Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.info:eu-repo/semantics/publishedVersio

Update of the spectrum of mucopolysaccharidoses type III in Tunisia: identification of three novel mutations and in silico structural analysis of the missense mutations

BACKGROUND: Mucopolysaccharidoses type III (MPS III) are a group of autosomal recessive lysosomal storage diseases, caused by mutations in genes that code for enzymes involved in the lysosomal degradation of heparan sulphate: heparan sulfate sulfamidase (SGSH), α-N-acetylglucosaminidase (NAGLU), heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetylglucosamine-6-sulfatase (GNS). METHODS: In this study, we have performed the molecular analysis of the SGSH, NAGLU and HGSNAT genes in 10 patients from 6 different MPS III Tunisian families. RESULTS: In the SGSH gene, two mutations were identified: one novel (p.D477N) and one already described (p.Q365X). In the NAGLU gene, two novel mutations were discovered (p.L550P and p.E153X). For the novel missense mutations found in these two genes we performed an in silico structural analysis and the results were consistent with the clinical course of the patients harboring those mutations. Finally, in HGSNAT gene, we found the splicesite mutation c.234+1G>A that had already been reported as relatively frequent in MPS IIIC patients from countries surrounding the basin of the Mediterranean sea. Its presence in two Tunisian MPS IIIC families points to the hypothesis of its peri Mediterranean origin. With the exception of the c.234+1G>A mutation, that was identified in two unrelated MPS IIIC families, the other identified mutations were family-specific and were always found in homozygosity in the patients studied, thus reflecting the existence of consanguinity in MPS III Tunisian families. CONCLUSION: Three novel mutations are reported here, further contributing to the knowledge of the molecular basis of these diseases. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to an improvement in genetic counseling.This study was partially supported by Millennium bcp Foundation (bcp/LIM/DGH/2014). Coutinho MF is grantee from the Foundation for Science and Technology Portugal (SFRH/BPD/101965/2014)info:eu-repo/semantics/publishedVersio

Mucopolysaccharidoses in Tunisia: a molecular portrait of allelic heterogeneity and consanguinity

There are 11 different enzymes involved in the stepwise degradation of glycosaminoglycans (GAGs). Deficiencies in each of those enzymes result in eight different Mucopolysaccharidoses (MPSs), all sharing a series of clinical features, though in variable degrees. Typical symptoms include organomegally, dysostosys multiplex and coarse facies. CNS, hearing, vision and cardiovascular function may also be compromised. Traditionally, MPSs are recognized through analysis of urinary GAGs. Still, initial screenings of urinary GAGs allow discrimination between broad classes of MPSs but cannot distinguish subgroups. In fact, a definitive diagnosis may only be accessed through a combination of enzymatic assays and molecular analyses. Currently, there are countless laboratories in Europe where those biochemical and genetic tests are carried out. Nevertheless, developing countries often lack the necessary resources/expertise for proper diagnosis of rare genetic diseases. Being one of the labs where molecular genetic testing for virtually all MPSs is available for research purposes, we receive several samples from other countries, whose clinicians and/or centers struggle to get a molecular characterization of affected individuals. Here we present our results on the molecular characterization of MPS patients we have been receiving from Tunisia. Nine families suffering from five different diseases were studied so far: 3 MPS II; 2 MPS IIIA; 2 MPS IIIB; 1 MPS IIIC and 1 MPS VI. We have identified 9 different mutations, 5 of which were novel: 1 in the IDS gene (c.1333delC); another in the SGSH gene (p.D477N); 2 in the NAGLU gene (p.L550P and p.E153X) and yet another in the ARSB gene (p.L82P). All detected mutations were further analyzed with the most suitable approaches. Special attention was paid to the novel alterations, particularly to the missense ones, whose impact on protein structure and function was evaluated in silico. In general, there was a strong correlation between the observed clinical phenotype and the genotype assessed through molecular analysis. Also noteworthy is the astonishing level of homozygosity in our sample (100%), with each family presenting its own molecular defect, a pattern consistent with the occurrence of consanguineous matings in Tunisia, where such marriages are thought to provide social, economic and cultural benefits. Altogether, our results provide a preliminary overview of the molecular basis, enzymatic defects and clinical manifestations of MPSs in Tunisia, further supporting previous reports on the high impact of inbreeding and regional endogamy on the occurrence of autosomal recessive disorders in that country. Hopefully, these results will not only contribute to improve genetic counseling for affected families, but also to highlight the need for reinforced and continuous information of general public and health professionals on the potential negative medical impact of intra-family marriages, particularly in Northern Africa, Middle East and South Asia.This work was supported by Fundação Millennium bcp (bcp/LIM/DGH/2014). Coutinho MF is grantee from the FCT (SFRH/BPD/101965/2014)

Genetic Heterogeneity of 72 Patients With Mucopolysaccharidosis In Tunisia

The mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases, presenting with a progressive multisystem disorder, with extreme clinical heterogeneity. The purpose of this study was to investigate six genes involved in different types of mucopolysaccharidosis (MPS), a group of lysosomal storage diseases. Mutation screening was performed for a total of 72 MPS Tunisian patients with MPS I (n=43), MPS II (n=5), MPS IIIA (n=7), MPS IIIB (n=3), MPS IIIC (n=2), or MPS IVA (n=12). The methodological approaches included genomic PCR sequencing, RT-PCR for MPS IVA and tetra-primer ARMS PCR assay for MPSI. The present study revealed one novel splice site mutation in one MPSI patient, in addition to the 11 previously Tunisian mutations reported for the first time by our research team: three in the IDUA gene (MPS I) including two missense, one nonsense four in the SGSH gene (MPS IIIA) including a mutation involving the start codon, one small duplication, one small deletion and a large deletion of exons 1 to 5 two in the NAGLU gene (MPS IIIB) including one missense mutation and one nonsense mutation two in the HGSNAT gene (MPS IIIC) including one missense and one nonsense mutation, two in the GALNS gene including one missense and one splicing mutation. These data demonstrate the remarkable mutational heterogeneity characterizing all types of MPS tested, although high consanguinity rate in the Tunisian population. We report biochemical and molecular aspects of these patients to prevent (e.g. genetic counseling, prenatal diagnosis) the recurrence of affected child since these diseases are still not rare in our area and as specific therapy is not available in our country. nbs