Littérature scientifique sur le sujet « R274W »

Mitochondrial fusion is essential to mitochondrial fitness and cellular health. Neurons of patients with genetic neurodegenerative diseases often exhibit mitochondrial fragmentation, reflecting an imbalance in mitochondrial fusion and fission (mitochondrial dysdynamism). Charcot–Marie–Tooth (CMT) disease type 2A is the prototypical disorder of impaired mitochondrial fusion caused by mutations in the fusion protein mitofusin (MFN)2. Yet, cultured CMT2A patient fibroblast mitochondria are often reported as morphologically normal. Metabolic stress might evoke pathological mitochondrial phenotypes in cultured patient fibroblasts, providing a platform for the pre-clinical individualized evaluation of investigational therapeutics. Here, substitution of galactose for glucose in culture media was used to redirect CMT2A patient fibroblasts (MFN2 T105M, R274W, H361Y, R364W) from glycolytic metabolism to mitochondrial oxidative phosphorylation, which provoked characteristic mitochondrial fragmentation and depolarization and induced a distinct transcriptional signature. Pharmacological MFN activation of metabolically reprogrammed fibroblasts partially reversed the mitochondrial abnormalities in CMT2A and CMT1 and a subset of Parkinson’s and Alzheimer’s disease patients, implicating addressable mitochondrial dysdynamism in these illnesses.

ObjectiveMutations in the aryl hydrocarbon receptor-interacting protein (AIP) were recently shown to confer a pituitary adenoma predisposition in patients with familial isolated pituitary adenomas (FIPA). We report a large Samoan FIPA kindred from Australia/New Zealand with an R271W mutation that was associated with aggressive pituitary tumors.Design and methodsCase series with germline screening of AIP and haplotype analyses among R271W families.ResultsThis previously unreported kindred consisted of three affected individuals that either presented with or had first symptoms of a pituitary macroadenoma in late childhood or adolescence. The index case, a 15-year-old male with incipient gigantism and his maternal aunt, had somatotropinomas, and the maternal uncle of the index case had a prolactinoma. All tumors were large (15, 40, and 60 mm maximum diameter) and two required transcranial surgery and radiotherapy. All three affected subjects and ten other unaffected relatives were found to be positive for a germline R271W AIP mutation. Comparison of the single nucleotide polymorphism patterns among this family and two previously reported European FIPA families with the same R271W mutation demonstrated no common ancestry.ConclusionsThis kindred exemplifies the aggressive features of pituitary adenomas associated with AIP mutations, while genetic analyses among three R271W FIPA families indicate that R271W represents a mutational hotspot that should be studied further in functional studies.

Abstract In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre–pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells.

Abstract Gain-of-function (GOF) mutations in the gene for signal transducer and activator of transcription 1 (STAT1) account for approximately one-half of patients with chronic mucocutaneous candidiasis (CMC) disease. Patients with GOF-STAT1 mutations display a broad variety of infectious and autoimmune manifestations in addition to CMC, and those with severe infections and/or autoimmunity have a poor prognosis. The establishment of safe and effective treatments based on a precise understanding of the molecular mechanisms of this disorder is required to improve patient care. To tackle this problem, we introduced the human R274Q GOF mutation into mice [GOF-Stat1 knock-in (GOF-Stat1R274Q)]. To investigate the immune responses, we focused on the small intestine (SI), which contains abundant Th17 cells. Stat1R274Q/R274Q mice showed excess phosphorylation of STAT1 in CD4+ T cells upon IFN-γ stimulation, consistent with the human phenotype in patients with the R274Q mutation. We identified two subpopulations of CD4+ T cells, those with ‘normal’ or ‘high’ level of basal STAT1 protein in Stat1R274Q/R274Q mice. Upon IFN-γ stimulation, the ‘normal’ level CD4+ T cells were more efficiently phosphorylated than those from WT mice, whereas the ‘high’ level CD4+ T cells were not, suggesting that the level of STAT1 protein does not directly correlate with the level of pSTAT1 in the SI. Inoculation of Stat1R274Q/R274Q mice with Candida albicans elicited decreased IL-17-producing CD4+RORγt+ cells. Stat1R274Q/R274Q mice also excreted larger amounts of C. albicans DNA in their feces than control mice. Under these conditions, there was up-regulation of T-bet in CD4+ T cells. GOF-Stat1R274Q mice thus should be a valuable model for functional analysis of this disorder.

AbstractObjectiveTo assess the relative validity of a new, web-based, self-administered 24 h dietary recall, the R24W, for assessment of energy and nutrient intakes among French Canadians.DesignEach participant completed a 3d food record (FR) and the R24W on three occasions over a 4-week period. Intakes of energy and of twenty-four selected nutrients assessed by both methods were compared.SettingQuébec City metropolitan area.SubjectsFifty-seven women and fifty men (mean (sd) age: 47·2 (13·3) years).ResultsEquivalent proportions of under-reporters were found with the R24W (15·0%) and the FR (23·4%). Mean (sd) energy intake from the R24W was 7·2% higher than that from the FR (10 857 (3184) kJ/d (2595 (761) kcal/d) v. 10 075 (2971) kJ/d (2408 (710) kcal/d); P<0·01). Significant differences in mean nutrient intakes between the R24W and the FR ranged from –54·8% (i.e. lower value with R24W) for niacin to +40·0% (i.e. higher value with R24W) for alcohol. Sex- and energy-adjusted deattenuated correlations between the two methods were significant for all nutrients except Zn (range: 0·35–0·72; P<0·01). Cross-classification demonstrated that 40·0% of participants were classified in the same quartile with both methods, while 40·0% were classified in the adjacent quartile and only 3·6% were grossly misclassified (1st v. 4th quartile). Analysis of Bland–Altman plots revealed proportional bias between the two assessment methods for 8/24 nutrients.ConclusionsThese data suggest that the R24W presents an acceptable relative validity as compared with the FR for estimating usual dietary intakes in a cohort of French Canadians.

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival &gt;180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.

Automated, self-administered, Web-based 24-h dietary recall tools are increasingly available for nutrition research in different settings, particularly in epidemiological studies and national surveys because of their practicality and efficiency. However, the usability of different 24-h dietary recall tools must be assessed and compared for use in specific populations as it is a major driver of the response rate and retention of participants. The primary aim of this study was to compare the usability of two validated, self-administered, web-based 24-h dietary recall tools available for the Canadian population: the R24W and the 2018 Canadian version of the ASA24. The R24W was developed in French for primary use in the province of Québec, Canada while the ASA24 was developed in English for primary use in the USA and recently adapted and translated for use in French-speaking Canadian adults. Whether the R24W and the ASA24-Canada-2018 yield similar nutritional data was also tested. In this randomized crossover study, 48 women and 20 men (mean age of 35 ± 14 years; range: 19–79 years) recruited in the province of Quebec completed the R24W and the ASA24-Canada-2018 in French twice on each occasion. Participants also completed the System Usability Scale (SUS), a reliable and valid scale giving a global view of subjective assessments of usability. Mean SUS score as well as mean dietary intakes of energy, nutrients and food groups generated by each tool were compared using mixed model analyses for repeated measures. On a scale of 0 to 100, the mean SUS scores (±SD) for the R24W and the ASA24-Canada-2018 were 81 ± 2 and 58 ± 2, respectively (p < 0.0001). 84% of participants stated that they would prefer to use the R24W if they were invited to complete additional 24-h dietary recalls. No significant difference was found between the R24W and the ASA24-Canada-2018 for the intake of energy, proteins, lipids, saturated fatty acids, carbohydrates, fibers, sodium and vegetables and fruits. In sum, while the R24W and the ASA24-Canada-2018 generate comparable self-reported dietary intake data, the R24W showed a better usability than the ASA24-Canada-2018 in a sample of French-speaking adults from the province of Quebec.

The objective of this study was to validate an automated self-administered 24-hour dietary recall web application (R24W) against recovery biomarkers for sodium, potassium and protein intakes and to identify individual characteristics associated with misreporting in a sample of 61 men and 69 women aged 20–65 years from Québec City, Canada. Each participant completed 3 dietary recalls using the R24W, provided two 24-hour urinary samples and completed questionnaires to document psychosocial factors. Mean reported intakes were 2.2%, 2.1% and 5.0% lower than the urinary reference values, respectively, for sodium, potassium and proteins (significant difference for proteins only (p = 0.04)). Deattenuated correlations between the self-reported intake and biomarkers were significant for sodium (r = 0.48), potassium (r = 0.56) and proteins (r = 0.68). Cross-classification showed that 39.7% (sodium), 42.9% (potassium) and 42.1% (proteins) of participants were ranked into the same quartile with both methods and only 4.8% (sodium), 3.2% (potassium) and 0.8% (proteins) were ranked in opposite quartiles. Lower body esteem related to appearance was associated with sodium underreporting in women (r = 0.33, p = 0.006). No other individual factor was found to be associated with misreporting. These results suggest that the R24W has a good validity for the assessment of sodium, potassium and protein intakes in a sample of French-speaking adults. Novelty: The validity of an automated self-administered 24-hour dietary recall web application named the R24W was tested using urinary biomarkers. According to 7 criteria, the R24W was found to have a good validity to assess self-reported intakes of sodium, potassium and proteins.

Abstract Introduction Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced slow 'passive leak' of red cell K+ into plasma, first described in a large Scottish family from Edinburgh (Stewart GW, et al., 1979). Although in freshly obtained blood samples plasma [K+] was normal, it was increased when measured in blood stored at or below room temperature. This trait was accompanied by mild abnormalities of red cell shape. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval in three multigenerational FP families with 20 affected individuals identified two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype (Andolfo I. et al., 2013). The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that in erythrocyte membranes bears the Langereis blood group antigen system (Krishnamurthy PC, et al., 2006; Helias V, et al., 2012). Recently, the ABCB6 mutation R723Q was found in two patients with FP (Bawazir W, et al.,2014). Of note, both patients presented as blood donors, and increased cold-induced potassium leak was demonstrated. The transfusion of pseudohyperkalemic blood has clinical implications especially for neonates and infants receiving large-volume RBC transfusions. In this study we analyzed one additional family and report the first functional characterization of an ABCB6 mutation, towards understanding the pathogenic mechanism of FP. Moreover, we screened an Italian blood donor population for the R276W variant of the ABCB6 gene. Methods DNA was obtained for genetic analysis from affected and unaffected family members, after signed informed consent, according to the Declaration of Helsinki. The search for mutations was performed by direct sequencing of the ABCB6 gene. cDNAs encoding full-length wildtype ABCB6 were cloned into pcDNA3.1. Our patients' novel point mutation (c.826C>T, p.R276W) was introduced into pcDNA3.1-ABCB6 by site-directed mutagenesis. WT and mutant constructs were transfected into HEK-293 cells and the cells were maintained at 30°C for 72 hrs to evaluate the effects of reduced temperature. After transfection, the cells were incubated in a medium containing 86rubidium (86Rb+) as a surrogate for K+. 86Rb+ was determined in cell lysate, and K content of extracellular medium was determined by atomic absorption spectrometry. Results We found the heterozygous mutation c.826G>T, p. R276W in an Irish family. This mutation is annotated in public databases as single nucleotide variants (SNVs), and is predicted by PolyPhen2 and SIFT to be damaging. Variant R276W showed a minor allele frequency (MAF) of 1.3:100 confirming that many patients with FP could be present in the blood donor population. R276W and previously identified ABCB6 variants R375Q and R375Wwere overexpressed in HEK-293 cells to characterize the functional properties of these variants. Expression of ABCB6 mutants showed no change in RNA or polypeptide levels. However, measurement of ouabain- and bumetanide-resistant net cation flux demonstrated a greater loss of cell K from mutant ABCB6-expressing cells than from WT ABCB6-expressing cells. The high allele frequency of ABCB6 variant R276W prompted a genetic screen of 327 blood donors. The variant was present in 0.3% (1/327) of the donor cohort, and this single subject with ABCB6 R276W exhibited slightly increased MCV and variably increased plasma K+ concentrations. Conclusions: Our findings demonstrate that missense mutations in ABCB6 lead to increased K+ efflux from RBCs in FP patients. Storage of FP blood can cause a significant increase in blood K+ levels, with especially serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Furthermore, the prevalence of FP might be underestimated, since patients with FP can be asymptomatic and thus undetected in the donor population. Screening for the most frequent ABCB6 variation, R276W, confirmed that FP patients are present in the Italian blood donor population. In the future, genetic tests for FP could be added to blood donor prescreening to improve the quality and safety of donor units. Disclosures No relevant conflicts of interest to declare.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic (DA) neurons and the formation of Lewy bodies. Despite most cases being idiopathic, mutations in several genes have been implicated in familial forms of PD. In particular, recessive mutations in Parkin gene (PARK2) are the most common cause of young-onset inherited parkinsonism. Parkin is an E3 ubiquitin ligase involved both in the control of mitochondrial turnover and in the proteasome-dependent degradation of proteins, two pathways that have been causally linked to PD development. Although initially described as a recessive disorder, experimental evidence suggests that heterozygous Parkin mutations can exert dominant toxic effects causing neurodegeneration. In 2012, Ruffmann and colleagues identified the first pure heterozygous R275W Parkin patient with clinical features of typical late-onset PD and a diffuse Lewy body pathology. To assess the impact of R275W Parkin, we generated the first mouse line carrying Parkin R274W mutation, which corresponds to the human R275W substitution. Unlike Parkin deficient mouse models, both homo- and heterozygous R274W mice show an age-related motor impairment, degeneration of dopaminergic neurons and neuroinflammation. We detected structural and functional mitochondrial abnormalities related to PARIS-PGC-1α axis impairment in R274W+/+ mice brain and skeletal muscle. Strikingly, we noticed signs of protein aggregation in both R274W+/- and +/+ mice, while we identified bona fide Lewy bodies only in the midbrain of heterozygous mice. Additionally, in the brains of R274W mice we discovered overt abnormalities of the glymphatic system, the main route for brain waste clearance. Our preliminary observations suggest that Parkin influences aquaporin-4 (AQP4) localization. Altogether, our data suggest that R274W Parkin substitution behaves both as a loss ofand a gain of toxic function, highlighting a link between Parkin dominant toxicity and age-dependent motor impairment, neuroinflammation, DA neurons loss, glymphatic system dysfunctions and α-synuclein aggregation in vivo. Hence, our study provides a new robust mouse model to explore PD pathogenesis and glymphatic dysfunctions, offering the possibility to test novel therapeutic strategies with great predictivity.

A pancreatite crônica é uma desordem complexa, na qual a interação entre fatores ambientais e genéticos resulta na enfermidade. O presente estudo incluiu 148 pacientes com diagnóstico de pancreatite crônica, 110 etilistas crônicos e 297 controles sadios com o objetivo de investigar a frequência de tabagismo e das mutações N34S e P55S do gene SPINK1 e R254W do gene CTRC nesta população. Foi aplicado questionário presencial e realizada reação de sequenciamento para a pesquisa das mutações genéticas, após assinatura do Termo de Consentimento Livre e Esclarecido. Os portadores de pancreatite crônica possuíam etiologia alcoólica em 74% das vezes e idiopática em 26%. A pancreatite alcoólica apresentou-se de maneira distinta da pancreatite crônica idiopática, sendo que o primeiro grupo é composto por maior prevalência do gênero masculino (88,18% versus 34,21%), por maior média de idade (55,64 anos versus 45,20 anos), menor frequência de caucasianos (63,89% versus 84,21%), menor escolaridade (23,30% concluíram ensino médio ou superior versus 57,89%) e maior frequência de repercussões da doença, como diarréia (54,21% versus 24,24%), emagrecimento (56,07% versus 24,24%), diabete melito (57,94% versus 36,36%) e ocorrência de pseudocistos pancreáticos (31,78% versus 12,12%), repercussões estas que não foram acompanhadas de maior frequência de alterações morfológicas, como calcificações pancreáticas ou dilatação do ducto pancreático principal. A frequência de tabagismo foi significativamente maior em pacientes com pancreatite crônica alcoólica do que em etilistas sem pancreatite crônica, podendo ser considerado cofator de risco para o desenvolvimento da pancreatite crônica entre alcoolistas (p = 0,002); a frequência da mutação N34S do gene SPINK1 em pacientes com pancreatite crônica foi de 3,38%, maior do que a frequência de 0,49% encontrada nos grupos controle (p = 0,016); a frequência de 2,03% da mutação P55S do gene SPINK1 e a frequência de 0,67% da mutação R254W do gene CTRC, encontradas nos pacientes com pancreatite crônica, não diferiram estatisticamente quando comparadas às frequências, de 0,49% de ambas mutações, encontradas nos grupos controle. (p = 0,120 e 0,751). Pela investigação da associação de tabagismo e da mutação N34S do gene SPINK1 com as características clínicas e morfológicas da pancreatite crônica, verificou-se que a mutação N34S não se associou a maior gravidade da apresentação clínica ou morfológica da pancreatite crônica; no entanto o tabagismo associou-se a maior frequência de diabete melito entre os portadores de pancreatite crônica. Concluiuse que o tabagismo e a mutação N34S do gene SPINK1 podem ser considerados cofatores de risco para o desenvolvimento da pancreatite crônica
Chronic pancreatitis is a complex disorder in which the interaction between environmental and genetic factors results in the disease. This study included 148 patients with chronic pancreatitis, 110 chronic alcoholics and 297 healthy controls in order to investigate the frequency of smoking and N34S and P55S mutation of SPINK1 gene and R254W of CTRC gene in this population. A questionnaire was applied and gene sequencing was done, after having the Informed Consent Statement. Those with chronic pancreatitis had alcoholic etiology in 74% of cases and idiopathic in 26%. Alcoholic pancreatitis presented in a distinct way of idiopathic chronic pancreatitis. The first group is composed of a higher prevalence of males (88.18% versus 34.21%), by higher mean age (55.64 years versus 45.20 years), lower frequency of Caucasians (63.89% versus 84.21%), lower education (23.30% completed secondary or higher education versus 57.89%) and worst impact from the disease such as diarrhea (54.21% versus 24.24%), weight loss (56.07% versus 24.24%), diabetes mellitus (57.94% versus 36.36%) and occurrence of pancreatic pseudocysts (31.78% versus 12 , 12%). These effects were not accompanied by increased frequency of morphological changes, such as pancreatic calcifications or dilation of the main pancreatic duct. The frequency of smoking was significantly higher in patients with alcoholic pancreatitis than in alcoholics without chronic pancreatitis, therefore tabagism may be considered as a cofactor for the development of chronic pancreatitis among alcoholics (p = 0.002); the frequency of N34S mutation of SPINK1 gene in patients with chronic pancreatitis was 3.38%, higher than the rate of 0.49% found in the control groups (p = 0.016); the frequency of 2.03% of the P55S mutation of SPINK1 gene and the frequency of 0.67% of the CTRC gene R254W mutation found in patients with chronic pancreatitis were not statistically different when compared to the frequencies of 0.49% of both mutations, found in the control groups. (p = 0.120 and 0.751) For the investigation of the association of smoking and N34S mutation of SPINK1 gene with the clinical and morphological features of chronic pancreatitis, it was noticed that the N34S mutation did not determine a greatest severity in the presentation of chronic pancreatitis, however smoking was associated with a higher frequency of diabetes mellitus in patients with chronic pancreatitis. It was concluded that smoking and the N34S mutation of SPINK1 gene are positively correlated with chronic pancreatitis

Deficient activity of $\beta$-hexosaminidase A (Hex A), resulting from mutations in the HEX4 gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e. individuals who are pseudodeficient) have been described. Most of these individuals have a C739T (R247W) mutation in compound heterozygosity with a Tay-Sachs disease-causing allele. We identified a second benign mutation, C745T (R249W) in a pseudodeficient subject and showed that it accounted for approximately 6% (4/63) of enzyme-defined non-Jewish Tay-Sachs disease carriers. Taken together, the two benign mutations accounted for approximately 38% of non-Jewish enzyme-defined carriers, making their identification an important component of Tay-Sachs disease prevention programs. To confirm the relationship between benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, each of the benign mutations and other mutations associated with infantile, juvenile, and adult-onset forms of Gm2 gangliosidosis were transiently expressed as Hex S $(\alpha\alpha)$ and Hex A $(\alpha\beta)$ in Cos-7 cells. The benign mutations decreased the expressed Hex A and Hex S activities toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-$\beta$-N-acetylglucosaminide (4-MUGS) by 60 to 80%, indicating they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations reduced enzymatic activity by reducing the $\alpha$-subunit protein level. No change in Hex A heat sensitivity, catalytic activity, or specificity toward 4-MUG and 4-MUGS, were detected in the studies. The effects of benign mutations on Hex A were further analysed in fibroblasts, and during transient expression, using pulse-chase metabolic labelling. These studies showed that (1) the benign mutations reduced the $\alpha$-subunit protein by affecting its stability in vivo, not by affecting the processing of the $\alpha$-subunit, ie. phosphorylation, targeting, or secretion, and (2) these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.