons of gray matter found in the central white matter between the cortex and the ventricular surface, which comprises the less severe end of the lissencephaly (agyria-pachygyria-band) spectrum of malformations. Mutations in DCX (also known as XLIS ) have previously been described in females with SBH. We have now identified mutations in either the DCX or LIS1 gene in three of 11 boys studied, demonstrating for the first time that mutations of either DCX or LIS1 can cause SBH or mixed pachygyria-SBH (PCH-SBH) in males. All three changes detected are missense mutations, predicted to be of germline origin. They include a missense mutation in exon 4 of DCX in a boy with PCH-SBH (R78H), a different missense mutation in exon 4 of DCX in a boy with mild SBH and in his mildly affected mother (R89G) and a missense mutation in exon 6 of LIS1 in a boy with SBH (S169P). The missense mutations probably account for the less severe brain malformations, although other patients with missense mutations in the same exons have had diffuse lissencephaly. Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. However, we expect that somatic mosaic mutations of both LIS1 and DCX will also prove to be an important mechanism in causing SBH in males.     

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.     

Identification of 11 novel mutations in 49 Korean patients with mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a rare lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS). As MPS II is X-linked, patients are usually males with heterogeneous mutations ranging from point mutations to gross deletions and recombination. In 2003, we reported a mutation analysis of 25 patients with MPS II. In this study, 31 mutations in another 49 Korean patients (45 families) with MPS II are reported: 12 missense, nine deletions, four splicing, two nonsense, two insertions, one deletion/insertion, and IDS-IDS2 recombination mutations. Among these mutations, 11 were novel ones (4 missense mutations: Ser61Pro, Pro97Arg, Pro228Ala, and Pro261Ala; 5 deletions: c.344delA, c.420delG, c.768delT, c.1112delC and c.1402delC; 1 deletion/insertion: c.1222delinsTA; and 1 insertion mutation: c.359_360insATCC). The IDS-IDS2 recombination mutations were most frequently observed; all patients with this mutation had the severe MPS II phenotype. However, most of the patients (5/7) with the G374G splicing mutation had an attenuated phenotype, except for two sibling cases with the severe phenotype. Except for a few recurrent mutations such as the G374G, R443X, L522P, and recombination mutations, each patient had a unique individual mutation. Therefore, careful interpretation of genotype-phenotype correlations is warranted.     

Mutational‐screening in the factor VIII gene resulting in the identification of three novel mutations,one of which is a donor splice mutation

Hemophilia A is an X‐linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1‐26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the a ®c transversion at position 3 of the donor‐splice‐site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A. Hum Mutat 13:504, 1999. © 1999 Wiley‐Liss, Inc.     

Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German L?nder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).     

Identification of 9 novel IDS gene mutations in 19 unrelated Hunter syndrome (Mucopolysaccharidosis type II) patients

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 of 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 (421insA and 499insA) were identified. In one patient a complete IDS deletion was found, extending from locus DXS1185 to locus DXS466. © 1998 Wiley-Liss, Inc.     

Mutations of the human tyrosinase gene associated with tyrosinase related oculocutaneous albinism (OCA1). Mutations in brief no. 204. Online

Mutations in the human tyrosinase gene produce tyrosinase-related oculocutaneous albinism (OCA1, MIM #203100). Tyrosinase is a copper containing enzyme and is responsible for catalyzing the rate limiting step in melanin biosynthesis, the hydroxylation of tyrosine to dopaquinone. We report 13 new mutations in the tyrosinase gene associated with OCA1A (without pigment) and OCA1B (with pigment) including 9 missense mutations (H19Q, R521, R77C, G97R, C289R, L312V, P313R, F340L and H404P), two nonsense mutations (W80X and R116X) and two frameshift mutations (53delG and 223 delG). Our previous work has defined clusters of missense mutations that appear to represent functional domains of the enzyme, and three of the missense mutations fall into these clusters including two (F340L and H404P) that flank the copper B bindng site and the missense mutation R52I that is located in the amino terminal end cluster of the protein. The G97R missense mutation is the first identified within the epidermal growth factor (EGF)-like sequence and the H19Q missense mutation alters the cleavage site of the signal peptide sequence. Mutational analysis can provide a definitive diagnosis of the type of OCA as well as help structure/function analysis.     

Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria.

Mutation analysis was performed on DNA samples of 965 individuals from four different ethnic groups in South Africa, in an attempt to determine the spectrum of sequence variants in the haemochromatosis ( HFE ) gene. This population screening approach, utilizing a combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) method, revealed three previously described and four novel missense mutations. Novel variants V53M and V59M were identified in exon 2, Q127H in exon 3 and R330M in exon 5. The exon 5 variant was identified in one of 13 patients referred for a molecular diagnosis of hereditary haemochromatosis (HH), who tested negative for the known C282Y and H63D mutations. Mutation Q127H was detected in exon 3 of the HFE gene together with mutation H63D in an apparently severely affected patient previously shown to carry the protoporphyrinogen oxidase ( PPOX ) gene mutation R59W, which accounts for dominantly inherited variegate porphyria (VP) in >80% of affected South Africans. The mutant allele frequency of the C282Y mutation was found to be significantly lower in 73 apparently unrelated VP patients with the R59W mutation than in 102 controls drawn from the same population ( P = 0.005). The population screening approach used in this study revealed considerable genotypic variation in the HFE gene and supports previous data on the involvement of this gene in the porphyria phenotype.     

Mutation screening of the fibrillin-1 (FBN1) gene in 76 unrelated patients with Marfan syndrome or Marfanoid features leads to the identification of 11 novel and three previously reported mutations

Mutations in the gene encoding fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study we performed SSCP to analyze all 65 exons of the FBN1 gene in 76 patients presenting with classical MFS or related phenotypes. We report 7 missense mutations, 3 splice site alterations, one indel mutation, one nonsense mutation and two mutations causing frameshifts: a 16bp deletion and a single nucleotide insertion. 5 of the missense mutations (Y1101C, C1806Y, T1908I, G1919D, C2251R) occur in calcium-binding Epidermal Growth Factor-like (EGFcb) domains of exons 26, 43, 46 and 55, respectively. One missense mutation (V449I) substitutes a valine residue in the non-calcium-binding epidermal growth factor like domain (EGFncb) of exon 11. One missense mutation (G880S) affects the "hybrid" motif in exon 21 by replacing glycine to serine. The 3 splice site mutations detected are: IVS1-1G>A in intron 1, IVS38-1G>A in intron 38 and IVS46+5G>A in intron 46. C628delinsK was identified in exon 15 leading to the substitution of a conserved cysteine residue. Furthermore two frameshift mutations were found in exon 15 (1904-1919del ) and exon 63 (8025insC) leading to premature termination codons (PTCs) in exon 17 and 64 respectively. Finally we identified a nonsense mutation (R429X) located in the proline rich domain in exon 10 of the FBN1 gene. Y1101C, IVS46+5G>A and R429X have been reported before.     

Identification of novel mutations in the MTM1 gene causing severe and mild forms of X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a congenital muscular disease characterized by severe hypotonia and generalized muscle weakness, leading in most cases to early postnatal death. The gene responsible for the disease, MTM1, encodes a dual specificity phosphatase, named myotubularin, which is highly conserved throughout evolution. To date, 139 MTM1 mutations in independent patients have been reported, corresponding to 93 different mutations. In this report we describe the identification of 21 mutations (14 novel) in XLMTM patients. Seventeen mutations are associated with a severe phenotype in males, with death occurring mainly before the first year of life. However, four mutations-three missense (R241C, I225T, and novel mutation P179S) and one single-amino acid deletion (G294del)-were found in patients with a much milder phenotype. These patients, while having a severe hypotonia at birth, are still alive at the age of 4, 7, 13, and 15 years, respectively, and display mild to moderate muscle weakness.     

Identification of four novel mutations of the XLRS1 gene in Japanese patients with X-linked juvenile retinoschisis. Mutation in brief no. 234. Online

The XLRS1 gene (HUGO-approved symbol, RS1) has been found to cause X-linked recessive retinoschisis (RS) which is characterized by splitting of the superficial layer of the retina. Recent mutation analysis of this gene revealed 82 different mutations in 214 patients with RS. We have now identified 10 mutations of the XLRS1 gene in 11 unrelated Japanese males with RS. Mutations found in these patients were; 1) a 20-kb deletion in exon 1 region; 2) mutations in the initiation sequence (M1V); 3) mutations in the splice donor site (IVS1 + 1 g-->a); 4) two nonsense mutations (Q88X, W163X); and 5) five missense mutations (E72K, Y89C, R182C, G109E, P203L). Four (M1V, Q88X, G109E, and W163X) of the 10 mutations were novel. The R182C mutation was identified in 2 unrelated patients. The 3 mutations found between exons 1 and 3 cause premature translation termination in the XLRS1 protein. The rest of the 7 mutations were clustered between exons 4 and 6. This region of the protein is homologous to the proteins implicated in cell-cell adhesion.     

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine- to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.      

Glycogen storage disease type IIIa: first report of a causative missense mutation (G1448R) of the glycogen debranching enzyme gene found in a homozygous patient

Several different mutations in the glycogen-debranching enzyme gene AGL have been found in patients with glycogen storage disease type III (GSD III) to date, but no missense mutations have been reported for GSD III, only nonsense, splicing, and deletion/insertion lesions. Here we describe a novel G1448R missense mutation in a Japanese GSD IIIa patient from a consanguineous family. Sequence analysis of cDNA from the patient' liver specimen revealed two separate nucleotide changes: a G-to-A transition at nucleotide 3737 in exon 26 (3737G>A) and a G-to-C transversion at nucleotide 4742 in exon 33 (4742G>C), both of which result in substitution of glycine by arginine (G1115R and G1448R). Because homo-zygotes for G1115R were found in healthy controls, G1115R seems to be a polymorphism. Restriction fragment length polymorphism analysis with Bsa JI showed that the patient was homozygous for G1448R and that none of the normal controls had the mutation. This missense mutation is located at a putative glycogen-binding site that is indispensable for enzyme activity. Thus, G1448R is likely to be the causative mutation in this patient. This is the first report of a missense mutation associated with GSD III.     

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.     

Differences in methylation patterns in the methylation boundary region of IDS gene in Hunter syndrome patients: implications for CpG hot spot mutations

Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.