Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each 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.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

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.     

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

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (ARSB) gene. These mutations result in a deficiency of ARSB activity. Ten MPS VI patients were involved in a phase II clinical study 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. Thirteen substitutions (c.215T>G [p.L72R] c.284G>A [p.R95Q], c.305G>A [p.R102H], c.323G>T [p.G108V], c.389C>T [p.P130L], c.511G>A [p.G171S], c.904G>A [p.G302R], c.944G>A [p.R315Q], c.1057T>C [p.W353R], c.1151G>A [p.S384N], c.1178A>C [p.H393P], c.1289A>G [p.H430R] and c.1336G>C [p.G446R]), one deletion (c.238delG), and two intronic mutations (c.1213+5G>A and c.1214-2A>G) were identified. Nine of the 16 mutations identified were novel (R102H, G108V, P130L, G171S, W353R, H430R, G446R, c.1213+5G>A and c.1214-2A>G). The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified in some of the patients, along with the silent mutations c.972A>G and c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient and, together with genotype information, used to predict the expected clinical severity of each patient.     

Molecular analysis and characterization of nine novel CTSK mutations in twelve patients affected by pycnodysostosis. Mutation in brief #961. Online

Molecular characterization of twelve unrelated patients affected by the autosomal recessive osteosclerotic skeletal dysplasia, Pycnodysostosis (cathepsin k deficiency), revealed 11 different genotypes. The mutational profile consisted of 12 different mutations, including nine previously unreported ones, spread throughout the whole gene. One mutation occurred in regions coding predomain, two affected the prodomain and nine others occurred in the mature domain. The novel lesions consisted in six missense mutations c.20T>C (p.L7P), c.494A>G (p.Q165R), c.580G>A (p.G194S), c.746T>C (p.I249T), c.749A>G (p.D250G), c.955G>T (p.G319C), two frameshifts c.60_61dupGA (p.I21RfsX29), c.282dupA (p.S95VfsX9) and a splicing mutation c.890G>A (r.785_890del). The six new missense mutations were examined by western blots of COS-7 cells transfected with mutant CTSK genes. The L7P, occurring within the predicted hydrophobic domain of signal peptide, showed a significantly reduced expression level compared to the wild type control. These findings suggested that the mutation affected targeting and translocation of the nascent lysosomal protein across the endoplasmatic reticulum membrane. The novel amino acid changes were also modeled into the three-dimensional structure that predicted incorrect protein folding for all of them. Molecular characterization of the patients is of particular value for genetic counseling of patients and their families as diagnosis of Pycnodysostosis based on enzyme assay is unpractical and thus not offered routinely.     

Identification of novel MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal, recessively inherited disease caused by mutations in the MLC1 gene. Most of the previously published studies have been carried out in ethnic populations other than the Chinese. In this study, the analysis of clinical features and MLC1 mutation screening were performed in 13 Chinese patients for the first time. A total of 10 MLC1 mutations were identified in these patients, including five novel missense mutations (c.65G>A, p.R22Q; c.95C>T, p.A32V; c.218G>A, p.G73E; c.823G>A, p.A275T; c.832T>C, p.Y278H), one novel splicing mutation (c.772-1G>C in IVS9-1), one novel small deletion (c.907_930del, p.V303_L310del), one known nonsense mutation (c.593delCTCA, p.Y198X) and two known missense mutations (c.206C>T, p.S69L; c.353C>T, p.T118M). Mutation c.772-1G>C in IVS9-1, accounting for 27.3% (3/11) of the total number of genetically confirmed patients found in this study, is thus a putative hot-spot mutation in the present study group. The existence of a unique MLC1 mutation spectrum in Chinese MLC patients was shown. A systemic study to assess the mutation spectra in different populations should be undertaken.     

Liver glycogen storage diseases due to phosphorylase system deficiencies: diagnosis thanks to non invasive blood enzymatic and molecular studies

Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501_502ins361pb, c.528+2T>C, c.856-29_c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573_577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells.     

Molecular analysis in Fabry disease in Spain: fifteen novel GLA mutations and identification of a homozygous female

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A gene (GLA). Here we report molecular studies in 22 unrelated Spanish patients with Fabry disease ( 20 males and two females). Fifteen novel mutations were identified. In addition 7 previously described mutations and two previously reported polymorphisms were detected. The 15 novel mutations comprise: eight missense E48K (c.142G>A), W81S (c.242G>C), D170H (c.508G>C), W226C (c.678G>T), Q279R (c.836A>G), C382Y (c.1145G>A), I407K (c.1220T>A), L414S (c.1241T>C); one nonsense W95X (c.284G>A); one insertion Y216fsX15 (c.646_647insT); two small deletions G346fsX1 (c.1037delG), K426fsX23 (c.1277_1278delAA); one gross deletion comprising exons 5, 6, 7; one complex mutation (insertion and deletion) A368fsX24 (c.1102delGinsTTATAC), and one splice-site mutation IVS4+1G>A (c.639+1G>A). One of the females was found homozygous for Q279R mutation and she presented with the classic phenotype since the age of 8 years, this case extending into women the severe phenotype observed in classically affected males. Mutation analysis provided precise identification for 30 heterozygotes among female relatives and detection of a de novo mutation. The molecular studies on Spanish Fabry patients here reported further contribute to the identification of new mutations in this disease, and allow reliable detection of heterozygotes which has consequences for genetic counselling and for treatment.     

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.     

The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).     

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.     

Molecular and functional characterization of eight novel GAA mutations in Italian infants with Pompe disease

We characterized 29 unrelated patients presenting with the severe form of Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic mutations divided over 28 different genotypes. Among the eight new mutations, five were exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A) leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A comprehensive evaluation, based on different methodological approaches, confirmed the detrimental effect of the eight mutations on the protein and its function. Structural alterations potentially induced by the five missense mutations were also predicted through visual inspection of the atomic model of the GAA protein, in terms of both function and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe disease was already known, our data demonstrate and confirm the power of molecular and functional analysis in predicting the natural course of Pompe disease.     

Kallmann syndrome: 14 novel mutations in KAL1 and FGFR1 (KAL2)

Kallmann syndrome (KAL) combines hypogonadotropic hypogonadism and anosmia. Hypogonadism is due to Gonadotropin Releasing Hormone (GnRH) deficiency and anosmia is related to hypoplasia of the olfactory bulbs. Occasional symptoms include renal agenesis, bimanual synkinesia, cleft lip palate, dental agenesis. KAL is genetically heterogeneous and two genes have so far been identified, namely KAL1 (Xp22.3) and FGFR1/KAL2 (8p12), which underlie the X chromosome‐linked form and an autosomal dominant form of the disease, respectively. We studied a cohort of 98 unrelated Caucasian KAL patients. We identified KAL1 mutations in 14 patients, of which 7 (c.3G>A (p.M1?), g.IVS1+1G>T, c.570_571insA (p.R191fsX14), c.784G>C (p.R262P), c.958G>T (p.E320X), c.1651_1654delinsAGCT (p.P551_E552delinsSX), c.1711T>A (p.W571R)) have not been previously reported. In addition, we found FGFR1 mutations in 7 patients, namely c.303G>A (p.V102I), C.385A>C (p.D129A), c.810G>A (p.V273M), c.1093_1094delAG (p.R365fsX41), c.1561G>A (p.A520T), c.1836_1837insT (p.Y613fsX42), c.2190C>G (p.Y730X), all of which were novel mutations. In this study, unilateral renal agenesis and bimanual synkinesia were exclusively found associated with KAL1mutations, cleft palate and dental agenesia with FGFR1mutations. © 2004 Wiley‐Liss, Inc.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Acid sphingomyelinase: identification of nine novel mutations among Italian Niemann Pick type B patients and characterization of in vivo functional in-frame start codon

Niemann Pick disease (NPD) is an autosomal recessive disorder due to the deficit of lysosomal acid sphingomyelinase, which results in intracellular accumulation of sphingomyelin. In the present work we studied 18 patients with NPD type B, including five individuals who presented an intermediate phenotype characterised by different levels of neurological involvement. We identified nine novel mutations in the SMPD1 gene including six single base changes c.2T>G, c.96G>A, c.308T>C, c.674T>C, c.732G>C, c.841G>A (p.M1_W32del, p.W32X, p.L103P, p.L225P, p.W244C, p.A281T) and three frameshift mutations c.100delC, c.565dupC, c.575dupC (p.G34fsX42, p.P189fsX1 and p.P192fsX14). The novel c.2T>G (p.M1_W32del) mutation inactivates the first in-frame translation start site of the SMPD1 gene and in the homozygous status causes NPD type B indicating that in'vivo translation of wild type SMPD1 initiates from the first in-frame ATG. Moreover, the new c.96G>A (p.W32X) introduces a premature stop codon before the second in-frame ATG. As a consequence of either c.2T>G (p.M1_W32del) or c.96G>A (p.W32X), impaired translation from the first in-frame ATG results in a mild NPD-B phenotype instead of the severe phenotype expected for a complete deficiency of the enzyme, suggesting that when the first ATG is not functional, the second initiation codon (ATG33) still produces a fairly functional sphingomyelinase. Analysis of the patients'clinical and molecular data demonstrated that all five patients with the intermediate phenotype carried at least one severe mutation. No association between the onset of pulmonary symptoms and genotype was observed. Finally, the presence of c.96G>A (p.W32X), the most frequent allele among Italian NPD type B population, and c.1799G>C (p.R600P) as compound heterozygotes in association with severe mutations suggested a beneficial effect for both mutations.     

Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations

In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non-denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild-type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild-type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild-type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non-denaturing PAGE revealed that the wild-type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations.     

Identification of novel mutations in classical galactosemia

Classical galactosemia is an autosomal recessive disorder of galactose metabolism due to galactose-1-phosphate uridyltransferase (GALT) deficiency. Treatment through restriction of dietary galactose intake is lifesaving, but, in spite of this diet, most patients develop abnormalities. In this paper we report the mutational spectrum of classical galactosemia in a cohort of 123 Dutch patients, all with biochemically proven classical galactosemia. In the human GALT gene, which is located on chromosome 9p13, we identified 24 different mutations, including nine mutations that have not been reported previously. The novel mutations include five missense mutations (c.152G>A/p.R51Q, c.404C>T/p.S135W, c.687G>T/p.K229N, c.756G>T/p.Q252H, and c.1140A>C/p.X380C), a frame shift mutation (c.410dupT), a splice site mutation (c.821-2A>G), a possible branch point mutation (c.508-29delT), and a large deletion encompassing at least exons 1-11. Six of these novel mutations were found in patients of Dutch descent: p.R51Q, p.S135W, p.K229N, p.Q252H, p.X380C, and c.410dupT.     

Novel LMNA mutations in patients with Emery‐Dreifuss muscular dystrophy and functional characterization of four LMNA mutations

Mutations in LMNA cause a variety of diseases affecting striated muscle including autosomal Emery‐Dreifuss muscular dystrophy (EDMD), LMNA‐associated congenital muscular dystrophy (L‐CMD), and limb‐girdle muscular dystrophy type 1B (LGMD1B). Here, we describe novel and recurrent LMNA mutations identified in 50 patients from the United States and Canada, which is the first report of the distribution of LMNA mutations from a large cohort outside Europe. This augments the number of LMNA mutations known to cause EDMD by 16.5%, equating to an increase of 5.9% in the total known LMNA mutations. Eight patients presented with either p.R249W/Q or p.E358K mutations and an early onset EDMD phenotype: two mutations recently associated with L‐CMD. Importantly, 15 mutations are novel and include eight missense mutations (p.R189P, p.F206L, p.S268P, p.S295P, p.E361K, p.G449D, p.L454P, and p.W467R), three splice site mutations (c.IVS4 + 1G>A, c.IVS6 ? 2A>G, and c.IVS8 + 1G>A), one duplication/in frame insertion (p.R190dup), one deletion (p.Q355del), and two silent mutations (p.R119R and p.K270K). Analysis of 4 of our lamin A mutations showed that some caused nuclear deformations and lamin B redistribution in a mutation specific manner. Together, this study significantly augments the number of EDMD patients on the database and describes 15 novel mutations that underlie EDMD, which will contribute to establishing genotype–phenotype correlations. Hum Mutat 31:–16, 2011. © 2011 Wiley‐Liss, Inc.