Clare E. Beesley

Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients.

In this study we have investigated a group of 29 Brazilian patients, who had been diagnosed with the lysosomal storage disorder, Mucopolysaccharidosis type I (MPS-I). MPS I is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase. Ninety percent of the MPS I patients in this study were genotyped and revealed 10 recurrent and thirteen novel IDUA gene mutations. Eight of these new mutations and three common mutations W402X, P533R, and R383H were individually expressed in CHO-K1 cells and analyzed for alpha-L-iduronidase protein and enzyme activity. A correlation was observed between the MPS I patient clinical phenotype and the associated mutant alpha-L-iduronidase protein/enzyme activity expressed in CHO-K1 cells. This was the first time that Brazilian MPS I patients had been thoroughly analyzed and highlighted the difficulties of mutation screening and clinical phenotype assessment in populations with high numbers of unique mutations.

Genotypes and phenotypes of patients in the UK with carbohydrate-deficient glycoprotein syndrome type 1.

Abstract18 UK patients (14 families) have been diagnosed with the carbohydrate-deficient glycoprotein syndrome (CDGS), type 1, on the basis of their clinical symptoms and/or abnormal electrophoretic patterns of serum transferrin. Eleven out of the 16 infants died before the age of 2 years. Patients from 12 families had a typical type 1 transferrin profile but one had a variant profile and another, who had many of the clinical features of CDGS type 1, had a normal profile. Eleven of the patients (10 families) with the typical type 1 profile had a deficiency of phosphomannomutase (PMM), (CDGS type 1a) but there was no correlation between residual enzyme activity and severity of disease. All these patients were compound heterozygotes for mutations in the phosphomannomutase (PMM2) gene, with 7 out of the 10 families having the common R141H mutation. Eight different mutations were found, including three novel ones. There was no correlation between genotype and phenotype, although siblings had similar phenotypes. Three patients, including the one with the normal transferrin profile, did not have a deficiency of phosphomannomutase or phosphomannose isomerase (CDGS 1b).

Mutations in TMEM76* Cause Mucopolysaccharidosis IIIC (Sanfilippo C Syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

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.

Molecular defects in Sanfilippo syndrome type B (mucopolysaccharidosis IIIB)

SummarySanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by the deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU). NAGLU is involved in the degradation of the glycosaminoglycan (GAG) heparan sulphate, and a deficiency results in the accumulation of partially degraded GAGs inside lysosomes. Early clinical symptoms include hyperactivity, aggressiveness and delayed development, followed by progressive mental deterioration, although there are a small number of late-onset attenuated cases. The gene for NAGLU has been fully characterized and we report the molecular analysis of 18 Sanfilippo B families. In total, 34 of the 36 mutant alleles were characterized in this study and 20 different mutations were identified including 8 novel changes (R38W, V77G, 407–410del4, 703delT, A246P, Y335C, 1487delT, E639X). The four novel missense mutations were transiently expressed in Chinese hamster ovary cells and all were shown to decrease the NAGLU activity markedly, although A246P did produce 12.7% residual enzyme activity.

Mutational analysis of Sanfilippo syndrome type A (MPS IIIA): identification of 13 novel mutations

Editor—Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) encompasses a group of four lysosomal storage disorders resulting from a failure to break down the glycosaminoglycan heparan sulphate. Each of the four subtypes, A, B, C, and D, is caused by the deficiency of a different enzyme in the degradative pathway of heparan sulphate: heparan-N-sulphatase (EC 3.10.1.1), α-N-acetylglucosaminidase (EC3.2.1.50), acetyl-CoA N-acetyl transferase (EC 2.3.1.3), and N-acetylglucosamine-6-sulphatase (EC 3.1.6.14), respectively.1 Clinical symptoms usually occur after two years of apparently normal development and include hyperactivity, aggressive behaviour, delayed development (particularly in speech), sleep disturbances, coarse hair, hirsutism, and diarrhoea. There are only relatively mild somatic manifestations. There then follows a period of progressive mental retardation with death usually between the second and third decade of life. In a small number of patients with Sanfilippo syndrome type B, there is a more slowly progressive form of the disease with later onset known as the attenuated phenotype.2-4 A late onset phenotype has also been described for Sanfilippo syndrome type A.5 Sanfilippo syndrome type A (MPS IIIA) is caused by a deficiency in the enzyme heparan-N-sulphatase (sulphamidase). The disease is autosomal recessive and the gene encoding the enzyme is situated on chromosome 17q25.3, contains eight exons, and encodes a protein of 502 amino acids.6 7 To date, 46 different mutations have been identified in Sanfilippo A patients,6 8-13 several of which have been found at high frequencies in particular populations. The R245H, R74C, 1091delC, and S66W were the most frequent mutations in the Dutch (56.7%),11 Polish (56%),8 Spanish (45.5%),13 and Italian (33%)12 populations, respectively. Several polymorphisms have been identified in the sulphamidase gene including R456H, which has a high frequency of 55% in the normal Australian population.9 In this study, mutational analysis has …

Sanfilippo syndrome type D: identification of the first mutation in the N-acetylglucosamine-6-sulphatase gene

Mucopolysaccharidosis type IIID is the least common of the four subtypes of Sanfilippo syndrome. It is caused by a deficiency of N-acetylglucosamine-6-sulphatase, which is one of the enzymes involved in the catabolism of heparan sulphate. We present the clinical, biochemical, and, for the first time, the molecular diagnosis of a patient with Sanfilippo D disease. The patient was found to be homozygous for a single base pair deletion (c1169delA), which will cause a frameshift and premature termination of the protein. Accurate carrier detection is now available for other members of this consanguineous family.

Mucopolysaccharidosis Type I in 21 Czech and Slovak Patients: Mutation Analysis Suggests a Functional Importance of C-Terminus of the IDUA Protein

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α‐L‐iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C‐terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C‐terminus of the IDUA protein.

Autophagic vacuolar myopathy in twin girls

Hereditary autophagic vacuolar myopathy (AVM) may occur in several diseases including the rimmed vacuolar myopathies, acid maltase deficiency, Danon disease, infantile autophagic vacuolar myopathy and X‐linked myopathy with excessive autophagy (XMEA). In the latter three conditions the vacuoles are lined by membranes with sarcolemmal features. We present two unusual cases of autophagic vacuolar myopathy in twin girls born at term with no family history of neurological disease. After initial normal developmental milestones they developed progressive leg weakness and wasting with contractures from the age of 12 years. Investigations showed raised CK, normal female karyotype, normal acid maltase activity, normal nerve conduction and myopathic EMG features. Frozen sections of skeletal muscle were stained using routine tinctorial and histochemical methods. Immunohistochemical staining for spectrin, merosin, dystrophin, complement membrane attack complex and sarcoglycans was performed and ultrastructural examination undertaken. Direct sequence analysis of the lamp‐2 gene using genomic DNA extracted from lymphocytes was performed. Histological analysis of the muscle biopsies demonstrated myofibres with vacuoles lacking glycogen and lipid many of which were delineated using immunohistochemistry for merosin, dystrophin and sarcoglycans. Ultrastructural examination showed duplication of the myofibre basal lamina with associated autophagic material. Vacuoles within myofibres were either membrane bound containing autophagic material or lined by plasma membrane and basal lamina. Intermyofibrillar glycogen was increased. Sequence analysis of the coding region and intron/exon boundaries of the lamp‐2 gene was normal. This is the first report of female cases of AVM with sarcolemmal features. We suggest that these patients may represent manifesting carriers of XMEA, or alternatively, a new form of disease with a similar phenotype having autosomal recessive inheritance.

Advantages and pitfalls of an extended gene panel for investigating complex neurometabolic phenotypes

Targeted gene panels can be used to establish molecular diagnoses in paediatric cohorts. Reid et al. report that this approach is accurate, efficient and can be preferred to whole-exome or genome sequencing for patients with neurological symptomatology and clues suggestive of an inherited metabolic disorder.