Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arrays.

Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.

Prevalence of Fabry's disease within hemodialysis patients in Spain.

AIMS: To identify patients with Fabry's disease (FD) within patients with kidney dysfunction, submitted to hemodialysis. SUBJECTS AND METHODS: Patients under hemodialysis were screened using a combined enzymatic α-Gal A assay and dried blood spot samples to determine GLA genotype. RESULTS: A total of 3,650 samples (18.5% of all patients under hemodialysis in Spain) were tested and 11 new unrelated FD patients (4 males and 7 females) were diagnosed. 66 relatives of 11 patients were tested and 23 new FD patients were identified. This study allowed the diagnosis of 34 FD patients. Among the 11 unrelated FD patients, 5 presented the same R118C mutation and one novel mutation was detected: D109G. CONCLUSIONS: This study reveals the need for screening for FD in all patients under hemodialysis for unknown causes and indicates that the incidence and prevalence of FD are underestimated so far.

Twenty-one novel mutations in the GLB1 gene identified in a large group of GM1-gangliosidosis and Morquio B patients: possible common origin for the prevalent p.R59H mutation among gypsies.

GM1-gangliosidosis and Morquio B disease are rare lysosomal storage disorders caused by beta-galactosidase deficiency due to mutations in the GLB1 gene. Three major clinical forms of GM1-gangliosidosis have been established on the basis of age of onset and severity of symptoms: infantile, late infantile/juvenile, and adult. We performed mutation analysis on 30 GM1-gangliosidosis and five Morquio B patients, mainly of Spanish origin, and all the causative mutations were identified. Thirty different mutations were found, 21 of which were novel. With the exception of two adults and one juvenile patient, all the GM1-gangliosidosis patients were affected by the infantile form. Clinical findings are presented for all patients. We report the association of the novel mutations p.T420K and p.L264S with the adult form and the juvenile form, respectively. In addition, the novel mutation p.Y83C was associated with Morquio B disease. Among the 30 GM1-gangliosidosis patients, 6 were of Gypsy origin (Roma). Moreover, those six Gypsy patients shared not only the same mutation (p.R59H) but also a common haplotype. This observation indicates a possible founder effect in this group and suggests that screening of the p.R59H mutation may be appropriate in GM1-gangliosidosis patients of Gypsy origin. This is the first exhaustive mutational analysis performed in a large group of Iberian GM1-gangliosidosis and Morquio B patients.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

Proliferation, activity, and osteogenic differentiation of bone marrow stromal cells cultured on calcium titanium phosphate microspheres.

In this study, the behavior of bone marrow stromal cells cultured on calcium titanium phosphate (CTP) microspheres was analyzed. Cell adhesion and proliferation were estimated by the neutral red assay and by total DNA quantification. Morphology and deposition of extracellular matrix were assessed by confocal laser scanning microscopy and/or scanning electron microscopy. The expression of the osteoblastic phenotype was evaluated by monitoring alkaline phosphatase activity and osteocalcin secretion. Results revealed that cells were able to attach and spread on the surface of CTP microspheres, and gradually grow into nearly confluent monolayers. Moreover, cells were able to bridge adjacent microspheres forming microsphere-cell clusters. Cells produced an abundant amount of fibrillar extracellular matrix that covered the substrate surface. Alkaline phosphatase activity peaked around days 7-14 and then decreased until day 21. Cells secreted osteocalcin, with higher levels being detected at day 14 than at day 21. Taken together, these results suggest that CTP microspheres are appropriate scaffolds for the growth and differentiation of cells along the osteoblastic lineage.