Elisabeth Young

Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3

Two different enzyme preparations are used for the treatment of Fabry disease patients, agalsidase alpha (Replagal, Shire) and agalsidase beta (Fabrazyme, Genzyme). Therapeutic efficacy of both products has been variable probably due to differences in gender, severity, age and other patient characteristics. We studied the occurrence of alpha-Gal A antibodies and their effect on urinary and plasma globotriaosylceramide (GL-3), plasma chitotriosidase and clinical outcome in 52 patients after 12 months of treatment with either 0.2mg/kg agalsidase alppha (10 males, 8 females) or beta (8 males, 5 females) or 1.0mg/kg agalsidase beta (10 males, 11 females). Antibodies were detected in 18/28 male patients after 6 months. None of the females developed antibodies. Following 12 months of 0.2mg/kg treatment, urinary GL-3 decreased in antibody negative (AB-) but increased in antibody positive (AB+) patients. Treatment with 1.0mg/kg gave a reduction in urinary GL-3 in both AB- and AB+ patients. Levels of plasma GL-3 and chitotriosidase decreased in all patient groups. Twelve months of 0.2mg/kg treatment did not change renal function or left ventricular mass. Further, no change in renal function was seen following 1.0mg/kg treatment and left ventricular mass decreased in both AB- and AB+ patients. In summary, alpha-Gal A antibodies frequently develop in male Fabry disease patients and interfere with urinary GL-3 excretion. Infusion of a dose of 1.0mg/kg results in a more robust decline in GL-3, less impact, if any of antibodies, stable renal function and reduction of LVMass.

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.

Prenatal diagnosis of the carbohydrate-deficient glycoprotein syndrome type 1A (CDG1A) by a combination of enzymology and genetic linkage analysis after amniocentesis or chorionic villus sampling

Two pregnancies at risk for the carbohydrate‐deficient glycoprotein syndrome Type 1A (CDG1A, phosphomannomutase deficient) were monitored by enzyme and genetic linkage analyses. The index case in both families had a proven deficiency of phosphomannomutase (PMM). An unaffected fetus was predicted in family 1 following amniocentesis. Normal PMM activity was found in cultured amniotic fluid cells and there was no elevation of lysosomal enzymes in the amniotic fluid. Genetic linkage analysis using microsatellite markers closely linked to the CDG1A gene confirmed this prediction. A healthy child was born. In the second family direct assay of chorionic villi showed a profound deficiency of PMM and genetic linkage analysis showed the fetus to have the same haplotype as the proband. The pregnancy was terminated and a deficiency of PMM was confirmed in cultured fibroblasts from the fetus. Reliable prenatal diagnosis of CDG Type 1A (PMM‐deficient) can be achieved by a combination of biochemical and molecular genetic tests.

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.

Laboratory Diagnosis of Fabry Disease

The definitive diagnosis of Fabry disease in male patients is normally made by demonstrating a deficiency of α-galactosidase A in a blood sample, which may be white blood cells, plasma/serum or a dried blood spot. The diagnosis is confirmed by mutational analysis. The enzymatic assay is unreliable for detecting female carriers, who can only be diagnosed reliably by mutational analysis. The measurement of the storage products, globotriaosylceramide (Gb3) in plasma and urine or globotriaosylsphingosine (lyso-Gb3) in plasma can often provide support for a diagnosis and is useful for monitoring treatment. Methods for mass or high-risk screening have been developed based on measuring the α-galactosidase A activity and/or protein in dried blood spots or the storage products in urine collected on filter paper. In the future the detection of mutations in the α-galactosidase A using high-throughput methods for analysing DNA might be the first step rather than a confirmatory one in the diagnosis of Fabry disease.