Molecular analysis of Turkish Gaucher disease patients: identification of novel mutations in glucocerebrosidase (GBA) gene

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to autosomal recessive deficiency of acid beta-glucosidase and is characterized by the accumulation of glucocerebroside. In this work we carried out molecular analysis of the glucocerebrosidase gene (GBA) in 57 unrelated patients and the alleleic frequencies of gene mutations in Turkish patients are reported. The most prevalent are L444P and N370S accounting for 42% and 30% in our patients. We identified three novel genetic alterations: two missense changes S356F, L296V that are associated with the severe phenotype of type 1 GD. 303-305delCAC was identified in a homozygous state in one patient type 1 or type 3.     

Functional analysis of 11 novel GBA alleles

Gaucher disease is the most frequent lysosomal storage disorder due to the deficiency of the acid β-glucosidase, encoded by the GBA gene. In this study, we report the structural and functional characterization of 11 novel GBA alleles. Seven single missense alleles, P159S, N188I, E235K, P245T, W312S, S366R and W381C, and two alleles carrying in cis mutations, (N188S; G265R) and (E326K; D380N), were studied for enzyme activity in transiently transfected cells. All mutants were inactive except the P159S, which retained 15% of wild-type activity. To further characterize the alleles carrying two in cis mutations, we expressed constructs bearing singly each mutation. The presence of G265R or D380N mutations completely abolished enzyme activity, while N188S and E326K mutants retained 25 and 54% of wild-type activity, respectively. Two mutations, affecting the acceptor splice site of introns 5 (c.589-1G>A) and 9 (c.1389-1G>A), led to the synthesis of aberrant mRNA. Unpredictably, family studies showed that two alleles resulted from germline or ‘de novo'' mutations. These results strengthen the importance of performing a complete and accurate molecular analysis of the GBA gene in order to avoid misleading conclusions and provide a comprehensive functional analysis of new GBA mutations.     

Glucocerebrosidase pseudogene variation and Gaucher disease: Recognizing pseudogene tracts in GBA alleles

We surveyed the genetic variability of the glucocerebrosidase pseudogene (psGBA) in a worldwide sample of 100 human chromosomes. psGBA is the non-functional duplicate of the gene responsible for Gaucher disease (GBA), the most common lipid storage disorder. The existence of only one psGBA allele described until now, together with the high homology between GBA and psGBA, often prevented recognition of the complex alleles formed by the combination of GBA and psGBA, because psGBA variants could be confused with GBA mutations. In order to determine the variability existent in psGBA, the whole psGBA DNA segment was PCR-amplified and sequenced, and the genotype for all samples was obtained. The ascertainment of the phase among the heterozygous sites was possible through cloning and sequencing a single allele. Eighteen variable sites were detected along psGBA. Two of the variants already have been reported as Gaucher-causing mutations when present in GBA alleles. The other variants were unknown. The knowledge of the psGBA variants described in this report will allow identification of psGBA-GBA complex alleles that may aid in understanding the intricate phenotype-genotype relationship in Gaucher disease.     

Haplotype analysis suggests a single Balkan origin for the Gaucher disease [D409H;H255Q] double mutant allele

Gaucher disease is an autosomal recessive lysosomal storage disease that is mainly due to mutations in the GBA gene. Most of the mutant alleles described so far bear a single mutation. However, there are a few alleles bearing two or more DNA changes. It has been reported that patients homozygous for the [D409H;H255Q] double mutant allele (HGVS-approved nomenclature, p.[D448H;H294Q]) present a more severe phenotype than patients homozygous for the relatively common D409H mutation. In this study, we confirmed the detrimental cumulative effect of these two mutations at the enzymatic activity level by the heterologous expression of the single and double mutant alleles. Additionally, we found a high frequency of the [D409H;H255Q] allele in patients from the Balkans and the Adriatic area of Italy. This prompted us to perform a haplotype analysis, using five microsatellite polymorphisms close to the GBA gene, to determine the origin of this allele. The results of the 37 chromosomes analysed showed that most of them share a common haplotype and are consistent with a single origin in the Balkans and the Adriatic area of Italy for the [D409H;H255Q] allele.     

Revisiting the diagnosis of Gaucher disease in a family with multiple GBA1 variants

Our ability to identify different variants in GBA1, the gene mutated in the lysosomal storage disorder Gaucher disease (GD), has greatly improved. We describe a multigenerational family with type 1 GD initially evaluated over three decades ago. Re-evaluating both the genotype and phenotype, we determined that one family member with genotype N370S/T369M (p.N409S/p.T408M), was likely erroneously diagnosed with GD. This case substantiates that GBA1 variant T369M, while mildly reducing glucocerebrosidase activity, does not result in GD. The observation has clinical relevance as cases with this genotype will increasingly be ascertained through screening programs in newborns and in movement disorder clinics.     

The identification of eight novel glucocerebrosidase (GBA) mutations in patients with Gaucher disease

Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gaucher disease. In this study, seven novel missense mutations in the glucocerebrosidase gene (A136E, H162P, K198E, Y205C, F251L, Q350X and I402F) and a splice site mutation (IVS10+2T-->A) were identified by direct sequencing of three amplified segments of the glucocerebrosidase gene. Five of the novel mutations were found in patients with neuronopathic forms of Gaucher disease, two of which, K198E and F251L, appear to be associated with type 2 Gaucher disease.     

Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA)

Gaucher disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Lysosomal storage of the substrate in cells of the reticuloendothelial system leads to multisystemic manifestations, including involvement of the liver, spleen, bone marrow, lungs, and nervous system. Patients with GD have highly variable presentations and symptoms that, in many cases, do not correlate well with specific genotypes. Almost 300 unique mutations have been reported in the glucocerebrosidase gene (GBA), with a distribution that spans the gene. These include 203 missense mutations, 18 nonsense mutations, 36 small insertions or deletions that lead to either frameshifts or in-frame alterations, 14 splice junction mutations, and 13 complex alleles carrying two or more mutations in cis. Recombination events with a highly homologous pseudogene downstream of the GBA locus also have been identified, resulting from gene conversion, fusion, or duplication. In this review we discuss the spectrum of GBA mutations and their distribution in the patient population, evolutionary conservation, clinical presentations, and how they may affect the structure and function of glucocerebrosidase.     

The GBA p.Trp378Gly mutation is a probable French‐Canadian founder mutation causing Gaucher disease and synucleinopathies

Biallelic GBA mutations cause Gaucher disease (GD), and heterozygous carriers are at risk for synucleinopathies. No founder GBA mutations in French‐Canadians are known. GBA was fully sequenced using targeted next generation and Sanger sequencing in French‐Canadian Parkinson disease (PD) patients (n = 436), rapid eye movement (REM)‐sleep behavior disorder (RBD) patients (n = 189) and controls (n = 891). Haplotype, identity‐by‐descent (IBD) and principal component analyses (PCA) were performed using single nucleotide polymorphism‐chip data. Data on GD patients from Toronto and Montreal were collected from patients' files. A GBA p.Trp378Gly mutation was identified in two RBD and four PD patients (1% of all patients combined), and not in controls. The two RBD patients had converted to DLB within 3 years of their diagnosis. Haplotype, IBD and PCA analysis demonstrated that this mutation is from a single founder. Out of 167 GD patients screened, 15 (9.0%) carried the p.Trp378Gly mutation, all in trans with p.Asn370Ser. Three (20%) of the GD patients with the p.Trp378Gly mutation had developed Parkinsonism, and 11 patients had family history of PD. The p.Trp378Gly mutation is the first French‐Canadian founder GBA mutation to be described, which leads to synucleinopathies and to GD type 1 when in compound heterozygosity with p.Asn370Ser.     

The D409H variant in GBA1: Challenges in predicting the Gaucher phenotype in the newborn screening era

Gaucher disease (GD) is an autosomal recessive disorder resulting from glucocerebrosidase deficiency due to pathologic variants in GBA1. While clinically heterogeneous, GD encompasses three types, non-neuronopathic (GD1), acute neuronopathic (GD2), and chronic neuronopathic (GD3). Newborn screening (NBS), which has made remarkable inroads in detecting certain diseases before detrimental health consequences and fatality ensues, is now being piloted for GD in several states and countries. Early on, clinical features of GD2 can overlap with GD3; hence, predicting outcome is challenging. As NBS for GD becomes more available, the increased detection of GD in neonates is inevitable. As a result, health care providers and families will be faced with uncertainty with respect to clinical management. Since more severe GBA1 variants are generally associated with neuronopathic GD, there has been an increased dependence on genotypic information. We present an infant detected by NBS with genotype D409H(p.Asp448His)/RecNciI (p.Leu483Pro; p.Ala495Pro;p.Val499=). To assist in genetic counseling, we performed a retrospective review of other patients in our cohort carrying D409H and reviewed the literature. The study illustrates the challenges faced in counseling for infants with neuronopathic GD, even with known GBA1 variants, and the tough management decisions that can ensue from detection in newborns.     

Gaucher disease in Colombia: mutation identification and comparison to other Hispanic populations

Gaucher disease is the most common of the lysosomal storage disorders, affecting all ethnic groups. The pathology of this recessively inherited disease arises from the accumulation of glucocerebroside in tissues due to deficient activity of the enzyme glucocerebrosidase (E.C. 3.2.1.45). The glucocerebrosidase (GBA) gene spans a 7.2kb fragment located on locus 1q 21, consisting of 11 exons and 10 introns. Located 16 kb downstream is a highly homologous pseudogene sequence [M. Horowitz, S. Wilder, Z. Horowitz, O. Reiner, T. Gelbart, E. Beutler, The Human Glucocerebrosidase gene and pseudogene: structure and evolution. Genomics 4 (1) (1989) 87-96.]. Fourteen fragments comprising 11 exons of the GBA gene were analyzed in DNA samples from 25 Colombian patients using denaturing High Pressure Liquid Chromatography (DHPLC). Sequencing of abnormal findings led to the discovery of three novel mutations (c.595_596 delCT, c.898 delG and c.1,255 G>C [p.D 419 H] in exons 6, 7, and 9 of the GBA gene) with high prevalence among Colombian patients. We have also found the presence of a double mutation p.L 483 P+p.E 355 K (L 444 P+E 326 K, traditional nomenclature) in two different families classified as Gaucher type 1. This mutation was previously reported in one patient with Gaucher type 2. We have found DHPLC to be a reliable and sensitive method for the detection of mutations and allelic variation in Gaucher patients.     

55-Base pair deletion in certain patients with Gaucher disease complicates screening for common Gaucher alleles

Mutations in the glucocerebrosidase gene which result in Gaucher disease can originate from the highly homologous glucocerebrosidase pseudogene. A 55-bp deletion in exon 9, which corresponds to a 55-bp segment absent from the pseudogene, has been identified in patients with Gaucher disease. We have developed a simple polymerase chain reaction (PCR)-based method to detect this 55-bp deletion, and have found this mutation in 3 of 75 DNA samples (4%) collected from patients with Gaucher disease. Commonly used PCR-based screening methods for specific Gaucher mutations frequently make use of primers either within or surrounding the 55-bp gap to selectively distinguish the glucocerebrosidase gene from the pseudogene. However, if the 55-bp deletion in exon 9 occurs, primers will either fail to produce an amplification product or will produce a shortened product which will be falsely attributed to the pseudogene. This could lead to inaccurate genotyping and genetic counseling for some Gaucher patients and their families. We therefore recommend that laboratories using PCR-based screening techniques involving primers in this region initially determine whether this 55-bp sequence is present. © 1996 Wiley-Liss, Inc.     

Mutation analysis of 28 Gaucher disease patients: The Australasian experience

Gaucher disease is the most common lysosomal storage disease. It is an autosomal recessive disorder that results from a deficiency of β-glucocerebrosidase. Three clinical phenotypes have been described: non-neuronopathic, acute neuronopathic, and subacute neuronopathic. Genomic DNA from 28 Australasian patients of diverse ethnic origin with Gaucher disease was screened for 3 common mutations (1226G, 1448C and 84GG) using the amplification refractory mutation system (ARMS), and one uncommon mutation (1504T) by restriction enzyme digestion. Thirty-eight of the 56 independent alleles in these patients were characterized, with 1448C present in 42% and 1226G in 28% of the alleles. The 1226G mutation was associated only with the non-neuronopathic phenotype and 7 of the 15 patients who carried the 1448C mutation developed neuronopathic disease. Three infants who died in the neonatal period following a rapidly progressive neurodegenerative course carried no identifiable mutations. The 84GG mutation was carried by 2 Jewish patients and 1504T was present in one patient. It is now possible to rapidly identify the common Gaucher mutations using ARMS and restriction enzyme digestion, and our findings confirm the heterogeneity of mutations in Gaucher disease. It is also possible to predict in part the phenotypic outcome when screening patients for these mutations. We consider mutation analysis to be of most use in prenatal diagnosis and for carrier detection within affected families. © 1994 Wiley-Liss, Inc.     

Altered level of plasma exosomes in patients with Gaucher disease

Mutations in the glucocerebrosidase gene (GBA) cause Gaucher disease (GD), the lysosomal storage disorder (LSD), and are the most common genetic risk factor of Parkinson's disease (PD). Lysosome functionality plays a critical role for secretion of extracellular vesicles (EVs) and their content. Here we compared EVs from the blood plasma of 8 GD patients and 8 controls in terms of amounts, size distribution, and composition of their protein cargo. EVs were isolated via sequential centrifugation and characterized by сryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). The presence of exosomal markers HSP70 and tetrasponins were analyzed by Western blot and flow cytometry. Protein profiling was performed by mass-spectrometry (shotgun analysis).Here, for the first time we reported an increased size and altered morphology in exosomes derived from blood plasma of GD patients. An increased size of plasma exosomes from GD patients compared to controls was demonstrated by cryo-EM and DLS (р<0.0001, p < 0.001, respectively) and confirmed by mode size detected by NTA (p < 0.02). Cryo-EM demonstrated an increased number of double and multilayer vesicles in plasma EVs from GD patients. We found that the EVs were enriched with the surface exosomal markers (CD9, СD63, CD81) and an exosome-associated protein HSP70 in case of the patients with the disease. Proteomic profiling of exosomal proteins did not reveal any proteins associated with PD pathogenesis. Thus, we showed that lysosomal dysfunction in GD patients lead to a striking alteration of plasma exosomes in size and morphology.     

Mutation analysis and genotype/phenotype relationships of Gaucher disease patients in Spain

Mutations in the glucocerebrosidase (GBA) gene cause Gaucher disease (GD). The aim of this study was to characterise the GBA mutations and analyze genotype/phenotype relationships in 193 unrelated patients from the Spanish GD Registry. We have identified 98.7% of the mutated GBA alleles, finding 56 different GBA mutations and 66 genotypes causing GD in Spain: 47 previously described mutations and 9 novel mutations (4 missense R395C, R463H, W312R and V398I, 1 nonsense R359X, 4 frameshift c.708delC, c.1214-1215delGC, c.1439-1445del7 and c.42-65del24). The most prevalent mutations were N370S and L444P, accounting for 68.7% of the mutated alleles. A wide phenotypic difference was observed within each genotypic group, and 9% of diagnosed type 1 patients developed neurological involvement including parkisonism, tremor, hypoacusia and eye movements. All of these findings indicate that there is a significant genotypic heterogeneity that explains the huge phenotypic variation among Spanish GD patients.     

Gaucher disease in Spanish patients: Analysis of eight mutations

Gaucher disease is particularly prevalent among Ashkenazi Jews; thus most studies have been reported on this ethnic group. We present the first data on Spanish patients with Gaucher disease and provide one of the first reports on a fairly well defined, large, non-Jewish population. Eight mutations were analyzed in 35 patients, with different clinical subtypes, by restriction enzyme digestion or allelespecific oligonucleotide (ASO) hybridization, after PCR amplification of genomic DNA. Analysis of the eight mutations allowed identification of 77.2% of the disease alleles, N370S and L444P alone accounting for 70%. Mutation N370S, carried by 31 alleles (44.3%), appeared to be the most prevalent in the Spanish population. The frequency of this mutation and of the N370S/N370S genotype is closer to those described for Ashkenazi Jews than to the frequencies found in other non-Jewish populations. Mutation L444P, the second most abundant mutation, occurred in 25.7% of the disease alleles. Four alleles carrying mutation D409H (5.7%) were detected in patients of different clinical expression and one RecNciI allele in a type I patient. Mutations 84GG, IVS2 + l, R463C, and RecTL were also screened but were not found in any of our patients. © 1995 Wiley-Liss, Inc.     

Molecular analysis and clinical findings in the Spanish Gaucher disease population: Putative haplotype of the N370S ancestral chromosome

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the lysosomal β-glucocerebrosidase (GBA) gene. As the disease is particularly prevalent among Ashkenazi Jews, most studies have been carried out on this ethnic group. In the current study, we present a mutation analysis of the GBA gene in Spanish patients together with the clinical findings. We conducted a systematic analysis in 53 unrelated GD patients. The GBA gene was initially scanned for nine previously described mutations by ASO hybridization or restriction analysis after PCR amplification. The remaining unidentified alleles were screened by nonisotopic PCR-SSCP analysis and sequenced. This approach allowed the identification of 101 of 106 GD alleles (95.3%) involving 24 different mutations, 11 of which are described for the first time: G113E (455G→A), T134P (517A→C), G389E (1283G→A), P391L (1289C→T), N392I (1292A→T), Y412H (1351T→G), W(-4)X (108G→A), Q169X (662C→T), R257X (886C→T), 500insT, and IVS5+1G→T. Most mutations are present in one or few GD chromosomes. However, two mutations, N370S (1226A→G) and L444P (1448T→C), are very frequent and account for 66.1% of the total number of alleles. Linkage disequilibrium was detected between these two mutations and an intragenic polymorphism, indicating that expansion of founder alleles occurred in both cases. Analysis of several microsatellite markers close to the GBA gene allowed us to establish the putative haplotype of the ancestral N370S chromosome. Hum Mutat 11:295–305, 1998. © 1998 Wiley-Liss, Inc.     

Intracellular cholesterol modifies the ERAD of glucocerebrosidase in Gaucher disease patients

Gaucher disease (GD) is a lysosomal storage disorder resulting from an inborn reduced activity or deficiency of glucocerebrosidase due mainly to mutations in the glucocerebrosidase gene. We have recently shown that mutant glucocerebrosidase variants present variable degrees of endoplasmic reticulum (ER) retention and undergo ER associated degradation (ERAD) in the proteasomes. The degree of ERAD is one of the factors that determine GD severity. In order to define what factors affect the ERAD process of glucocerebrosidase in GD, we focused on two brothers with GD, carrying the same mutations but presenting extremely different clinical manifestations. One is mildly affected while the other developed severe GD with nervous system complications. Our results strongly indicated that both brothers presented variable degrees of ERAD, which was more extensive in the severely affected brother. Measurement of cholesterol demonstrated high intracellular levels in cells that derived from the severely affected brother. Growing the cells in cholesterol depleted medium led to lessening in the degree of ERAD in cells that derived from the severely affected brother and thus to improvement in stabilization, maturation, lysosomal localization and activity of the mutant glucocerebrosidase variants. The same effect was achieved by treating the cells with the HMG CoA reductase inhibitor mevastatin. None of the treatments had a significant effect on glucocerebrosidase properties in normal cells or in cells that derived from the mildly affected brother, indicating that intracellular cholesterol is one of the factors that affect the ERAD process of glucocerebrosidase and may influence the severity of GD.     

Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression

We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).