Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic,clinical, and biological implications

Mucopolysaccharidosis (MPS) types IIIA, B, C, and D are a group of autosomal recessive lysosomal storage diseases caused by mutations in one of four genes which encode enzyme activities required for the lysosomal degradation of heparan sulfate. The progressive lysosomal storage of heparan sulfate eventually results in the clinical onset of disease, which is predominantly characterized by severe central nervous system degeneration. MPS‐IIIA and MPS‐IIIB involve deficiencies of heparan sulfate sulfamidase (SGSH) and α‐N‐acetylglucosaminidase (NAGLU), respectively. Both the SGSH and NAGLU genes have been cloned and characterized, thereby permitting mutation analysis of MPS‐IIIA and MPS‐IIIB patients. A total of 62 mutations have now been defined for MPS‐IIIA consisting of 46 missense/nonsense mutations, 15 small insertions/deletions, and one splice site mutation. A total of 86 mutations have been identified in the NAGLU gene of MPS‐IIIB patients; 58 missense/nonsense mutations, 27 insertions/deletions, and one splice site mutation. Most of the identified mutations in the SGSH and NAGLU genes are associated with severe clinical phenotypes. Many of the missense, nonsense, and insertion/deletion mutations have been expressed in mammalian cell lines to permit the characterization of their effects on SGSH and NAGLU activity and intracellular processing and trafficking. For MPS‐IIIA and MPS‐IIIB many of the reported mutations are unique making screening the general population difficult. However, molecular characterization of MPS‐IIIA patients has revealed a high incidence of particular mutations of different geographical origins, which will be beneficial for the molecular diagnosis of MPS‐IIIA. Hum Mutat 18:264–281, 2001. © 2001 Wiley‐Liss, Inc.     

Mutational analysis of the HGSNAT gene in Italian patients with mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis (MPS) describes any inherited lysosomal storage disorder resulting from an inability to catabolize glycosaminoglycans. MPS III (or Sanfilippo syndrome) is an autosomal recessive disease caused by a failure to degrade heparan sulphate. There are four subtypes of MPS III, each categorized by a deficiency in a specific enzyme involved in the heparan sulphate degradation pathway. The genes mutated in three of these (MPS IIIA, MPS IIIB, and MPS IIID) have been cloned for some time. However, only very recently has the gene for MPS IIIC (heparin acetyl CoA: α‐glucosaminide N‐acetyltransferase, or HGSNAT) been identified. Its product (previously termed transmembrane protein 76, or TMEM76) has little sequence similarity to other proteins of known function, although it is well conserved among all species. In this study, a group of MPS IIIC patients, who are mainly of Italian origin, have been clinically characterized. Furthermore, mutational analysis of the HGSNAT gene in these patients resulted in the identification of nine alleles, of which eight are novel. Three splice‐site mutations, three frameshift deletions resulting in premature stop codons, one nonsense mutation, and two missense mutations were identified. The latter are of particular interest as they are located in regions which are predicted to be of functional significance. This research will aid in determining the molecular basis of HGSNAT protein function, and the mechanisms underlying MPS IIIC. © 2007 Wiley‐Liss, Inc.     

Mutational analysis of the HGSNAT gene in Italian patients with mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Mutation in brief #959. Online

Mucopolysaccharidosis (MPS) describes any inherited lysosomal storage disorder resulting from an inability to catabolize glycosaminoglycans. MPS III (or Sanfilippo syndrome) is an autosomal recessive disease caused by a failure to degrade heparan sulphate. There are four subtypes of MPS III, each categorized by a deficiency in a specific enzyme involved in the heparan sulphate degradation pathway. The genes mutated in three of these (MPS IIIA, MPS IIIB, and MPS IIID) have been cloned for some time. However, only very recently has the gene for MPS IIIC (heparin acetyl CoA: alpha-glucosaminide N-acetyltransferase, or HGSNAT) been identified. Its product (previously termed transmembrane protein 76, or TMEM76) has little sequence similarity to other proteins of known function, although it is well conserved among all species. In this study, a group of MPS IIIC patients, who are mainly of Italian origin, have been clinically characterized. Furthermore, mutational analysis of the HGSNAT gene in these patients resulted in the identification of nine alleles, of which eight are novel. Three splice-site mutations, three frameshift deletions resulting in premature stop codons, one nonsense mutation, and two missense mutations were identified. The latter are of particular interest as they are located in regions which are predicted to be of functional significance. This research will aid in determining the molecular basis of HGSNAT protein function, and the mechanisms underlying MPS IIIC.     

Fabry disease: 20 novel GLA mutations in 35 families

Thirty two mutations have been found in 35 unrelated patients of European origin with Fabry disease, including 8 females. Twenty of the mutations are novel and comprise of 13 missense: H46Y, W47G, R49P, C94S, F113S, G258R, P259R, Q279H, Q280H, R363H, A377D, P409A, P409T; 1 nonsense: L294X; 5 small deletions: 154delT, 520delT, 909‐918del10, 1152‐1153delCA, 1235‐1236delCT and 1 splice site mutation: IVS5+2t→c. The remaining 12 mutations have all been reported previously. All patients with deletions had the classic form of the disease but it was not possible to predict the phenotype from the missense mutations. © Wiley‐Liss, Inc.     

Clinical,biochemical, and molecular characterization of mucopolysaccharidosis type III in 34 Egyptian patients

Mucopolysaccharidosis type III (MPS III) is a rare autosomal recessive lysosomal storage disorder characterized by progressive neurocognitive deterioration. There are four MPS III subtypes (A, B, C, and D) that are clinically indistinguishable with variable rates of progression. A retrospective analysis was carried out on 34 patients with MPS III types at Cairo University Children's Hospital. We described the clinical, biochemical, and molecular spectrum of MPS III patients. Of 34 patients, 22 patients had MPS IIIB, 7/34 had MPS IIIC, 4/34 had MPS IIIA, and only 1 had MPS IIID. All patients presented with developmental delay/intellectual disability, and speech delay. Ataxia was reported in a patient with MPS IIIC, and cerebellar atrophy in a patient with MPS IIIA. We reported 25 variants in the 4 MPS III genes, 11 of which were not previously reported. This is the first study to analyze the clinical and genetic spectrum of MPS III patients in Egypt. This study explores the genetic map of MPS III in the Egyptian population. It will pave the way for a national registry for rare diseases in Egypt, a country with a high rate of consanguineous marriage and consequently a high rate of autosomal recessive disorders.     

Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc‐1‐Phosphotransferase

The lysosomal storage disorder ML III γ is caused by defects in the γ subunit of UDP‐GlcNAc:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase, the enzyme that tags lysosomal enzymes with the mannose 6‐phosphate lysosomal targeting signal. In patients with this disorder, most of the newly synthesized lysosomal enzymes are secreted rather than being sorted to lysosomes, resulting in increased levels of these enzymes in the plasma. Several missense mutations in GNPTG, the gene encoding the γ subunit, have been reported in mucolipidosis III γ patients. However, in most cases, the impact of these mutations on γ subunit function has remained unclear. Here, we report that the variants c.316G>A (p.G106S), c.376G>A (p.G126S), and c.425G>A (p.C142Y) cause misfolding of the γ subunit, whereas another variant, c.857C>T (p.T286M), does not appear to alter γ subunit function. The misfolded γ subunits were retained in the ER and failed to rescue the lysosomal targeting of lysosomal acid glycosidases.     

Aberrant splicing at catalytic site as cause of infantile onset glycogen storage disease type II (GSDII): Molecular identification of a novel IVS9 (+2GT→GC) in combination with rare IVS10 (+1GT→CT)

Glycogen storage disease type II (GSDII) results from deleterious mutations in acid α‐glucosidase gene. To date several mutant alleles have been studied including missense and nonsense mutations, insertions, small and large deletions as well as splice site mutations. Apart from IVS1 (? 13→G), 525delT, and Δ18, the other mutations are rare and often unique to single patients. Moreover, the molecular findings also observed in the different ethnic groups makes it difficult to attempt to correlate genotype and phenotype to explain the origin of clinical variability. Even though there are no conclusive genotype phenotype correlations, the in frame splice site mutations identified up until now have been found associated with the juvenile/adult onset of GSDII. In this study we describe a novel in frame splicing defect, IVS9 (+2GT→GC), identified in combination with the rare IVS10 (+1GT→CT) mutation in a patient with classic infantile GSDII disease. Because both mutations occur at the catalytic site region, it is likely that the alteration of both catalytic function and steric conformation of the enzyme may be responsible for the most severe form of the disease. © 2001 Wiley‐Liss, Inc.     

Scanning of estrogen receptor α (ERα) and thyroid hormone receptor α (TRα) genes in patients with psychiatric diseases: Four missense mutations identified in ERα gene

Estrogen and thyroid hormones exert effects on growth, development, and differentiation of the nervous system. Hormone administration can lead to changes in behavior, suggesting that genetic variants of the estrogen receptor α (ERα) and the thyroid hormone receptor α (TRα) genes may predispose to psychiatric diseases. To investigate this possibility, regions of likely functional significance (all coding exons and flanking splice junctions) of the ERα and TRα genes were scanned in patients with schizophrenia (113), along with pilot studies in patients with bipolar illness (BPI), puerperal psychosis, autism, attention‐deficit hyperactivity disorder (ADHD), and alcoholism. A total of 1.18 megabases of the ERα gene and 1.16 megabases of the TRα gene were scanned with Detection of Virtually All Mutations‐SSCP (DOVAM‐S), a method that detects virtually all mutations. Four missense mutations, seven silent mutations and one deletion were identified in the ERα gene, while only four silent mutations were present in the TRα gene. Two of the missense mutations in ERα are conserved in the six available mammalian and bird species (H6Y, K299R) and a third sequence variant (P146Q) is conserved in mammals, birds, and Xenopus laevis, hinting that these sequence changes will be of functional significance. These changes were found in one patient each with BPI, puerperal psychosis, and alcoholism, respectively. Analysis of the ERα and TRα genes in 240 subjects reveals that missense changes and splice site variants are uncommon (1.7% and 0%, respectively). Further analyses are necessary to determine if the missense mutations identified in this study are associated with predisposition or outcome for either psychiatric or nonpsychiatric diseases. © 2001 Wiley‐Liss, Inc.     

αIIbβ3 integrin: new allelic variants in Glanzmann thrombasthenia,effects on ITGA2B and ITGB3 mRNA splicing,expression, and structure–function

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin αIIbβ3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North‐African and Asian were characterized. Promoter and exon sequences of αIIb and β3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The αIIb p.S926L, p.V903F, and β3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS‐7 cells by altering the αIIb or the β3 subunit structure. As shown by free energy analyses applied on the resolved structure of αIIbβ3 and structural modeling of the mutant, the p.K253M substitution of β3 helped to define a key role of the K253 in the interaction of the αIIb β‐propeller and the β3 β‐I domains. finally, the αIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of αIIb for the αIIbβ3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect. Hum Mutat 30:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

Mucopolysaccharidosis I mutations in Chinese patients: identification of 27 novel mutations and 6 cases involving prenatal diagnosis

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease that results from the deficiency of α-l-iduronidase and is transmitted in an autosomally recessive manner. This report describes the first systematic screening for mutations in Chinese MPS I patients from mainland China, wherein we have summarized the phenotype/genotype correlation of the individuals in Chinese MPS I patients. Mutational analyses were performed in 57 unrelated Chinese MPS I patients. Overall, 105 mutant alleles were identified from a set of 41 different mutations. Notably, of these 41 mutations, 27 were novel mutations that consisted of 8 splicing mutations (c.1-2C>G, c.296+4G>A, c.300-1G>C, c.792+1G>C, c.973-4G>A, c.1189+5G>T, c.1402+1G>T and c.1402+2T>G), 1 nonsense mutation (p.W41X), 1 insertion (c.668-670ins GCG), 5 duplications (c.531dupT, c.657dupG, c.883dupC, c.1147dupG and c.1225dupG), 3 deletions (c.349delT, c.1593delG and c.1244-1271del27),1 nucleotide substitution c.2T>C and 8 missense mutations (p.H33P,p.F52L, p.G168V,p.T179R,p. E182D, p.L237R, p.L238R and p.L421P). The missense mutations p.A79V and p.L346R, which accounted for 16.7% (19/114) and 12.3% (14/114) respectively, were the common mutations in Chinese patients but were rare in the mutational profiles reported for other populations. These results indicate that Chinese MPS I patients may have a different mutational spectrum compared to those of other populations. Moreover, for the first time in China, molecular genetic methods were used for prenatal diagnosis of six cases in five families.     

Cystathionine γ-lyase: Clinical, metabolic, genetic, and structural studies

We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine γ-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B₆ administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects.     

Mutations at the ataxia‐telangiectasia locus and clinical phenotypes of A–T patients

Mutations at the ataxia‐telangiectasia (A–T) locus on chromosome band 11q22 cause a distinctive autosomal recessive syndrome in homozygotes and predispose heterozygotes to cancer, ischemic heart disease, and early mortality. PCR amplification from genomic DNA and automated sequencing of the entire coding region (66 exons) and splice junctions detected 77 mutations (85%) in 90 A–T chromosomes. Heteroduplex analysis detected another 42 mutations at the A–T locus. Out of a total of 71 unique mutations, 50 were found only in a single family, and 51 had not been reported previously. Most (58/71, 82%) mutations were frameshift and nonsense mutations that are predicted to cause truncation of the A–T protein; the less common mutation types were missense (9/71, 13%), splicing (3/71, 4%) and one in‐frame deletion, 2546 3 (1/71, 1%). The mean survival and height distribution of 134 A–T patients correlated significantly with the specific mutations present in the patients. Patients homozygous for a single truncating mutation, typically near the N‐terminal end of the gene, or heterozygous for the in‐frame deletion 2546 3, were shorter and had significantly shorter survival than those heterozygous for a splice site or missense mutation, or heterozygous for two truncating mutations. Alterations of the length or amino acid composition of the A–T gene product affect the A–T clinical phenotype in different ways. Mutation analysis at the A–T locus may help estimate the prognosis of A–T patients. Am. J. Med. Genet. 92:170–177, 2000. © 2000 Wiley‐Liss, Inc.     

Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients

Cystinuria is an autosomal recessive disorder that affects luminal transport of cystine and dibasic amino acids in the kidneys and the small intestine. Three subtypes of cystinuria can be defined biochemically, and the classical form (type I) has been associated with mutations in the amino acid transporter gene SLC3A1. The mutations detected in SLC3A1 tend to be population specific and have not been previously investigated in Sweden. We have screened the entire coding sequence and the intron/exon boundaries of the SLC3A1 gene in 53 cystinuria patients by means of single strand conformation polymorphism (SSCP) and DNA sequencing. We identified 12 novel mutations (a 2 bp deletion, one splice site mutation, and 10 missense mutations) and detected another three mutations that were previously reported. Five polymorphisms were also identified, four of which were formerly described. The most frequent mutation in this study was the previously reported M467T and it was also detected in the normal population with an allelic frequency of 0.5%. Thirty‐seven patients were homozygous for mutations in the SLC3A1 gene and another seven were heterozygous which implies that other genes may be involved in cystinuria. Future investigation of the non‐type I cystinuria gene SLC7A9 may complement our results but recent studies also suggest the presence of other potential disease genes. Hum Mutat 18:516–525, 2001. © 2001 Wiley‐Liss, Inc.     

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999. © 1999 Wiley‐Liss, Inc.     

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N‐acetylgalactosamine‐4‐sulfatase

Mucopolysaccharidosis type VI (MPS‐VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N‐acetylgalactosamine‐4‐sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4‐sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS‐VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS‐VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS‐VI alleles will be difficult. Hum Mutat 18:282–295, 2001. © 2001 Wiley‐Liss, Inc.     

Disease‐causing missense mutations within the N‐terminal transmembrane domain of GlcNAc‐1‐phosphotransferase impair endoplasmic reticulum translocation or Golgi retention

Transport of newly synthesized lysosomal enzymes to the lysosome requires tagging of these enzymes with the mannose 6‐phosphate moiety by UDP‐GlcNAc:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase (GlcNAc‐1‐phosphotransferase), encoded by two genes, GNPTAB and GNPTG. GNPTAB encodes the α and β subunits, which are initially synthesized as a single precursor that is cleaved by Site‐1 protease in the Golgi. Mutations in this gene cause the lysosomal storage disorders mucolipidosis II (MLII) and mucolipidosis III αβ (MLIII αβ). Two recent studies have reported the first patient mutations within the N‐terminal transmembrane domain (TMD) of the α subunit of GlcNAc‐1‐phosphotransferase that cause either MLII or MLIII αβ. Here, we demonstrate that two of the MLII missense mutations, c.80T>A (p.Val27Asp) and c.83T>A (p.Val28Asp), prevent the cotranslational insertion of the nascent GlcNAc‐1‐phosphotransferase polypeptide chain into the endoplasmic reticulum. The remaining four mutations, one of which is associated with MLII, c.100G>C (p.Ala34Pro), and the other three with MLIII αβ, c.70T>G (p.Phe24Val), c.77G>A (p.Gly26Asp), and c.107A>C (p.Glu36Pro), impair retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes. Our results uncover the basis for the disease phenotypes of these patient mutations and establish the N‐terminal TMD of GlcNAc‐1‐phosphotransferase as an important determinant of Golgi localization.     

Missense mutation of TRPS1 in a family of tricho‐rhino‐phalangeal syndrome type III

We report a new Japanese family with tricho‐rhino‐phalangeal syndrome type III (TRPS III) who have a missense mutation (Arg908Gln) of theTRPS1 gene (TRPS1) in affected individuals of the family. This study supports the notion that TRPS III results from missense mutations in exon 6 of TRPS1. © 2001 Wiley‐Liss, Inc.     

Identification of 16 sulfamidase gene mutations including the common R74C in patients with mucopolysaccharidosis type IIIA (Sanfilippo A)

Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo A disease) is a storage disorder caused by deficiency of the lysosomal enzyme sulfamidase. Mutation screening, using SSCP/heteroduplex analyses on cDNA and genomic DNA fragments, was performed in a group of 42 European patients. Sixteen of the 17 different gene mutations characterized have not been previously described. The spectrum of gene lesions consists of two 1-bp deletions (1091delC, 1093delG), an 18-bp duplication (421ins18), a splice site mutation (IVS2-2A→G), and 13 different missense point mutations. As in other lysosomal storage disorders, the phenotypic heterogeneity is associated with a considerable genetic heterogeneity. The missense mutation R74C, which alters an evolutionary conserved amino acid in the active site of the enzyme, was found on 56% of alleles of 16 Polish patients, whereas it was less frequent among German patients (21% of disease alleles). R245H, a previously reported common mutation, represents 35% of disease alleles in German patients, but only 3% in Polish patients. As the combined frequency of the common mutations (R74C and R245H) in German and Polish populations exceeds 55%, screening for these two mutations will assist molecular genetic diagnosis of MPS IIIA and allow heterozygote testing in these populations. Hum Mutat 10:479–485, 1997. © 1997 Wiley-Liss, Inc.