Maleic acid is a biomarker for maleylacetoacetate isomerase deficiency; implications for newborn screening of tyrosinemia type 1.

Dried blood spot succinylacetone (SA) is often used as a biomarker for newborn screening (NBS) for tyrosinemia type 1 (TT1). However, false-positive SA results are often observed. Elevated SA may also be due to maleylacetoacetate isomerase deficiency (MAAI-D), which appears to be clinically insignificant. This study investigated whether urine organic acid (uOA) and quantitative urine maleic acid (Q-uMA) analyses can distinguish between TT1 and MAAI-D. We reevaluated/measured uOA (GC-MS) and/or Q-uMA (LC-MS/MS) in available urine samples of nine referred newborns (2 TT1, 7 false-positive), eight genetically confirmed MAAI-D children, and 66 controls. Maleic acid was elevated in uOA of 5/7 false-positive newborns and in the three available samples of confirmed MAAI-D children, but not in TT1 patients. Q-uMA ranged from not detectable to 1.16 mmol/mol creatinine in controls (n = 66) and from 0.95 to 192.06 mmol/mol creatinine in false-positive newborns and MAAI-D children (n = 10). MAAI-D was genetically confirmed in 4/7 false-positive newborns, all with elevated Q-uMA, and rejected in the two newborns with normal Q-uMA. No sample was available for genetic analysis of the last false-positive infant with elevated Q-uMA. Our study shows that MAAI-D is a recognizable cause of false-positive TT1 NBS results. Elevated urine maleic acid excretion seems highly effective in discriminating MAAI-D from TT1.

Evaluating genetic susceptibility to Staphylococcus aureus bacteremia in African Americans using admixture mapping.

The incidence of Staphylococcus aureus bacteremia (SAB) is significantly higher in African American (AA) than in European-descended populations. We used admixture mapping (AM) to test the hypothesis that genomic variations with different frequencies in European and African ancestral genomes influence susceptibility to SAB in AAs. A total of 565 adult AAs (390 cases with SAB; 175 age-matched controls) were genotyped for AM analysis. A case-only admixture score and a mixed χ2(1df) score (MIX) to jointly evaluate both single-nucleotide polymorphism (SNP) and admixture association (P<5.00e-08) were computed using MIXSCORE. In addition, a permutation scheme was implemented to derive multiplicity adjusted P-values (genome-wide 0.05 significance threshold: P<9.46e-05). After empirical multiplicity adjustment, one region on chromosome 6 (52 SNPs, P=4.56e-05) in the HLA class II region was found to exhibit a genome-wide statistically significant increase in European ancestry. This region encodes genes involved in HLA-mediated immune response and these results provide additional evidence for genetic variation influencing HLA-mediated immunity, modulating susceptibility to SAB.

Socioeconomic disparities in gait speed and associated characteristics in early old age.

BACKGROUND: A few studies have documented associations between socioeconomic position and gait speed, but the knowledge about factors from various domains (personal factors, lifestyle, occupation…) which contribute to these disparities is limited. Our objective was to assess socioeconomic disparities in usual gait speed in a general population in early old age in France, and to identify potential contributors to the observed disparities, including occupational factors. METHODS: The study population comprised 397 men and 339 women, aged 55 to 69, recruited throughout France for the field pilot of the CONSTANCES cohort. Gait speed was measured in meters/second. Socioeconomic position was based on self-reported occupational class. Information on personal characteristics, lifestyle, comorbidities and past or current occupational physical exposure came either from the health examination, from interview or from self-administered questionnaire. Four groups were considered according to sex-specific distributions of speed (the two slowest thirds versus the fastest third, for each gender). Logistic regression models adjusted for health screening center and age allowed to the study of cross-sectional associations between: 1- slower speed and occupational class; 2- slower speed and each potential contributor; 3- occupational class and selected potential contributors. The association between speed and occupational class was then further adjusted for the factors significantly associated both with speed and occupational class, in order to assess the potential contribution of these factors to disparities. RESULTS: With reference to managers/executives, gait speed was reduced in less skilled categories among men (OR 1.21 [0.72-2.05] for Intermediate/Tradesmen, 1.95 [0.80-4.76] for Clerks, Sale/service workers, 2.09 [1.14-3.82] for Blue collar/Craftsmen) and among women (OR 1.12 [0.55-2.28] for Intermediate/Tradesmen, 2.33 [1.09-4.97] for Clerks, 2.48 [1.18-5.24] for Sale/service workers/Blue collar/Craftsmen). Among men, occupational exposure to carrying heavy loads explained a large part of socioeconomic disparities. Among women, obesity and occupational exposure to repetitive work contributed independently to the disparities. CONCLUSIONS: This study suggests that some potentially modifiable occupational and personal factors explain at least part of the differences in gait speed between occupational classes, and that these factors differ between men and women. Longitudinal studies are needed to confirm and complement these findings.

Musculoskeletal pain at various anatomical sites and socioeconomic position: Results of a national survey.

BACKGROUND: Prevalence of musculoskeletal pain according to sites of pain and associated factors in the community has not been thoroughly documented. The association between pain and socioeconomic position has been studied by several authors, but without details in most studies regarding sites of pain, whereas the relations with social position could differ according to the site of pain. The objective of this study was to explore these differences in the community in France. METHODS: The national Health and Occupational History survey was conducted in France in 2006 in subjects aged 20-74 years. Self-assessment of pain at various sites in the previous year was recorded. Five sites were considered here: back, neck, shoulder, upper limb, and lower limb. After a description of prevalence according to gender and age, the associations with socioeconomic position at the beginning of the subjects' working life, in seven categories, were studied with logistic models adjusted for age. The analyses were limited to those aged 30-74 years and were conducted separately for men and women. RESULTS: Of the 5520 males and 6643 females studied, prevalence was the highest for back pain (35% for males, 37% for females). Pain was globally more frequent for women. For all sites of pain an increase with age was significant for women. This was not observed in men for back pain (highest prevalence in the 40- to 49-year-old age group) or neck pain. Overall, prevalence of pain was the lowest for professionals (reference category in the analyses). For males, the first occupation as a farmer or blue-collar worker was associated with an increased prevalence for most sites of pain, with odds ratios close to 2. For females, prevalence was increased for more socioeconomic categories, as compared to professionals. Among the five sites, neck pain was an exception: for both men and women, no association was observed between neck pain and socioeconomic position. CONCLUSION: Although exploratory, these results are consistent with the available knowledge on occupational and personal risk factors for pain, which differ according to the site of pain. Other studies are needed to better understand the causal mechanisms underlying the associations observed.

Heavy smoking and lung cancer: are women at higher risk? Result of the ICARE study.

BACKGROUND: Whether women are more or equally susceptible to the carcinogenic effects of cigarette smoke on the lungs compared with men is a matter of controversy. Using a large French population-based case-control study, we compared the lung cancer risk associated with cigarette smoking by gender. METHODS: The study included 2276 male and 650 female cases and 2780 male and 775 female controls. Lifetime smoking exposure was represented by the comprehensive smoking index (CSI), which combines the duration, intensity and time since cessation of smoking habits. The analysis was conducted among the ever smokers. All of the models were adjusted for age, department (a regional administrative unit), education and occupational exposures. RESULTS: Overall, we found that the lung cancer risk was similar among men and women. However, we found that women had a two-fold greater risk associated with a one-unit increase in CSI than men of developing either small cell carcinoma (OR=15.9, 95% confidence interval (95% CI) 7.6, 33.3 and 6.6, 95% CI 5.1, 8.5, respectively; P<0.05) or squamous cell carcinoma (OR=13.1, 95% CI 6.3, 27.3 and 6.1, 95% CI 5.0, 7.3, respectively; P<0.05). The association was similar between men and women for adenocarcinoma. CONCLUSION: Our findings suggest that heavy smoking might confer to women a higher risk of lung cancer as compared with men.

The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation.

The folding of both wild-type and mutant forms of the cystic-fibrosis transmembrane-conductance regulator (CFTR), a plasma-membrane chloride-ion channel, is inefficient. Most nascent CFTR is retained in the endoplasmic reticulum and degraded by the ubiquitin proteasome pathway. Aberrant folding and defective trafficking of CFTRDeltaF508 is the principal cause of cystic fibrosis, but how the endoplasmic-reticulum quality-control system targets CFTR for degradation remains unknown. CHIP is a cytosolic U-box protein that interacts with Hsc70 through a set of tetratricorepeat motifs. The U-box represents a modified form of the ring-finger motif that is found in ubiquitin ligases and that defines the E4 family of polyubiquitination factors. Here we show that CHIP functions with Hsc70 to sense the folded state of CFTR and targets aberrant forms for proteasomal degradation by promoting their ubiquitination. The U-box appeared essential for this process because overexpresion of CHIPDeltaU-box inhibited the action of endogenous CHIP and blocked CFTR ubiquitination and degradation. CHIP is a co-chaperone that converts Hsc70 from a protein-folding machine into a degradation factor that functions in endoplasmic-reticulum quality control.

Clinical and dermoscopic features of 88 scalp naevi in 39 children.

BACKGROUND: Paediatric scalp naevi may represent a source of anxiety for practitioners and parents, as the clinical and dermoscopic features of typical naevi have yet to be defined. Prompted by concern about the large size, irregular borders and colour variation of scalp naevi, clinicians and parents may request unnecessary excision of these naevi. OBJECTIVES: To establish the typical clinical and dermoscopic patterns of scalp naevi in children younger than 18 years old to help optimize clinical care and management. METHODS: Scalp naevi were imaged with a camera (Canon Rebel, XSi; Canon, Tokyo, Japan) and dermoscopic attachment (Dermlite Foto, 30 mm lens; 3Gen, San Juan Capistrano, CA, U.S.A.) to the camera. The clinical and dermoscopic images were reviewed and analysed. Both acquired and congenital scalp naevi were included but were not further differentiated from each other. RESULTS: We obtained clinical and dermoscopic images of 88 scalp naevi in 39 white children. Two subjects had received chronic immunosuppressive medication. Nineteen children had a family history of melanoma. Boys (18/39 subjects, 46%) possessed 68% (60 naevi) of scalp naevi imaged. Younger (< 10 years old) subjects (24/39 subjects, 62%) possessed 42% (37 naevi) of scalp naevi. The main clinical patterns included eclipse (n=18), cockade (n = 3), solid brown (n=42) and solid pink (n=25) naevi. Solid-coloured naevi showed the following dermoscopic patterns: globular (57%), complex (reticular-globular) (27%), reticular (9%), homogeneous (6%) and fibrillar (1%). The majority of naevi had a unifying feature - perifollicular hypopigmentation, which caused the appearance of scalloped, irregular borders if occurring on the periphery, or variegation in pigmentation, if occurring within the naevi. CONCLUSIONS: Older subjects and boys tend to harbour a larger proportion of scalp naevi. The main clinical patterns include solid-coloured and eclipse naevi. The most common dermoscopic pattern of scalp naevi is the globular pattern. Perifollicular hypopigmentation is a hallmark feature of signature scalp naevi. Dermoscopy is a noninvasive tool in the evaluation of cutaneous melanocytic lesions in children and may decrease the number of unnecessary excisions.

Male reproductive system defects and subfertility in a mutant mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a dysmorphogenesis syndrome resulting from mutations in the GJA1 gene encoding the gap junction protein, connexin43 (CX43). In the testis this connexin localizes between Leydig cells, Sertoli cells and between Sertoli cells and germ cells. It is essential for Sertoli cell differentiation and spermatogenesis. This study explored male fertility in Gja1(Jrt) /+ mice which carry a dominant mutation that causes an amino acid substitution (G60S) in CX43. Gja1(Jrt) /+ mice mimic the phenotype of ODDD. Immunodetection methods revealed a reduction of both total CX43 and CX43 in membrane plaques in mutant testes. Correspondingly, intercellular coupling along the tubules was diminished as revealed by fluorescent dye transfer. Light and electron microscopy revealed loss of germ cells and sloughing of germ cells into the tubular lumen. There were also irregularities in size and shape of Sertoli cell nuclei. Analyses of cauda epididymal sperm indicated significant decreases in sperm count and sperm velocity parameters associated with sperm vigour, and significantly lower sperm head movement parameters associated with progressiveness. A significant decrease was also observed in total per cent motility. These results further confirm a critical role for CX43 in spermatogenesis and sperm maturation and support the possibility of subfertility in ODDD human males.

The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria.

The entry of segments of preproteins of defined lengths into the matrix space of mitochondria was studied. The mitochondrial chaperone Hsp70 (mtHsp70) interacted with proteins emerging from the protein import channel and stabilized translocation intermediates across the membranes in an adenosine triphosphate-dependent fashion. The chaperone bound to the presequence and mature parts of preproteins. In the absence of mtHsp70 binding, preproteins with less than 30 to 40 residues in the matrix diffused out of mitochondria. Thus, protein translocation was reversible up to a late stage. The import channels in both mitochondrial membranes constitute a passive pore that interacts weakly with polypeptide chains entering the matrix.

Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.

DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70.

The folding of proteins and the assembly of protein complexes within subcompartments of the eukaryotic cell is catalysed by different members of the Hsp70 protein family. The chaperone function of Hsp70 proteins in these events is regulated by members of the DnaJ-like protein family, which occurs through direct interaction of different Hsp70 and DnaJ-like protein pairs that appear to be specifically adapted to each other. This review highlights the diversity of functions of DnaJ-like proteins by using specific examples of DnaJ-Hsp70 interactions with polypeptides in yeast protein-biogenesis pathways.

Mitochondrial molecular chaperones: their role in protein translocation.

After synthesis in the cytosol, most mitochondrial proteins must traverse mitochondrial membranes to reach their functional location. During this process, proteins become unfolded and then refold to attain their native conformation after crossing the lipid bilayers. Mitochondrial molecular chaperones play an essential mechanistic role at various steps of this process. They facilitate presequence translocation, unfolding of the cytosol-localized domains of precursor proteins, movement across the mitochondrial membranes and, finally, folding of newly imported proteins within the matrix.

Tissue regeneration and the epididymal stem cell.

BACKGROUND: In most pseudostratified epithelia, basal cells represent a multipotent adult stem cell population. These cells generally remain in a quiescent state, until they are stimulated to respond to tissue damage by initiating epithelial regeneration. In the epididymis, cell proliferation occurs at a relatively slow rate under normal physiological conditions. Epididymal basal cells have been shown to share common properties with multipotent adult stem cells. The development of organoids from stem cells represents a novel approach for understanding cellular differentiation and characterization of stem cells. OBJECTIVE: To review the literature on tissue regeneration in the epididymis and demonstrate the presence of an epididymal stem cell population. METHODS: PubMed database was searched for studies reporting on cell proliferation, regeneration, and stem cells in the epididymis. Three-dimensional cell culture of epididymal cells was used to determine whether these can develop into organoids in a similar fashion to stem cells from other tissues. RESULTS: The epididymal epithelium can rapidly regenerate following orchidectomy or efferent duct ligation, in order to maintain epithelial integrity. Studies have isolated a highly purified fraction of rat epididymal basal cells and reported that these cells displayed properties similar to those of multipotent adult stem cells. In two-dimensional cell culture conditions, these cells differentiated into cells which expressed connexin 26, a marker of columnar cells, and cytokeratin 8. Furthermore, three-dimensional cell culture of epididymal cells resulted in the formation of organoids, a phenomenon associated with the proliferation and differentiation of stem cells in vitro. CONCLUSIONS: The rapid proliferation and tissue regeneration of the epididymal epithelium to preserve its integrity following tissue damage as well as the ability of cells to differentiate into organoids in vitro support the notion of a resident progenitor/stem cell population in the adult epididymis.

Prenatal exposure to betamethasone causes intergenerational impairment of epididymal development in the rat.

BACKGROUND: Studies on epididymal toxicology are scarce. Betamethasone (BM) is a glucocorticoid used in clinical practice for antenatal therapy. We previously reported changes to testicular morphology, altered sperm quality, and fertility in adult rats following intrauterine administration of BM. OBJECTIVES: Given that high levels of corticosteroids during gestation lead to fetal androgen depletion, and the essential role of testosterone during epididymal development, here we investigated epididymal morphology and physiology in the F1 and F2 male offspring of female rats treated with BM during gestation. MATERIALS AND METHODS: Pregnant rats were randomly divided into two experimental groups: control (saline vehicle, n = 11) and BM-treated group (0.1 mg/kg betamethasone 21-phosphate disodium, n = 13). Rats received an intramuscular injection of vehicle or BM on gestational days 12, 13, 18, and 19. This encompasses the beginning of the critical window of male rat reproductive tract development. A subset of three males from each litter (n = 5 litters/group) was used: One rat per litter was euthanized at puberty, one was euthanized at adulthood, while the others were mated with a non-treated female to obtain the F2 generation. The same protocol described for the F1 was applied for F2, except for the mating protocol. RESULTS: In both F1 and F2 generations, prenatal BM exposure resulted in delayed differentiation of the cauda epididymal epithelium, characterized by increased cribriform appearance on PND 45, and displayed weaker or non-detectable Cx43 immunostaining. Furthermore, in the F1 generation only, immunostaining of TP63, a transcription factor expressed in basal cells, appeared more intense with a greater number of TP63-positive cells observed in the cauda epididymis. In adults, the epithelial area was reduced in the F1 BM rats. The contractile activity of isolated epididymal ducts was comparable between groups. DISCUSSION AND CONCLUSION: Prenatal BM exposure leads to intergenerational impairment in the development and structure of the rat epididymis.

Impaired plasma nonesterified fatty acid tolerance is an early defect in the natural history of type 2 diabetes.

CONTEXT: Abnormal plasma nonesterified fatty acid (NEFA) metabolism may play a role in the development of type 2 diabetes. OBJECTIVES: Our objectives were to demonstrate whether there is a defect in insulin-mediated suppression of plasma NEFA appearance (RaNEFA) and oxidation (OxNEFA) during enhanced intravascular triacylglycerol lipolysis early in the natural history of type 2 diabetes, and if so, to determine whether other mechanisms than reduced insulin-mediated suppression of intracellular lipolysis are involved. DESIGN: These are cross-sectional studies. SETTING: The studies were performed at an academic clinical research center. PARTICIPANTS: Nine healthy subjects with both parents with type 2 diabetes (FH+) and nine healthy subjects with no first-degree relatives with type 2 diabetes (FH-) with similar anthropometric features were included in the studies. INTERVENTIONS: Pancreatic clamps and iv infusion of stable isotopic tracers ([1,1,2,3,3-(2)H5]-glycerol and [U-(13)C]-palmitate or [1,2-(13)C]-acetate) were performed while intravascular triacylglycerol lipolysis was simultaneously clamped by iv infusion of heparin plus Intralipid at low (fasting) and high insulin levels. Oral nicotinic acid (NA) was used to inhibit intracellular lipolysis. MAIN OUTCOME MEASURES: RaNEFA and OxNEFA were determined. RESULTS: During heparin plus Intralipid infusion at high plasma insulin levels, and despite similar intravascular lipolytic rates, FH+ had higher RaNEFA and OxNEFA than FH- (RaNEFA: 17.4+/-6.3 vs. 9.2+/-4.2; OxNEFA: 4.5+/-1.8 vs. 2.3+/-1.5 micromol/kg lean body mass/min), independent of NA intake, gender, age, and body composition. In the presence of NA, insulin-mediated suppression of RaNEFA was still observed in FH-, but not in FH+. CONCLUSIONS: Increased RaNEFA and OxNEFA during intravascular lipolysis at high insulin levels occur early in the natural history of type 2 diabetes.

Do workers with self-reported symptoms have an elevated risk of developing upper extremity musculoskeletal disorders three years later?

OBJECTIVES: Few prospective studies have evaluated outcomes of workers with self-reported symptoms of upper extremity musculoskeletal disorders (UEMSD). The objective was to study the three-year outcomes of workers with self-reported symptoms, with or without a positive physical examination. METHODS: In 1993-4, 598 subjects highly exposed to repetitive work filled out a Nordic-style questionnaire. They underwent a standardised physical examination at that time and again in 1996-7 by the same occupational physician. The three-year outcomes (based on physical examination) of workers with a self-administered questionnaire positive at baseline for UEMSD, with or without a positive physical examination, were studied. RESULTS: The three-year incidence rate was 44.1%; one third of these incident cases had self-reported symptoms in 1993-4. Workers with a positive questionnaire had a significantly higher risk of UEMSD at physical examination three years later (80.1% UEMSD cases with positive questionnaires n = 354, vs 44.2% cases without positive questionnaires n = 69, p<0.001). Moreover, workers with positive questionnaires but without UEMSD diagnosed in 1993-4 (n = 177) also had a significantly higher risk of UEMSD at physical examination three years later (60.5% cases with positive questionnaires n = 26, vs 38.8% cases without positive questionnaires n = 52, p = 0.01). Results were similar when gender and age were taken into account. CONCLUSION: Workers highly exposed to repetitive movements have a high risk of developing UEMSD and should be followed closely in surveillance programmes. Workers with self-reported symptoms without UEMSD diagnosed in physical examination represented only one third of new cases three years later. However, their risk of developing UEMSD was significantly increased, compared with those without symptoms.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast.

BACKGROUND: In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated. RESULTS: The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain. CONCLUSIONS: These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.

Quebec neonatal mass urinary screening programme: from micromolecules to macromolecules.

The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2,500,000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb(3) on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb(3) in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb(3) in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.