Detection of mosaic variants in mothers of MPS II patients by next generation sequencing

Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event

Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications for neonatal screening and diagnostic confirmation of lysosomal disorders

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting.Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS).Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample.Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since its introduction for the neonatal screening of phenylketonuria almost 50 years ago. From that time onwards, the range of its applications has been extended until today, including next-generation sequencing (NGS) for molecular genetics diagnosis. This study aimed to evaluate a standardized organic method to extract DNA from DBS samples, for its use in the diagnostic setting. Methods: The clinical applicability of the method was tested using three samples collected in a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from three 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and its performance was assessed through standard PCR, Restriction Length Polymorphism, Sanger sequencing, and Targeted Next-generation sequencing (TNGS). Results: Results were compared with the ones obtained with DNA samples extracted following the internally validated in-house extraction protocol that used six 3-mm punches of DBS and samples extracted from whole blood. Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to presenting a lower cost per sample

Difficulties in the diagnosis of Gaucher disease in a low-income country : a case report from Mozambique

Introduction: Gaucher disease (GD) is one of the common lysosomal storage disorder (LSD) with an estimated frequency of one in 40,000 newborns globally. GD is an autosomal recessive condition, which results from mutations in the GBA1 gene, causing partial or complete deficiency of β-glucocerebrosidase enzyme activity, which leads to the widespread accumulation of the substrate glucosylceramide. Aims: This report presents different challenges of clinical management and communication between medical specialties to reach diagnose of any rare disease in Mozambique, a low-income country, which health system has limited infrastructure, trained personnel, and budget for diagnosis and to provide treatment for rare genetic disorders such as GD. Case Presentation: The patient was a 15-year old black female patient of Mozambican nationality born from non-consanguineous parents. Three of the four patient’s siblings were healthy; one sister had died of a disease with a similar clinical features. Our patient presented with abdominal distention and hepatosplenomegaly. Blood tests revealed pancytopenia and a high level of ferritin. Liver biopsy and histologic examination revealed infiltration of the splenic parenchyma and portal area of the liver as well as enlarged histiocytic cells with granular cytoplasm. Magnetic resonance imaging showed liver enlargement, changes in the femoral heads without osteonecrosis, a pathological fracture of the third thoracic vertebrae (T3), with absence of brain and spinal cord neurological abnormalities. The biochemical investigation disclosed low levels of β-glucocerebrosidase (0.223 nmol/h/ml; normal: above 0.98) and increased levels of lyso-Gb1 (0.43 µg/ml; normal: up to 0.003). Genotyping of the GBA1 gene indicated the presence of the pathogenic variant p.Arg87Trp (R48W) in homozygosis. Discussion and Conclusion: To the best of our knowledge, this report describes the first case of GD type 1 confirmed via biochemical and molecular genetic testing in Mozambique. As awareness of the GD and rare genetic diseases among Mozambican health professionals is very limited, and resources for diagnosis are scarce in the national health system, it is possible that other cases remain undiagnosed in this low-income country

Fast and robust protocol for prenatal diagnosis of mucopolysaccharidosis Type II

Introduction: Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal disorder caused by deficiency of iduronate-2-sulfatase (IDS). In this study, we proposed a new protocol for prenatal diagnosis, using DNA obtained from amniotic fluid cells that did not attach to the bottom of the culture flask after the first medium change. Methods: Four pregnant MPS II carriers were referred to the Medical Genetics Service of Hospital de Clinicas de Porto Alegre for a prenatal diagnosis and identification of the disease, which were performed by polymerase chain reaction (PCR) amplification, restriction fragment length polymorphism, and sequencing according to the mutation previously found in the family. Results: The analysis indicated the absence of mutation in three fetal materials and the presence of mutation in one case. Concomitantly, cytogenetic and biochemical analyses were performed after 12 days of cell culture, and only one case showed absence of enzyme activity, confirming the molecular analysis. Conclusions: This diagnostic protocol designed to provide more robust results and safer genetic counseling suggests that DNA obtained from floating amniotic fluid cells can be used as a source of fetal material to allow a faster alternative for prenatal care through molecular analysis. Determination of IDS gene mutation in fetal amniotic fluid cells together with IDS enzyme activity testing is a rapid, sensitive and accurate method for prenatal diagnosis of MPS II for high-risk pregnant women