A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

Givosiran in acute intermittent porphyria: A personalized medicine approach.

BACKGROUND: In patients with acute intermittent porphyria (AIP), induction of delta aminolevulinic acid synthase 1 (ALAS1) leads to haem precursor accumulation that may cause recurring acute attacks. In a recent phase III trial, givosiran significantly reduced the attack rate in severe AIP patients. Frequent adverse events were injection-site reaction, fatigue, nausea, chronic kidney disease and increased alanine aminotransferase. OBJECTIVES: To describe the efficacy and safety of givosiran based on a personalized medical approach. METHODS: We conducted a retrospective patient file study in 25 severe AIP patients treated with givosiran in France. We collected data on clinical and biochemical efficacy along with reports of adverse events. RESULTS: Givosiran drastically reduced the attack rate in our cohort, as 96% were attack-free at the time of the study. The sustained efficacy of givosiran in most patients allowed us to personalize dosing frequency. In 42%, givosiran was only given when haem precursor levels were increasing. Our data suggest that givosiran is most effective when given early in the disease course. We confirmed a high prevalence of adverse events. One patient discontinued treatment due to acute pancreatitis. All patients had hyperhomocysteinemia, and all patients with initial homocysteine levels available showed an increase under treatment. In this context, one patient was diagnosed with pulmonary embolism. CONCLUSION: The sustained effect of givosiran allowed a decrease in dosing frequency without compromising treatment efficacy. The high prevalence of adverse events emphasizes the importance of restricting the treatment to severe AIP and administering the minimum effective dose for each patient.

Vitamin C improves microvascular reactivity and peripheral tissue perfusion in septic shock patients.

BACKGROUND: Vitamin C has potential protective effects through antioxidant and anti-inflammatory properties. However, the effect of vitamin C supplementation on microvascular function and peripheral tissue perfusion in human sepsis remains unknown. We aimed to determine vitamin C effect on microvascular endothelial dysfunction and peripheral tissue perfusion in septic shock patients. METHODS: Patients with septic shock were prospectively included after initial resuscitation. Bedside peripheral tissue perfusion and skin microvascular reactivity in response to acetylcholine iontophoresis in the forearm area were measured before and 1 h after intravenous vitamin C supplementation (40 mg/kg). Norepinephrine dose was not modified during the studied period. RESULTS: We included 30 patients with septic shock. SOFA score was 11 [8-14], SAPS II was 66 [54-79], and in-hospital mortality was 33%. Half of these patients had vitamin C deficiency at inclusion. Vitamin C supplementation strongly improved microvascular reactivity (AUC 2263 [430-4246] vs 5362 [1744-10585] UI, p = 0.0004). In addition, vitamin C supplementation improved mottling score (p = 0.06), finger-tip (p = 0.0003) and knee capillary refill time (3.7 [2.6-5.5] vs 2.9 [1.9-4.7] s, p < 0.0001), as well as and central-to-periphery temperature gradient (6.1 [4.9-7.4] vs 4.6 [3.4-7.0] °C, p < 0.0001). The beneficial effects of vitamin C were observed both in patients with or without vitamin C deficiency. CONCLUSION: In septic shock patients being resuscitated, vitamin C supplementation improved peripheral tissue perfusion and microvascular reactivity whatever plasma levels of vitamin C. ClinicalTrials.gov Identifier: NCT04778605 registered 26 January 2021.

Expanding the phenotype of X-linked SSR4-CDG: Connective tissue implications.

Signal sequence receptor protein 4 (SSR4) is a subunit of the translocon-associated protein complex, which participates in the translocation of proteins across the endoplasmic reticulum membrane, enhancing the efficiency of N-linked glycosylation. Pathogenic variants in SSR4 cause a congenital disorder of glycosylation: SSR4-congenital disorders of glycosylation (CDG). We describe three SSR4-CDG boys and review the previously reported. All subjects presented with hypotonia, failure to thrive, developmental delay, and dysmorphic traits and showed a type 1 serum sialotransferrin profile, facilitating the diagnosis. Genetic confirmation of this X-linked CDG revealed one de novo hemizygous deletion, one maternally inherited deletion, and one de novo nonsense mutation of SSR4. The present subjects highlight the similarities with a connective tissue disorder (redundant skin, joint laxity, blue sclerae, and vascular tortuosity). The connective tissue problems are relevant, and require preventive rehabilitation measures. As an X-linked disorder, genetic counseling is essential.

A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection.IMPORTANCE Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.

Long term outcome of MPI-CDG patients on D-mannose therapy.

Mannose phosphate isomerase MPI-CDG (formerly CDG-1b) is a potentially fatal inherited metabolic disease which is readily treatable with oral D-mannose. We retrospectively reviewed long-term outcomes of patients with MPI-CDG, all but one of whom were treated with D-mannose. Clinical, biological, and histological data were reviewed at diagnosis and on D-mannose treatment. Nine patients were diagnosed with MPI-CDG at a median age of 3 months. The presenting symptoms were diarrhea (n = 9), hepatomegaly (n = 9), hypoglycemia (n = 8), and protein loosing enteropathy (n = 7). All patients survived except the untreated one who died at 2 years of age. Oral D-mannose was started in eight patients at a median age of 7 months (mean 38 months), with a median follow-up on treatment of 14 years 9 months (1.5-20 years). On treatment, two patients developed severe portal hypertension, two developed venous thrombosis, and 1 displayed altered kidney function. Poor compliance with D-mannose was correlated with recurrence of diarrhea, thrombosis, and abnormal biological parameters including coagulation factors and transferrin profiles. Liver fibrosis persisted despite treatment, but two patients showed improved liver architecture during follow-up. This study highlights (i) the efficacy and safety of D-mannose treatment with a median follow-up on treatment of almost 15 years (ii) the need for life-long treatment (iii) the risk of relapse with poor compliance, (iii) the importance of portal hypertension screening (iv) the need to be aware of venous and renal complications in adulthood.

Novel variants and clinical symptoms in four new ALG3-CDG patients, review of the literature, and identification of AAGRP-ALG3 as a novel ALG3 variant with alanine and glycine-rich N-terminus.

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man5 GlcNAc2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect.

Wide clinical spectrum in ALG8-CDG: clues from molecular findings suggest an explanation for a milder phenotype in the first-described patient.

BACKGROUND: Congenital disorders of glycosylation (CDG) includes ALG8 deficiency, a protein N-glycosylation defect with a broad clinical spectrum. If most of the 15 previously reported patients present an early-onset multisystem severe disease and early death, three patients including the cas princeps, present long-term survival and less severe symptoms. METHODS: In order to further characterize ALG8-CDG, two new ALG8 patients are described and mRNA analyses of the ALG8-CDG cas princeps were effected. RESULTS: One new patient exhibited a hepato-intestinal and neurological phenotype with two novel variants (c.91A > C p.Thr31Pro; c.139dup p.Thr47Asnfs*12). The other new patient, homozygous for a known variant (c.845C > T p.Ala282Val), presented a neurological phenotype with epilepsy, intellectual disability and retinis pigmentosa. The cas princeps ALG8-CDG patient was reported to have two heterozygous frameshift variants predicted to be without activity. We now described a novel ALG8 transcript variant in this patient and the 3D model of the putative encoded protein reveals no major difference with that of the normal ALG8 protein. CONCLUSION: The description of the two new ALG8 patients affirms that ALG8-CDG is a severe disease. In the cas princeps, as the originally described frameshift variants are degraded, the novel variant is promoted and could explain a milder phenotype.

CCDC115-CDG: A new rare and misleading inherited cause of liver disease.

Congenital disorders of glycosylation (CDG) linked to defects in Golgi apparatus homeostasis constitute an increasing part of these rare inherited diseases. Among them, COG-CDG, ATP6V0A2-CDG, TMEM199-CDG and CCDC115-CDG have been shown to disturb Golgi vesicular trafficking and/or lumen pH acidification. Here, we report 3 new unrelated cases of CCDC115-CDG with emphasis on diagnosis difficulties related to strong phenotypic similarities with mitochondriopathies, Niemann-Pick disease C and Wilson Disease. Indeed, while two individuals clinically presented with early and severe liver fibrosis and cirrhosis associated with neurological symptoms, the other one "only" showed isolated and late severe liver involvement. Biological results were similar to previously described patients, including hypercholesterolemia, elevated alkaline phosphatases and defects in copper metabolism. CDG screening and glycosylation study finally led to the molecular diagnosis of CCDC115-CDG. Besides pointing to the importance of CDG screening in patients with unexplained and severe liver disease, these reports expand the clinical and molecular phenotypes of CCDC115-CDG. The hepatic involvement is particularly addressed. Furthermore, hypothesis concerning the pathogenesis of the liver disease and of major biological abnormalities are proposed.

Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N- and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG, and SLC35A3-CDG, are described as illustrative examples.

Clinical, laboratory and molecular findings and long-term follow-up data in 96 French patients with PMM2-CDG (phosphomannomutase 2-congenital disorder of glycosylation) and review of the literature.

BACKGROUND: Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a multisystem inborn error of metabolism. OBJECTIVES: To better characterise the natural history of PMM2-CDG. METHODS: Medical charts of 96 patients with PMM2-CDG (86 families, 41 males, 55 females) were retrospectively reviewed. Data on clinical, laboratory and molecular parameters at diagnosis were analysed. Follow-up data at last examination were reported for 25 patients. RESULTS: The patients were born between 1963 and 2011. Diagnosis of PMM2-CDG was made at a mean (SD) age of 6.8 (8.5) years. The presenting signs were mostly neurological (hypotonia, intellectual disability, cerebellar syndrome) and observed in almost all the patients. A total of 38 patients (14 males, 24 females) exhibited, in addition to neurological signs, visceral features including at least one of these: feeding difficulty requiring a nutritional support (n=23), cardiac features (n=20; pericarditis: 14, cardiac malformation: 9, cardiomyopathy: 2), hepato-gastrointestinal features (n=12; chronic diarrhoea: 7, protein-losing enteropathy: 1, ascites: 3, liver failure: 1, portal hypertension: 1), kidney features (n=4; nephrotic syndrome: 2, tubulopathy: 2) and hydrops fetalis (n=1). Twelve patients died at a mean age of 3.8 years (especially from pericarditis and other cardiac issues). Laboratory abnormalities mostly included elevated transaminases and abnormal coagulation parameters. High thyreostimulin levels, hypocholesterolemia, hypoalbuminemia and elevated transaminases were associated with the visceral phenotype. Besides the common Arg141His PMM2 variant harboured by half of the patients, 45 different variants were observed. CONCLUSIONS: PMM2-CDG clinical phenotype is heterogeneous in terms of clinical course, with no clear division between neurological and visceral presentations.

Associations between serum lipids and breast cancer incidence and survival in the E3N prospective cohort study.

PURPOSE: Several mechanistic studies support a role of cholesterol or its metabolites in breast cancer etiology, but associations have been inconsistent in epidemiological studies. In observational studies, possible reverse causation must be accounted for using a prospective design. We investigated prospective associations between pre-diagnostic serum lipid concentrations [total cholesterol (TC), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides], and both breast cancer risk and survival in the E3N cohort study. METHODS: Analyses were performed on 583 cases from the E3N prospective cohort diagnosed between 1994 and 2005, and 1,043 controls matched on date, age, recruitment center and menopausal status at blood collection. Odds ratios (OR) and 95% confidence intervals were estimated using conditional logistic regression. Risks of recurrence were estimated among cases using Cox proportional hazards model. Models were adjusted for lifestyle risk factors and mutually adjusted for lipid concentrations. Survival analyses were additionally adjusted for tumor characteristics. RESULTS: Overall, there was no association between any serum lipid and breast cancer risk or survival. In stratified analyses, statistically significant interaction was observed between TC and menopausal status (P interaction = 0.05) and between TC and waist circumference (P interaction = 0.03), although the ORs did not reach statistical significance in any of the strata. There was no statistically significant effect modification by BMI, time between blood donation and diagnosis or ER status. CONCLUSIONS: Our results suggest that serum lipids are not associated with breast cancer risk overall, but that menopausal status and waist circumference should be considered in further studies.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae-associated hemolytic uremic syndrome and hemolytic anemia.

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.

Long-term evolution of nutritional deficiencies after gastric bypass: an assessment according to compliance to medical care.

OBJECTIVE: To study long-term nutritional deficits based on adherence to a standardized nutritional care after gastric bypass (GBP). BACKGROUND: Long-term prospective data on nutritional complications after GBP are missing. It is not known whether severe deficiencies are prevented by standard multivitamin supplementation and what parameters are influenced by patient adherence to nutritional care. DESIGN: One hundred forty-four consecutive subjects from our prospective database (90% women, initial body mass index: 48 ± 15 kg/m2, age: 43 ± 10 years) who underwent GBP more than 3 years before the study were assessed. Multivitamins were systematically prescribed after GBP, and additional supplements were introduced if deficiencies were recorded during follow-up. We identified a group of 66 compliant subjects who attended yearly medical visits and a group of 32 noncompliant subjects who were recalled because they had not attended any visit for more than 2 years. RESULTS: Weight loss was 42 ± 14 kg at 3 years or later. The number of nutritional deficits per subject was 3.2 ± 2.3 before surgery and did not significantly increase between 1 and 3 years or later after GBP (3.4 ± 2.0 and 3.5 ± 2.3, respectively). However, specific nutritional deficits occurred despite long-term multivitamin supplementation, including vitamins B1, B12, and D and iron. Noncompliant subjects had more deficits than compliant subjects (4.2 ± 1.9 vs 2.9 ± 2.0 deficits per patient, P < 0.01) and the number of deficits correlated with the time from last visit (r = 0.285, P < 0.01). CONCLUSIONS: Lifelong medical care is required to maintain a good nutritional status after GBP. Monitoring of nutritional parameters is necessary to add supplementation for deficits that are not prevented by multivitamin preparations.

C-reactive protein and postmenopausal breast cancer risk: results from the E3N cohort study.

BACKGROUND: C-reactive protein (CRP), a marker of low-grade inflammation, has been associated with breast cancer risk, but results are scarce and inconsistent. METHODS: A case-control study nested within the E3N prospective cohort included 549 postmenopausal breast cancer cases and 1,040 matched controls, all free of breast cancer at baseline. Serum levels of CRP were measured in samples collected between 1995 and 1999. Unconditional logistic regression models were used to evaluate the association between CRP and breast cancer risk, adjusting for matching factors and known breast cancer risk factors. RESULTS: No association was observed between CRP levels and breast cancer risk overall. However, a significant interaction was observed between CRP levels and body mass index (BMI). A statistically significant increase in breast cancer risk was observed in overweight and obese women (BMI ≥ 25 kg/m(2)) (OR 1.92, 95 % CI 1.20-3.08 for CRP ≥ 2.5 mg/L compared with CRP < 1.5 mg/l, p trend = 0.003, p interaction between CRP and BMI = 0.03). Similar results were observed in women with waist circumference (WC) ≥ 88 cm (p trend = 0.01, p interaction = 0.06) and waist-to-hip ratio (WHR) ≥ 0.80 (p trend = 0.06, p interaction = 0.35). CRP levels were not associated with breast cancer risk in women with normal BMI, WC, or WHR. CONCLUSIONS: We found a positive association between CRP levels and postmenopausal breast cancer risk restricted to women with excess adiposity. The suggested relationship between low-grade inflammation, abdominal adiposity, and postmenopausal breast cancer risk deserves further investigation.

Agreement of seven 25-hydroxy vitamin D3 immunoassays and three high performance liquid chromatography methods with liquid chromatography tandem mass spectrometry.

BACKGROUND: Several recent studies have shown some discrepancies between 25-hydroxyvitamin D [25(OH)D] assay methods, despite some improvement in the past few years. The accuracy of 25(OH)D assay methods is still a real challenge for clinical laboratories. The aim of this study was to assess the agreement between a large panel of routine assays and a two-dimensional liquid chromatography/tandem mass spectrometry (2D LC-MS/MS) method, selected as the reference method. METHODS: Forty-nine human plasma samples with only endogenous 25(OH)D3 were analyzed with 11 different methods, especially with three LC-UV methods that differed in the extraction step. Seven routine immunoassays were also tested: two manual (RIA and EIA from IDS) and five fully-automated methods. The results of the 25(OH)D3 assays were compared with those of the 2D LC-MS/MS method using weighted Deming regression analysis, Bland-Altman plots and concordance correlation coefficient (CCC). The ability of these methods to properly classify patients was evaluated by sorting results depending on vitamin D status. RESULTS: The CCC was >0.90 for the three LC-UV methods and for most of the automated IA, meaning substantial agreement with 2D LC-MS/MS results. The ability to properly classify patients according to their vitamin D status was overall satisfactory for most of the methods tested (concordance >90%). CONCLUSIONS: The immunoassays available on Liaison, Isys, Architect and Elecsys, together with our in-house LC-UV method preceded by an SLE step met the minimum requirements for the assessment of vitamin D status in clinical laboratories.

Progression of aortic valve stenosis is associated with bone remodelling and secondary hyperparathyroidism in elderly patients--the COFRASA study.

AIMS: There is currently no medical therapy that can prevent the progression of aortic valve stenosis (AS). Recent data highlight a possible relationship between bone metabolism and AS progression but prospective data are lacking. METHODS AND RESULTS: Serum levels of calcium, phosphorus, creatinine, 25-OH vitamin D, intact parathyroid hormon (iPTH), C-terminal-telopeptide of type-1-collagen (CTX) and osteocalcin were assessed at baseline in 110 elderly patients (age ≥70 years) with at least mild AS. CTX/osteocalcin ratio was calculated as a marker of bone remodelling balance. AS severity was assessed at baseline and 1-year based on the mean gradient. Two-thirds of patients had low 25-OH vitamin D and 20% had secondary hyperparathyroidism. AS progression was not associated with age, glomerular filtration rate (GFR), calcium and phosphorus levels, calcium-phosphorus product, but significantly with iPTH, CTX/osteocalcin and vitamin D status (all P < 0.01). There was no correlation between iPTH and CTX/osteocalcin (R = 0.04, P = 0.70) and AS progression was associated with CTX/osteocalcin (R = 0.42, P = 0.009), but not with iPTH (R = 0.10, P = 0.55) in patients with normal vitamin D levels, whereas it was associated with iPTH (R = 0.47, P < 0.001) and not with CTX/osteocalcin (R = 0.04, P = 0.73) in those with low vitamin D levels, especially if mild renal insufficiency was present (R = 0.61, P < 0.001). CONCLUSION: In elderly patients with AS, we observed an association between AS progression and vitamin D, iPTH and CTX/osteocalcin ratio and their respective influence varied according to the vitamin D status. In patients with normal vitamin D levels, AS progression was associated with a bone resorptive balance, whereas in patients with low vitamin D levels, AS progression was associated with iPTH and secondary hyperparathyroidism, especially if mild renal insufficiency was present. These findings may have important prognostic and therapeutic implications. Trial registration information: Clinicaltrials.gov identifier number: NCT00338676, funded by AP-HP, the COFRASA study.

Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.

Biomarkers for the diagnosis and monitoring of nitrous oxide intoxication: objectives and methodology of the SFBC Working Group.

The recreational use of nitrous oxide (N2O) is an emerging public health issue. Chronic N2O abuse may result in various clinical symptoms, encompassing neurological, psychiatric and cardiovascular outcomes. Despite the difficulties for the laboratory investigation of N2O intoxication, there is currently no guidelines in France to help both clinicians and biologists use appropriate biomarkers for the diagnosis and monitoring of patients with clinical symptoms potentially related to N2O intoxication. A multi-disciplinary Working Group, carried out under the auspices of the French Society of Clinical Biology (SFBC) and in collaboration with the French Societies of Emergency Medicine (SFMU), Analytical Toxicology (SFTA), Hemostasis and Thrombosis (SFTH), Vitamins and Biofactors (SFVB), and the French Federation of Neurology (FFN), was recently implemented to elaborate practical guidelines. The methodology of the Working Group is based on the critical analysis of the literature, and raising concerns and objectives are grouped into five working packages. The present manuscript primarily aims to expound upon the methodology and objectives of the ongoing SFBC Working Group on N2O.