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.

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.

Two Novel Homozygous Mutations in Phosphoglucomutase 3 Leading to Severe Combined Immunodeficiency, Skeletal Dysplasia, and Malformations.

Phosphoglucomutase 3 (PGM3) deficiency is a rare congenital disorder of glycosylation. Most of patients with autosomal recessive hypomorphic mutations in PGM3 encoding for phosphoglucomutase 3 present with eczema, skin and lung infections, elevated serum IgE, as well as neurological and skeletal features. A few PGM3-deficient patients suffer from a more severe disease with nearly absent T cells and severe skeletal dysplasia. We performed targeted next-generation sequencing on two kindred to identify the underlying genetic etiology of a severe combined immunodeficiency with developmental defect. We report here two novel homozygous missense variants (p.Gly359Asp and p.Met423Thr) in PGM3 identified in three patients from two unrelated kindreds with severe combined immunodeficiency, neurological impairment, and skeletal dysplasia. Both variants segregated with the disease in the two families. They were predicted to be deleterious by in silico analysis. PGM3 enzymatic activity was found to be severely impaired in primary fibroblasts and Epstein-Barr virus immortalized B cells from the kindred carrying the p.Met423Thr variant. Our findings support the pathogenicity of these two novel variants in severe PGM3 deficiency.

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.

Influence of the environmental relative humidity on the inflammatory response of skin model after exposure to various environmental pollutants.

The skin is an essential barrier, protecting the body against the environment and its numerous pollutants. Several environmental pollutants are known to affect the skin, inducing premature aging through mechanisms including oxidative stress, inflammation, and impairment of skin functions. Even climate conditions can impact the skin. Therefore, using a Reconstructed Human Epidermis (RHE), we tested the effect of two samples of fine particulate matters (PM0.3-2.5 - one metals-rich sample and the other organic compounds-rich), two Volatile Organic Compounds mixtures (VOCs - from a solvent-based paint and a water-based paint) and Tobacco Smoke (TS). All pollutants affected cellular functionality, but to a lesser extent for the water-based paint VOC. This effect was enhanced when RHE were preconditioned for 2 h by a semi-dry airflow (45% relative humidity) before pollutants application, compared to preconditioning by a humid airflow (90% relative humidity). In the absence of preconditioning, IL-1α, IL-6, IL-8, and RANTES were almost systematically induced by pollutants. When RHE were preconditioned by a semi-dry or humid airflow before being subjected to pollutants, the increase of IL-1α, IL-8, and RANTES falls into two groups. Similarly to RHE not treated with pollutants, RHE treated with VOCs after preconditioning by a semi-dry airflow showed increased IL-1α, IL-8, and RANTES release. On the contrary, RHE treated with PM or TS after preconditioning by a semi-dry airflow show a lower increase in IL-1α, IL-8, and RANTES compared to preconditioning by a humid airflow. The effect of real environmental relative humidity conditions of the air, combined with acute exposure to various environmental pollutants, seemed to relate mainly to structural changes of the skin, determining the outcome of the inflammatory response depending on the physicochemical characteristics of pollutants.

Serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies.

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.

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.

Long-term follow-up in PMM2-CDG: are we ready to start treatment trials?

PURPOSE: PMM2-CDG is the most common congenital disorder of glycosylation (CDG), which presents with either a neurologic or multisystem phenotype. Little is known about the longitudinal evolution. METHODS: We performed data analysis on PMM2-CDG patients' clinical features according to the Nijmegen CDG severity score and laboratory data. Seventy-five patients (28 males) were followed up from 11.0 ± 6.91 years for an average of 7.4 ± 4.5 years. RESULTS: On a group level, there was no significant evolution in overall clinical severity. There was some improvement in mobility and communication, liver and endocrine function, and strabismus and eye movements. Educational achievement and thyroid function worsened in some patients. Overall, the current clinical function, the system-specific involvement, and the current clinical assessment remained unchanged. On follow-up there was improvement of biochemical variables with (near) normalization of activated partial thromboplastin time (aPTT), factor XI, protein C, antithrombin, thyroid stimulating hormone, and liver transaminases. CONCLUSION: PMM2-CDG patients show a spontaneous biochemical improvement and stable clinical course based on the Nijmegen CDG severity score. This information is crucial for the definition of endpoints in clinical trials.

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.

International clinical guidelines for the management of phosphomannomutase 2-congenital disorders of glycosylation: Diagnosis, treatment and follow up.

Phosphomannomutase 2 (PMM2-CDG) is the most common congenital disorder of N-glycosylation and is caused by a deficient PMM2 activity. The clinical presentation and the onset of PMM2-CDG vary among affected individuals ranging from a severe antenatal presentation with multisystem involvement to mild adulthood presentation limited to minor neurological involvement. Management of affected patients requires a multidisciplinary approach. In this article, a systematic review of the literature on PMM2-CDG was conducted by a group of international experts in different aspects of CDG. Our managment guidelines were initiated based on the available evidence-based data and experts' opinions. This guideline mainly addresses the clinical evaluation of each system/organ involved in PMM2-CDG, and the recommended management approach. It is the first systematic review of current practices in PMM2-CDG and the first guidelines aiming at establishing a practical approach to the recognition, diagnosis and management of PMM2-CDG patients.

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.

Early polysensitization is associated with allergic multimorbidity in PARIS birth cohort infants.

BACKGROUND: Profiles of allergic sensitization are poorly documented in infancy. Relations between early sensitization and allergic morbidity need to be clarified. METHODS: This study dealt with children involved in the Pollution and Asthma Risk: an Infant Study (PARIS), a population-based prospective birth cohort. Allergic sensitization to twelve food and four inhalant allergens was assessed at 18 months and defined by a specific immunoglobulin E (IgE) level ≥0.35 kUA /l. Health data were collected by standardized questionnaires at 2 and 6 years. Early allergic profiles were identified by an unsupervised cluster analysis based on health data at 2 years and IgE measurements. Profiles were compared with regard to allergic morbidity and multimorbidity at 6 years. RESULTS: Sensitization to any allergen concerned 13.6% of infants. By cluster analysis, 1525 infants were grouped into three profiles: 89.2% not or rarely sensitized (only 3.7% of sensitized), 9.2% mainly sensitized to one or few allergens (45.2% of monosensitized and 45.9% of paucisensitized) and 1.6% all polysensitized. The prevalence of doctor-diagnosed asthma, rhinitis, eczema, food allergy and multimorbidity at 2 years increased from profile one to profile three (p-trend <0.001). At 6 years, symptoms of current asthma, rhinitis, eczema and multimorbidity were significantly more frequent in the last two profiles. CONCLUSIONS: This study highlights, as early as 18 months of age, three profiles of increasing severity with regard to allergic sensitization and diseases. These profiles also differ in terms of allergic morbidity at 6 years. Early sensitization can predict allergic multimorbidity in childhood, and in the case of early polysensitization, multimorbidity is more frequent as soon as infancy.

ALG6-CDG: a recognizable phenotype with epilepsy, proximal muscle weakness, ataxia and behavioral and limb anomalies.

INTRODUCTION: Alpha-1,3-glucosyltransferase congenital disorder of glycosylation (ALG6-CDG) is a congenital disorder of glycosylation. The original patients were described with hypotonia, developmental disability, epilepsy, and increased bleeding tendency. METHODS: Based on Euroglycan database registration, we approached referring clinicians and collected comprehensive data on 41 patients. RESULTS: We found hypotonia and developmental delay in all ALG6-CDG patients and epilepsy, ataxia, proximal muscle weakness, and, in the majority of cases, failure to thrive. Nine patients developed intractable seizures. Coagulation anomalies were present in <50 % of cases, without spontaneous bleedings. Facial dysmorphism was rare, but seven patients showed missing phalanges and brachydactyly. Cyclic behavioral change, with autistic features and depressive episodes, was one of the most significant complaints. Eleven children died before the age of 4 years due to protein losing enteropathy (PLE), sepsis, or seizures. The oldest patient was a 40 year-old Dutch woman. The most common pathogenic protein alterations were p.A333V and p.I299Del, without any clear genotype-phenotype correlation. DISCUSSION: ALG6-CDG has been now described in 89 patients, making it the second most common type of CDG. It has a recognizable phenotype and a primary neurologic presentation.

Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly.

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a "cobblestone" brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.

Catalase activity, allelic variations in the catalase gene and risk of kidney complications in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Oxidative stress is involved in the pathogenesis of diabetic nephropathy. The antioxidant enzyme catalase plays a key role in redox regulation in the kidney. We investigated associations of catalase gene (CAT) polymorphisms and plasma catalase activity with diabetic nephropathy in type 1 diabetic patients. METHODS: We genotyped nine single nucleotide polymorphisms (SNPs) in the CAT region in participants from the Survival Genetic Nephropathy (SURGENE) (340 French participants, 10 year follow-up) and the Génétique de la Néphropathie Diabétique (GENEDIAB) (444 Belgian and French participants, 8 year follow-up) study cohorts. Replication was performed in a Brazilian cross-sectional cohort (n = 451). Baseline plasma catalase activity was measured in SURGENE (n = 120) and GENEDIAB (n = 391) participants. RESULTS: The A allele of rs7947841 was associated with the prevalence of incipient (OR 2.79, 95% CI 1.21, 6.24, p = 0.01) and established or advanced nephropathy (OR 5.72, 95% CI 1.62, 22.03, p = 0.007), and with the incidence of renal events, which were defined as new cases of microalbuminuria or progression to a more severe stage of nephropathy during follow-up (HR 1.82, 95% CI 1.13, 2.81, p = 0.01) in SURGENE participants. The same risk allele was associated with incipient nephropathy (OR 3.13, 95% CI 1.42, 7.24, p = 0.004) and with the incidence of end-stage renal disease (ESRD) (HR 2.11, 95% CI 1.23, 3.60, p = 0.008) in GENEDIAB participants. In both cohorts, the risk allele was associated with lower catalase activity. Associations with incipient and established or advanced nephropathy were confirmed in the replication cohort. CONCLUSIONS/INTERPRETATION: CAT variants were associated with the prevalence and incidence of diabetic nephropathy and ESRD in type 1 diabetic patients. Our results confirm the protective role of catalase against oxidative stress in the kidney.

Cobblestone lissencephaly: neuropathological subtypes and correlations with genes of dystroglycanopathies.

Cobblestone lissencephaly represents a peculiar brain malformation with characteristic radiological anomalies, defined as cortical dysplasia combined with dysmyelination, dysplastic cerebellum with cysts and brainstem hypoplasia. Cortical dysplasia results from neuroglial overmigration into the arachnoid space, forming an extracortical layer, responsible for agyria and/or 'cobblestone' brain surface and ventricular enlargement. The underlying mechanism is a disruption of the glia limitans, the outermost layer of the brain. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal recessive diseases with cerebral, ocular and muscular deficits, Walker-Warburg syndrome, muscle-eye-brain and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN and FKRP genes attributed these diseases to α-dystroglycanopathies. However, studies have not been able to identify causal mutations in the majority of patients and to establish a clear phenotype/genotype correlation. Therefore, we decided to perform a detailed neuropathological survey and molecular screenings in 65 foetal cases selected on the basis of histopathological criteria. After sequencing the six genes of α-dystroglycanopathies, a causal mutation was observed in 66% of cases. On the basis of a ratio of severity, three subtypes clearly emerged. The most severe, which we called cobblestone lissencephaly A, was linked to mutations in POMT1 (34%), POMT2 (8%) and FKRP (1.5%). The least severe, cobblestone lissencephaly C, was linked to POMGNT1 mutations (18%). An intermediary type, cobblestone lissencephaly B, was linked to LARGE mutations (4.5%) identified for the first time in foetuses. We conclude that cobblestone lissencephaly encompasses three distinct subtypes of cortical malformations with different degrees of neuroglial ectopia into the arachnoid space and cortical plate disorganization regardless of gestational age. In the cerebellum, histopathological changes support the novel hypothesis that abnormal lamination arises from a deficiency in granule cells. Our studies demonstrate the positive impact of histoneuropathology on the identification of α-dystroglycanopathies found in 66% of cases, while with neuroimaging criteria and biological values, mutations are found in 32-50% of patients. Interestingly, our morphological classification was central in the orientation of genetic screening of POMT1, POMT2, POMGNT1, LARGE and FKRP. Despite intensive research, one-third of our cases remained unexplained; suggesting that other genes and/or pathways may be involved. This material offers a rich resource for studies on the affected neurodevelopmental processes of cobblestone lissencephaly and on the identification of other responsible gene(s)/pathway(s).