A versatile fluorescence-quenched substrate for quantitative measurement of glucocerebrosidase activity within live cells.

Loss of activity of the lysosomal glycosidase ß-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

Enfermedad de Gaucher, importancia de una sospecha precoz / Gaucher disease: importance of early suspicion

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Selective Fluorogenic ß-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates.

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme ß-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher's disease (GD) and a major risk factor for Parkinson's disease (PD). Humans harbor two other functionally related ß-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin ß-d-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.

Path mediation analysis reveals GBA impacts Lewy body disease status by increasing α-synuclein levels.

Synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal α-synuclein in intraneuronal inclusions, named Lewy bodies. Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase, have been identified as the most common genetic risk factor for PD and DLB. However, despite extensive research, the mechanism by which glucocerebrosidase dysfunction increases the risk for PD or DLB still remains elusive. In our study we expand the toolbox for PD-DLB post-mortem studies by introducing new quantitative biochemical assays for glucocerebrosidase and α-synuclein. Applying causal modelling, we determine how these parameters are interrelated and ultimately impact disease manifestation. We developed quantitative immuno-based assays for glucocerebrosidase and α-synuclein (total and phosphorylated at Serine 129) protein levels, as well as a liquid chromatography-mass spectrometry method for the detection of the glucocerebrosidase lipid substrate glucosylsphingosine. These assays were applied on tissue samples from frontal cortex, putamen and substantia nigra of PD (n = 15) and DLB (n = 15) patients and age-matched non-demented controls (n = 15). Our results confirm elevated p-129 over total α-synuclein levels in the insoluble fraction of PD and DLB post-mortem brain tissue and we found significantly increased α-synuclein levels in the soluble fractions in PD and DLB. Furthermore, we identified an inverse correlation between reduced glucocerebrosidase enzyme activity and protein levels with increased glucosylsphingosine levels. In the substantia nigra, a brain region particularly vulnerable in Parkinson's disease, we found a significant correlation between glucocerebrosidase protein reduction and increased p129/total α-synuclein ratios. We assessed the direction and strength of the interrelation between all measured parameters by confirmatory path analysis. Interestingly, we found that glucocerebrosidase dysfunction impacts the PD-DLB status by increasing α-synuclein ratios in the substantia nigra, which was partly mediated by increasing glucosylsphingosine levels. In conclusion, we show that the introduced immuno-based assays enable the quantitative assessment of glucocerebrosidase and α-synuclein parameters in post-mortem brain. In the substantia nigra, reduced glucocerebrosidase levels contribute to the increase in α-synuclein levels and to PD-DLB disease manifestation partly by increasing its glycolipid substrate glucosylsphingosine. This interrelation between glucocerebrosidase, glucosylsphingosine and α-synuclein parameters supports the hypothesis that glucocerebrosidase acts as a modulator of PD-DLB.

Reduced glucocerebrosidase activity in monocytes from patients with Parkinson's disease.

Missense mutations in glucocerebrosidase (GBA1) that impair the activity of the encoded lysosomal lipid metabolism enzyme (GCase) are linked to an increased risk of Parkinson's disease. However, reduced GCase activity is also found in brain tissue from Parkinson's disease patients without GBA1 mutations, implicating GCase dysfunction in the more common idiopathic form of Parkinson's disease. GCase is very highly expressed in monocytes, and thus we measured GCase activity in blood samples from recently diagnosed Parkinson's disease patients. Flow cytometry and immunoblotting assays were used to measure levels of GCase activity and protein in monocytes and lymphocytes from patients with Parkinson's disease (n = 48) and matched controls (n = 44). Gene sequencing was performed to screen participants for GBA1 missense mutations. In the Parkinson's disease patients, GCase activity was significantly reduced in monocytes, but not lymphocytes, compared to controls, even when GBA1 mutation carriers were excluded. Monocyte GCase activity correlated with plasma ceramide levels in the Parkinson's disease patients. Our results add to evidence for GCase dysfunction in idiopathic Parkinson's disease and warrant further work to determine if monocyte GCase activity associates with Parkinson's disease progression.

Relationship Between Glucocerebrosidase Activity and Clinical Response to Enzyme Replacement Therapy in Patients With Gaucher Disease Type I.

The quantification of enzyme activity in the patient treated with enzyme replacement therapy (ERT) has been suggested as a tool for dosage individualization, so we conducted a study to evaluate the relationship between glucocerebrosidase activity and clinical response in patients with Gaucher disease type I (GD1) to ERT. The study included patients diagnosed with GD1, who were being treated with ERT, and healthy individuals. Markers based on glucocerebrosidase activity measurement in patients' leucocytes were studied: enzyme activity at 15 min. post-infusion (Act75 ) reflects the amount of enzyme that is distributed in the body post-ERT infusion, and accumulated glucocerebrosidase activity during ERT infusion (Act75-0 ) indicates the total drug exposure during infusion. The clinical response was evaluated based on criteria established by Pastores et al. and Gaucher Severity Score Index. Statistical analysis included ROC analysis and area under the curve test. Act75 and Act75-0 were found to be moderate predictive markers of an optimal clinical response (area under the ROC of Act75 was 0.733 and Act75-0 was 0.817). Act75-0 showed statistical significance in its discriminative capacity (p < 0.05) for obtaining an optimal response to ERT. The cut-off point was 58% (RR = 1.800; 95% CI: 1.003-3.229; p < 0.05). Moreover, Act75 showed a significant and inverse correlation with the Gaucher Severity Score Index, and Act75 and Act75-0 presented a significant correlation with residual enzyme activity at diagnosis. Markers based on glucocerebrosidase activity have a good correlation with clinical response to ERT. Therefore, it could provide supporting clinical data for dose management in GD1 patients.

Fluorescence-Quenched Substrates for Quantitative Live Cell Imaging of Glucocerebrosidase Activity.

Glucocerebrosidase (GCase) is a lysosomal glycoside hydrolase that cleaves the glycolipid glucosylceramide (GlcCer). Deficiencies of this enzyme lead to accumulation of GlcCer and the development of the lysosomal storage disease known as Gaucher's disease. Recently, loss-of-function mutations in the GBA1 gene that encodes GCase have been linked to Parkinson's disease. Currently pursued therapeutic strategies to increase GCase involve enzyme replacement therapy, chemical chaperone therapy, and GCase activators. A challenge associated with advancing such strategies is to efficiently monitor GCase activity within the lysosomes of live cells. In this chapter, we review the design and use of the fluorescent-quenched probe GBA1-FQ2 to quantitatively measure GCase activity in lysosomes of live cells.

Activity-Based Probes for Glycosidases: Profiling and Other Applications.

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal ß-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl-enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for ß-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.

Lactate Dehydrogenase and ß-Glucuronidase as Salivary Biochemical Markers of Periodontitis Among Smokers and Non-Smokers.

OBJECTIVES: This study aimed to establish lactate dehydrogenase (LDH) and ß-glucuronidase as salivary biomarkers of periodontitis among smokers and non-smokers. METHODS: This cross-sectional case-control study was conducted at the Aligarh Muslim University, Aligarh, India, between January and June 2017. A total of 200 participants were divided into four groups based on their periodontal and smoking statuses. Unstimulated mixed saliva samples were collected to estimate LDH and ß-glucuronidase levels. In addition, total protein was estimated using Lowry's method. RESULTS: There was a significant increase in enzyme activity in the periodontitis groups compared to the non-periodontitis groups (P <0.001). However, significantly lower enzyme activity was observed among smokers, irrespective of periodontal status (P <0.001). Nevertheless, a receiver operating characteristic curve analysis indicated the diagnostic potential of both enzymes to be fair-to-excellent. CONCLUSION: Although smoking was found to significantly alter enzyme activity, LDH and ß-glucuronidase were reliable salivary biomarkers of periodontitis among both smokers and non-smokers.

In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling.

Epidermal ß-glucocerebrosidase (GBA1), an acid ß-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-ß-D-glucopyranoside or resorufin-ß-D-glucopyranoside; and ii) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various ß-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.

Validez de la prueba de actividad enzimática de la glucocerebrosidasa para el diagnóstico de enfermedad de Gaucher / Validez de la prueba de actividad enzimática de la glucocerebrosidasa para el diagnóstico de enfermedad de Gaucher

INTRODUCCIÓN: La enfermedad de Gaucher es ocasionada por deficiencia o ausencia de enzima Glucocerebrosidasa. Esta deficiencia favorece la acumulación del sustrato glucocerebrósido en los lisosomas de macrófagos (células de Gaucher) y monocitos, causando daño celular y disfunción orgánica. Existen tres tipos, según la ausencia (I) o presencia (II y III) de afección neurológica. En Colombia actualmente hay 139 enfermos con diagnóstico de enfermedad de Gaucher (Asociación Colombiana de Pacientes con Enfermedad Lisosomal - ACOPEL, Informe verbal 2014). La sospecha clínica se confirma midiendo actividad enzimática de Glucocerebrosidasa en leucocitos o sangre seca. En individuos con EG, la actividad enzimática está entre 0%-15% de su actividad normal. El tratamiento con terapia de reemplazo enzimático suele ser eficaz. No hay tratamiento para daño cerebral. Esta evaluación hace parte del esfuerzo del Ministerio de Salud y Protección Social de actualizar las tecnologías disponibles para el diagnóstico de enfermedades huérfanas como parte del proceso de actualización del plan obligatorio de salud. OBJETIVO: Evaluar la validez diagnóstica de la prueba de actividad enzimática de la glucocerebrosidasa en sangre seca y en leucocitos, en pacientes sospechosos de la enfermedad de Gaucher. METODOLOGÍA: Búsqueda sistemática y exhaustiva de literatura (MEDLINE, EMBASE, LILACS, CDSR y DARE), según estándares de la Colaboración Cochrane. Usando criterios QUADAS-2, dos revisores independientes evaluaron la calidad de la literatura. La información básica de los artículos seleccionados e incluidos se extrajo usando un formato estándar diseñado en Excel®. RESULTADOS: Se realizaron 5 búsquedas encontrándose 75 referencias, una vez se removieron los duplicados quedaron 47 referencias. Se excluyeron 46 artículos obtenidos de la búsqueda. Los resultados están basados en el estudio de validez diagnostica de Stroppiano y cols., que tuvo una calidad de 16/18. Este estudio reportó una sensibilidad del 88,2% (IC 95% 72.9­100%), especificidad del 88,5% (IC 95% 85.5­91.5%), valor predictivo positivo 23.4% y valor predictivo negativo de 99.5% para un punto de corte ajustado de 4.4, para la actividad enzimática de glucocerebrosidasa en sangre seca comparada con la prueba en leucocitos. CONCLUSIONES: La evidencia sobre la medición enzimática de glucocerebrosidasa en sangre seca es escasa, sin embargo el único artículo encontrado es de alta calidad, de acuerdo con la escala QUADAS-2. Esta evidencia nos permitió establecer que la prueba tiene muy buen desempeño como método diagnóstico inicial, pero no es concluyente debido a la alta proporción de falsos positivos. Esta prueba es de utilidad por su facilidad en la toma y transporte, pero sus resultados deben confirmarse con la medición de la actividad enzimática de la glucocerebrosidasa en leucocitos.(AU)

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.

Diagnóstico, biomarcadores y alteraciones bioquímicasde la enfermedad de Gaucher / Diagnosis, biomarkers and biochemical alterations in Gaucher's disease

La enfermedad de Gaucher (EG) es una enfermedad metabólica hereditaria debida al déficit de actividad de la enzima beta-glucocerebrosidasa. El diagnóstico de la EG habitualmente se realiza mediante el análisis fluorimétrico de la enzima beta-glucocerebrosidasa. También se puede realizar midiendo la actividad por espectrometría de masas en tándem. Esta enzima se puede analizar en diferentes materiales como leucocitos, fibroblastos cultivados, sangre seca sobre papel y, en caso del diagnóstico prenatal, en vellosidades coriales o en amniocitos cultivados. Se han descrito distintos biomarcadores para el seguimiento de la EG una vez diagnosticada, para evaluar la respuesta a posibles tratamientos. La actividad quitotriosidasa es el biomarcador más utilizado para la evaluación de la EG y, en caso de pacientes homocigotos para variantes nulas del gen CHIT1, en general, se sustituye por el análisis del biomarcador CCL18 (AU)

Gaucher’s disease (GD) is an inherited metabolic disease due to lack of activity of the enzyme betaglucocerebrosidase. The diagnosis of GD is usually performed by fluorimetric analysis of the enzyme betaglucocerebrosidase. It can also be done by measuring the activity of tandem mass spectrometry. This enzyme can be analyzed in different samples such as leukocytes, fibroblasts, blood dried on paper and in case of prenatal diagnosis in chorionic villi or culture of amniocytes. Various biomarkers have been described for monitoring GD once diagnosed, to evaluate the response to potential treatments. Chitotriosidase activity is the most widely used biomarker for the assessment of GD, and for patients homozygous for the null CHIT1 gene variants, in general, is replaced by the analysis of the biomarker CCL18 (AU)

[Diagnosis, biomarkers and biochemical alterations in Gaucher's disease]. / Diagnóstico, biomarcadores y alteraciones bioquímicasde la enfermedad de Gaucher.

Gaucher's disease (GD) is an inherited metabolic disease due to lack of activity of the enzyme betaglucocerebrosidase. The diagnosis of GD is usually performed by fluorimetric analysis of the enzyme betaglucocerebrosidase. It can also be done by measuring the activity of tandem mass spectrometry. This enzyme can be analyzed in different samples such as leukocytes, fibroblasts, blood dried on paper and in case of prenatal diagnosis in chorionic villi or culture of amniocytes. Various biomarkers have been described for monitoring GD once diagnosed, to evaluate the response to potential treatments. Chitotriosidase activity is the most widely used biomarker for the assessment of GD, and for patients homozygous for the null CHIT1 gene variants, in general, is replaced by the analysis of the biomarker CCL18.

Gaucher disease in sheep.

Gaucher disease, an autosomal recessive lysosomal storage disorder caused by mutations in the ß-glucocerebrosidase gene, was recently discovered in sheep on a "Southdown" sheep stud in Victoria, Australia. Clinical signs include neuropathy, thickened leathery skin, and ichthyosis, with lambs unable to stand from birth. Affected lambs were found to be deficient in glucocerebrosidase activity, and mutational analysis found them to be homozygous for the missense mutations c.1142G>A (p.C381Y) and c.1400C>T (p.P467L). In addition, four silent mutations were detected (c.777C>A [p.Y259Y], c1203A>G [p.Q401Q], c.1335T>C [p.I445I], c.1464C>G [p.L488L]). The human equivalent [C342Y] to the C381Y mutation leads to an acute neuronopathic phenotype in patients. Identification of an acute neuronopathic form of Gaucher disease in sheep provides a large animal model that will enable studies of pathology and evaluation of therapies to treat this common lysosomal storage disorder.

Nanomolar affinity, iminosugar-based chemical probes for specific labeling of lysosomal glucocerebrosidase.

Three different photoprobes were synthesized to label beta-glucosidases; one probe was based on glucose, two probes on the iminosugar deoxynojirimycin. The affinity of the probes for three different beta-glucosidases was determined. Furthermore, their labeling efficiencies, binding specificities through competition with deoxynojirimycin, and binding specificities in the presence of cell lysate, were evaluated. Especially one showed very high affinity towards non-lysosomal glucoceramidase (IC(50)=20 nM).

Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening.

Glucocerebrosidase (GC) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl beta-D-glucopyranoside and resorufin beta-D-glucopyranoside, have K(m) values of 768 microM and 33 microM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value.

Dependence of reversibility and progression of mouse neuronopathic Gaucher disease on acid beta-glucosidase residual activity levels.

Genetic and chemically induced neuronopathic mouse models of Gaucher disease were developed to facilitate understanding of the reversibility and/or progression of CNS involvement. The lethality of the skin permeability barrier defect of the complete gene knock out [gba, (glucocerebrosidase) GCase] was avoided by conditional reactivation of a low activity allele (D409H) in keratinocytes (kn-9H). In kn-9H mice, progressive CNS disease and massive glucosylceramide storage in tissues led to death from CNS involvement by the age of 14 days. Conduritol B epoxide (CBE, a covalent inhibitor of GCase) treatment (for 8-12 days) of wild type, D409H, D409V or V394L homozygotes recapitulated the CNS phenotype of the kn-9H mice with seizures, tail arching, shaking, tremor, quadriparesis, extensive neuronal degeneration loss and apoptosis, and death by the age of 14 days. Minor CNS abnormalities occurred after daily CBE injections of 100 mg/kg/day for 6 doses, but neuronal degeneration was progressive and glucosylceramide storage persisted in D409V homozygotes in the 2 to 5 months after CBE cessation; wild type and D409H mice had persistent neurological damage without progression. The persistent CNS deterioration, histologic abnormalities, and glucosylceramide storage in the CBE-treated D409V mice revealed a threshold level of GCase activity necessary for the prevention of progression of CNS involvement.

[Distribution of mutations of acid beta-D-glucosidase gene (GBA) among 68 Russian patients with Gaucher's disease].

Gaucher disease (GD) is the most frequent lysosomal storage disease presenting in all populations. Mutations in the acid beta-D-glucosidase gene (GBA) cause development of GD, resulting in a decrease or full loss of activity of this enzyme. We report here the results of the molecular-genetic analysis in 68 Russian GD patients from 65 families with the three types of the disease. We have identified 126 mutation alleles from 136 investigate alleles. In addition to known mutations p.N370S, c.1263-1317del (del55), p.L444P, p.R463C, Rec NciI, we identified rare mutations p.R120W, p.R170C, p.W184R, p.G202R, Rec C, presenting in other populations and mutations p.P236T, p.L249Q, p.L288P, p.P319S, p.V352M, p.W381X, p.A384D which are had not been described before.