Prenatal diagnosis of Gaucher disease using next-generation sequencing.

In the prenatal diagnosis of Gaucher disease (GD), glucocerebrosidase (GBA) activity is measured with fetal cells, and gene analysis is performed when pathogenic mutations in GBA are identified in advance. Herein is described prenatal diagnosis in a family in which two children had GD. Although prior genetic information for this GD family was not obtained, next-generation sequencing (NGS) was carried out for this family because immediate prenatal diagnosis was necessary. Three mutations were identified in this GD family. The father had one mutation in intron 3 (IVS2 + 1), the mother had two mutations in exons 3 (I[-20]V) and 5 (M85T), and child 1 had all three of these mutations; child 3 had none of these mutations. On NGS the present fetus (child 3) was not a carrier of GD-related mutations. NGS may facilitate early detection and treatment before disease onset.

Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations.

BACKGROUND: Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population. METHODS: Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid ß-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease-associated founder mutation-flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods. RESULTS: Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation-flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles. CONCLUSIONS: The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations. FUNDING: SZMC, Protalix Biotherapeutics Inc., and Centogene AG.

Glucosylsphingosine accumulation in mice and patients with type 2 Gaucher disease begins early in gestation.

Gaucher disease, the most common of the sphingolipidoses, results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although type 2 (acute neuronopathic) Gaucher disease is associated with rapidly progressive and fatal neurologic deterioration, the pathophysiologic mechanisms leading to the neurologic symptoms and early demise remain uncharacterized. While the pathology encountered in Gaucher disease has been attributed to glucocerebroside storage, glucosylsphingosine (Glc-sph), a cytotoxic compound, also accumulates in the tissues. Elevations of brain Glc-sph have been reported in patients with types 2 and 3 Gaucher disease. In this study, Glc-sph levels were measured using HPLC in tissues from mice with type 2 Gaucher disease created with a null glucocerebrosidase allele. Compared with unaffected littermates, homozygous mice with type 2 Gaucher disease had approximately a 100-fold elevation of Glc-sph in brain, as well as elevated levels in other tissues. This accumulation was detected in utero by E 13 and increased progressively throughout gestation. Similarly, elevated Glc-sph levels were seen in human fetuses with type 2 Gaucher disease, indicating that therapy initiated after birth may be too late to prevent the sequelae of progressive neurologic damage that begins early in gestation. These findings suggest that the accumulation of Glc-sph may be responsible for the rapid demise of mice with type 2 Gaucher disease and the devastating clinical course seen in patients with type 2 Gaucher disease.

Glucocerebrosidase gene mutations in patients with type 2 Gaucher disease.

Gaucher disease, the most common lysosomal storage disorder, results from the inherited deficiency of the enzyme glucocerebrosidase. Three clinical types are recognized: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. Type 2 Gaucher disease, the rarest type, is progressive and fatal. We have performed molecular analyses of a cohort of 31 patients with type 2 Gaucher disease. The cases studied included fetuses presenting prenatally with hydrops fetalis, infants with the collodion baby phenotype, and infants diagnosed after several months of life. All 62 mutant glucocerebrosidase (GBA) alleles were identified. Thirty-three different mutant alleles were found, including point mutations, splice junction mutations, deletions, fusion alleles and recombinant alleles. Eleven novel mutations were identified in these patients: R131L, H255Q, R285H, S196P, H311R, c.330delA, V398F, F259L, c.533delC, Y304C and A190E. Mutation L444P was found on 25 patient alleles. Southern blots and direct sequencing demonstrated that mutation L444P occurred alone on 9 alleles, with E326K on one allele and as part of a recombinant allele on 15 alleles. There were no homozygotes for point mutation L444P. The recombinant alleles that included L444P resulted from either reciprocal recombination or gene conversion with the nearby glucocerebrosidase pseudogene, and seven different sites of recombination were identified. Homozygosity for a recombinant allele was associated with early lethality. We have also summarized the literature describing mutations associated with type 2 disease, and list 50 different mutations. This report constitutes the most comprehensive molecular study to date of type 2 Gaucher disease, and it demonstrates that there is significant phenotypic and genotypic heterogeneity among patients with type 2 Gaucher disease. Hum Mutat 15:181-188, 2000. Published 2000 Wiley-Liss, Inc.

An autopsy case of fetal Gaucher disease.

BACKGROUND: A case of fetal form of Gaucher disease in a Japanese fetus is presented. RESULTS: A macerated baby showing hydrops fetalis was dissected at 29 weeks of gestation. The fetus was heavier in the body, liver and spleen than a normal fetus at the same gestation period. It also suffered from pericardial effusion and ascites. The diagnosis of Gaucher disease was made by histological and biochemical findings. In microscopical examinations 'Gaucher cells', which were periodic acid-Shiff (PAS)-positive, alcian blue-positive and CD68-positive, existed in the lungs, liver, spleen, thymus, adrenal glands, bone marrow and brain. In thin layer chromatography, a large quantity of glucocerebroside was seen to have accumulated in the patient's organs.

Reliable co-segregation analysis for prenatal diagnosis and heterozygote detection in Gaucher disease.

Mutations in the gene encoding beta-glucocerebrosidase are the main cause of Gaucher disease. The identification of some of these mutations in prenatal tests is a good complement to enzymatic assay and allows diagnosis and, in some cases, prognosis of the disease to be made. DNA analysis is particularly useful for carrier detection since the results of biochemical analyses are often ambiguous. The main drawback of mutation analysis for prenatal diagnosis and carrier detection in Gaucher disease is that rare mutations account for more than 30 per cent of the mutant alleles in most populations. The individual detection of these mutations is too expensive and time-consuming for routine use. Here we present a diagnostic protocol based on co-segregation analysis, using highly polymorphic markers, to be applied when at least one disease allele does not correspond to the most common mutations. Because of the frequency of the N370S mutation and its relevance for prognosis, an improved PCR detection method is included.

Prenatal lethality of a homozygous null mutation in the human glucocerebrosidase gene.

The complete spectrum of clinical phenotypes resulting from glucocerebrosidase deficiency continues to evolve. While most patients with Gaucher disease have residual glucocerebrosidase activity, we describe a fetus with severe prenatal lethal type 2 (acute neuronopathic) Gaucher disease lacking glucocerebrosidase activity. This 22-week fetus was the result of a first cousin marriage and had hydrops, external abnormalities, hepatosplenomegaly, and Gaucher cells in several organs. Fetal fibroblast DNA was screened for common Gaucher mutations, none of which was detected. Southern blot analysis using the restriction enzymes SstII and SspI ruled out a fusion gene, deletion, or duplication of either allele, and quantitative studies of SspI digested genomic DNA indicated that both alleles were present. Northern blot analysis of total RNA from fetal fibroblasts demonstrated no detectable transcription, although RT-PCR successfully amplified several exons, suggesting the presence of a very unstable mRNA. Direct PCR sequencing of all exons demonstrated a homozygous frameshift mutation (deletion of a C) on codon 139 in exon 5, thereby introducing a premature termination codon in exon 6. The absence of glucocerebrosidase protein was confirmed by Western analysis. This unique case confirms the essential role of glucocerebrosidase in human development and, like the null allele Gaucher mouse, demonstrates the lethality of a homozygous null mutation. The presence of this novel mutation and the resulting unstable mRNA accounts for the severity of the phenotype observed in this fetus, and contributes to the understanding of genotype/phenotype correlation in Gaucher disease.

Diagnosing sphingolipidoses in murine and human embryos.

The aim of this study was to diagnose lipid storage diseases in embryos at the preimplantation stage. Two parallel approaches were employed. Firstly, activities of several sphingolipid hydrolases were determined in extracts of murine embryos and also human oocytes and polyspermic embryos. Sensitive fluorescent or fluorogenic procedures provided indications that Tay-Sachs, Gaucher and Krabbe diseases might be diagnosed in one human blastomere, while for Niemann-Pick disease two might be required. Secondly, pyrene lipids were administered into murine embryos and their fluorescence was quantified by computerized imaging microscopy. As a model of Gaucher disease, the fluorescent substrate pyrene glucosylceramide was administered into murine embryos in the presence or absence of an inhibitor of the enzyme beta-glucosidase. Because of decreased degradation of the substrate in enzyme-inhibited cells, the fluorescence per blastomere was considerably greater relative to those which received no inhibitor. The results indicated that lipid storage diseases might be diagnosed in single human blastomeres at the preimplantation stage, obviating the need for pre-natal diagnosis and abortion of affected foetuses.