Retinal Proteomics of a Mouse Model of Dystroglycanopathies Reveals Molecular Alterations in Photoreceptors.

Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for α-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, here we used comparative mass spectrometry-based proteomics and immunofluorescence microscopy and investigated the changes in the retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to the effects related to impaired α-DG O-mannosylation, we observed morphological alterations in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins, and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.

Adults with familial hypercholesterolaemia have healthier dietary and lifestyle habits compared with their non-affected relatives: the SAFEHEART study.

OBJECTIVE: Healthy lifestyle habits are the cornerstone in the management of familial hypercholesterolaemia (FH). Nevertheless, dietary studies on FH-affected populations are scarce. The present study analyses dietary habits, adherence to a Mediterranean diet pattern and physical activity in an adult population with FH and compares them with their non-affected relatives. DESIGN: Cross-sectional study. SETTING: Data came from SAFEHEART, a nationwide study in Spain.ParticipantsIndividuals (n 3714) aged ≥18 years with a genetic diagnosis of FH (n2736) and their non-affected relatives (n 978). Food consumption was evaluated using a validated FFQ. RESULTS: Total energy intake was lower in FH patients v. non-affected relatives (P<0·005). Percentage of energy from fats was also lower in the FH population (35 % in men, 36 % in women) v. those non-affected (38 % in both sexes, P<0·005), due to the lower consumption of saturated fats (12·1 % in FH patients, 13·2 % in non-affected, P<0·005). Consumption of sugars was lower in FH patients v. non-affected relatives (P<0·05). Consumption of vegetables, fish and skimmed milk was higher in the FH population (P<0·005). Patients with FH showed greater adherence to a Mediterranean diet pattern v. non-affected relatives (P<0·005). Active smoking was lower and moderate physical activity was higher in people with FH, especially women (P<0·005). CONCLUSIONS: Adult patients with FH report healthier lifestyles than their non-affected family members. They eat a healthier diet, perform more physical activity and smoke less. However, this patient group's consumption of saturated fats and sugars still exceeds guidelines.

Expression in retinal neurons of fukutin and FKRP, the protein products of two dystroglycanopathy-causative genes.

Purpose: Dystroglycanopathies are a heterogeneous group of recessive neuromuscular dystrophies that affect the muscle, brain and retina, and are caused by deficiencies in the O-glycosylation of α-dystroglycan. This post-translational modification is essential for the formation and maintenance of ribbon synapses in the retina. Fukutin and fukutin-related protein (FKRP) are two glycosyltransferases whose deficiency is associated with severe dystroglycanopathies. These enzymes carry out in vitro the addition of a tandem ribitol 5-phosphate moiety to the so-called core M3 phosphotrisaccharide of α-dystroglycan. However, their expression pattern and function in the healthy mammalian retina has not so far been investigated. In this work, we have addressed the expression of the FKTN (fukutin) and FKRP genes in the retina of mammals, and characterized the distribution pattern of their protein products in the adult mouse retina and the 661W photoreceptor cell line. Methods: By means of reverse transcription (RT)-PCR and immunoblotting, we have studied the expression at the mRNA and protein levels of the fukutin and FKRP genes in different mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of their protein products in mouse retinal sections and in 661W cultured cells. Results: Both genes were expressed at the mRNA and protein levels in the neural retina of all mammals studied. Fukutin was present in the cytoplasmic and nuclear fractions in the mouse retina and 661W cells, and accumulated in the endoplasmic reticulum. FKRP was located in the cytoplasmic fraction in the mouse retina and concentrated in the Golgi complex. However, and in contrast to retinal tissue, FKRP additionally accumulated in the nucleus of the 661W photoreceptors. Conclusions: Our results suggest that fukutin and FKRP not only participate in the synthesis of O-mannosyl glycans added to α-dystroglycan in the endoplasmic reticulum and Golgi complex, but that they could also play a role, that remains to be established, in the nucleus of retinal neurons.

Gallus gallus orthologous to human alpha-dystroglycanopathies candidate genes: Gene expression and characterization during chicken embryogenesis.

Alpha-dystroglycanopathies are a heterogenic group of human rare diseases that have in common defects of α-dystroglycan O-glycosylation. These congenital disorders share common features as muscular dystrophy, malformations on central nervous system and more rarely altered ocular development, as well as mutations on a set of candidate genes involved on those syndromes. Severity of the syndromes is variable, appearing Walker-Warburg as the most severe where mutations at protein O-mannosyl transferases POMT1 and POMT2 genes are frequently described. When studying the lack of MmPomt1 in mouse embryonic development, as a murine model of Walker-Warburg syndrome, MmPomt1 null phenotype was lethal because Reitchert's membrane fails during embryonic development. Here, we report gene expression from Gallus gallus orthologous genes to human candidates on alpha-dystroglycanopathies POMT1, POMT2, POMGnT1, FKTN, FKRP and LARGE, making special emphasis in expression and localization of GgPomt1. Results obtained by quantitative RT-PCR, western-blot and immunochemistry revealed close gene expression patterns among human and chicken at key tissues affected during development when suffering an alpha-dystroglycanopathy, leading us to stand chicken as a useful animal model for molecular characterization of glycosyltransferases involved in the O-glycosylation of α-Dystroglycan and its role in embryonic development.

Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease.

PURPOSE: The POMGNT1 gene, encoding protein O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene's lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. METHODS: Using reverse transcription (RT)-PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. RESULTS: POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. CONCLUSIONS: Our results indicate that POMGnT1 participates not only in the synthesis of O-mannosyl glycans added to α-DG in the Golgi complex but also in the glycosylation of other yet-to-be-identified proteins in the nucleus of mouse photoreceptors.

Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1ß, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

O-Mannose and O-N-acetyl galactosamine glycosylation of mammalian α-dystroglycan is conserved in a region-specific manner.

Defects in the O-linked glycosylation of the peripheral membrane protein α-dystroglycan (α-DG) are the main cause of several forms of congenital muscular dystrophies and thus the characterization of the glycosylation of α-DG is of great medical importance. A detailed investigation of the glycosylation pattern of the native α-DG protein is essential for the understanding of the biological processes related to human disease in which the protein is involved. To date, several studies have reported novel O-glycans and attachment sites on the mucin-like domain of mammalian α-DG with both similar and contradicting glycosylation patterns, indicating the species-specific O-glycosylation of mammalian α-DG. By applying a standardized purification scheme and subsequent glycoproteomic analysis of native α-DG from rabbit and human skeletal muscle biopsies and from cultured mouse C2C12 myotubes, we show that the O-glycosylation patterns of the mucin-like domain of native α-DG are conserved among mammalians in a region-specific manner.

Relationship between seasonal influenza rates and hospitalization and mortality rates due to acute cardiovascular diseases in a Spanish region. / Relación entre las tasas de gripe estacional y las tasas de hospitalización y mortalidad hospitalaria por enfermedades cardiovasculares agudas en una región española.

INTRODUCTION AND OBJECTIVE: Influenza virus infection can contribute to cardiovascular morbidity and mortality. The purpose of this study is to confirm if the increase in seasonal influenza rates is associated with a growth in hospitalisation and mortality rates for acute cardiovascular diseases (ACVD). METHODS: Retrospective cohort study of hospital discharges due to ACVD (myocardial infarction, unstable angina, heart failure and ischemic stroke) in the Castilla y León (Spain) hospital system between 2001 and 2015. Hospitalisation and hospital mortality rates due to ACVD, and influenza rates in Castilla y León between 2001 and 2015 were studied. To calculate hospitalisation and mortality rates, the hospital discharges database was used; for influenza rates, the weekly reports of the Sentinel System for the surveillance of influenza in Spain (Carlos III Health Institute) were used. A statistical analysis of linear and multivariate logistic regressions was performed. RESULTS: 239,586 ACVD (myocardial infarction: 55,004; unstable angina: 15,406; heart failure: 11,1647; ischemic stroke: 57,529) were studied. Increasing rates of influenza were associated with increased mortality due to ACVD and all the diseases studied, except unstable angina. A linear correlation was observed between influenza rates and hospitalisation (r2=0.03; p=0.02) and mortality (r2=0.14; p<0.001) rates by ACVD. Virtually all influenza rates were associated, as independent variables, to an increase in mortality due to ACVD, being higher in rates>139/100,000 inhabitants (OR: 1.25; p<0.001). CONCLUSIONS: The rates of hospitalisation and in-hospital mortality due to ACVD in the period 2001-2015 increased in relation to infection rates due to the influenza virus.

Adequacy of antibiotic prophylaxis protocol in appendectomies in children.

OBJECTIVE: To assess compliance of antibiotic prophylaxis in surgery for acute appendicitis in children and its effect on surgical site infection. METHODS: We carried out a prospective cohort study to evaluate compliance of antibiotic prophylaxis in appendectomies in children. An assessment of the level of compliance with prophylaxis was made, as well as the causes of non-compliance. The effect of non-compliance of antibiotic prophylaxis on the incidence of surgical site infection was studied with the adjusted relative risk (RR) with a backstep logistic regression model. RESULTS: The study included a total of 412 patients. Antibiotic prophylaxis was indicated in 348 patients, and administered in 95.7% of cases, with an overall protocol compliance of 90.7%. The principal cause of non-compliance was time of initiation. Cumulative incidence of surgical site infection was 2.7%. No relationship was found between inadequate prophylaxis compliance and infection (RR: 1.01; 95% confidence interval: 0.95-1.11; p = 0.61). CONCLUSIONS: Compliance of antibiotic prophylaxis was high, but could be improved. No relationship was found between prophylaxis compliance and surgical site infection rate.

OBJETIVO: Evaluar la adecuación de la profilaxis antibiótica en la cirugía de apendicitis aguda en niños y su efecto en la infección del sitio quirúrgico. MÉTODO: Estudio de cohortes prospectivo para evaluar la adecuación al protocolo de la profilaxis antibiótica en apendicectomías en población infantil. Se evaluaron la administración de la profilaxis y las causas de la inadecuación. Se estudió el efecto de la inadecuación en la incidencia de infección del sitio quirúrgico con el riesgo relativo (RR) ajustado con un modelo de regresión logística por pasos hacia atrás. RESULTADOS: Se estudiaron 412 pacientes. La profilaxis antibiótica estaba indicada en 348 pacientes y se administró en el 95.7% de los casos, con una adecuación global al protocolo del 90.7%. La causa principal del incumplimiento fue la hora de inicio. La incidencia acumulada de infección del sitio quirúrgico fue del 2.7%. No se encontró relación entre la adecuación de la profilaxis y la infección del sitio quirúrgico (RR: 1.01; intervalo de confianza del 95%: 0.95-1.11; p = 0.61). CONCLUSIONES: La adecuación de la profilaxis antibiótica fue alta, pero puede mejorarse. No se encontró relación entre la adecuación de la profilaxis antibiótica y la incidencia de infección del sitio quirúrgico.

Long-term effect of 2 intensive statin regimens on treatment and incidence of cardiovascular events in familial hypercholesterolemia: The SAFEHEART study.

BACKGROUND: Maximal doses of potent statins are the basement of treatment of familial hypercholesterolemia (FH). Little is known about the use of different statin regimens in FH. OBJECTIVES: The objectives of the study were to describe the treatment changes and low-density lipoprotein cholesterol (LDL-C) goal achievement with atorvastatin (ATV) and rosuvastatin (RV) in the SAFEHEART cohort, as well as to analyze the incidence of atherosclerotic cardiovascular events (ACVEs) and changes in the cardiovascular risk. METHODS: SAFEHEART is a prospective follow-up nationwide cohort study in a molecularly defined FH population. The patients were contacted on a yearly basis to obtain relevant changes in life habits, medication, and ACVEs. RESULTS: A total of 1939 patients were analyzed. Median follow-up was 6.6 years (5-10). The estimated 10-year risk according the SAFEHEART risk equation was 1.61 (0.67-3.39) and 1.22 (0.54-2.93) at enrollment for ATV and RV, respectively (P < .001). There were no significant differences at the follow-up: 1.29 (0.54-2.82) and 1.22 (0.54-2.76) in the ATV and RV groups, respectively (P = .51). Sixteen percent of patients in primary prevention with ATV and 18% with RV achieved an LDL-C <100 mg/dL and 4% in secondary prevention with ATV and 5% with RV achieved an LDL-C <70 mg/dL. The use of ezetimibe was marginally greater in the RV group. One hundred sixty ACVEs occurred during follow-up, being its incidence rate 1.1 events/100 patient-years in the ATV group and 1.2 in the RV group (P = .58). CONCLUSION: ATV and RV are 2 high-potency statins widely used in FH. Although the reduction in LDL-C levels was greater with RV than with ATV, the superiority of RV for reducing ACVEs was not demonstrated.

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy.

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3-4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of ß-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.

Trends in Hospitalization and Mortality Rates Due to Acute Cardiovascular Disease in Castile and León, 2001 to 2015.

INTRODUCTION AND OBJECTIVES: To analyze hospitalization and mortality rates due to acute cardiovascular disease (ACVD). METHODS: We conducted a cross-sectional study of the hospital discharge database of Castile and León from 2001 to 2015, selecting patients with a principal discharge diagnosis of acute myocardial infarction (AMI), unstable angina, heart failure, or acute ischemic stroke (AIS). Trends in the rates of hospitalization/100 000 inhabitants/y and hospital mortality/1000 hospitalizations/y, overall and by sex, were studied by joinpoint regression analysis. RESULTS: A total of 239 586 ACVD cases (AMI 55 004; unstable angina 15 406; heart failure 111 647; AIS 57 529) were studied. The following statistically significant trends were observed: hospitalization: ACVD, upward from 2001 to 2007 (5.14; 95%CI, 3.5-6.8; P < .005), downward from 2011 to 2015 (3.7; 95%CI, 1.0-6.4; P < .05); unstable angina, downward from 2001 to 2010 (-12.73; 95%CI, -14.8 to -10.6; P < .05); AMI, upward from 2001 to 2003 (15.6; 95%CI, 3.8-28.9; P < .05), downward from 2003 to 2015 (-1.20; 95%CI, -1.8 to -0.6; P < .05); heart failure, upward from 2001 to 2007 (10.70; 95%CI, 8.7-12.8; P < .05), upward from 2007 to 2015 (1.10; 95%CI, 0.1-2.1; P < .05); AIS, upward from 2001 to 2007 (4.44; 95%CI, 2.9-6.0; P < .05). Mortality rates: downward from 2001 to 2015 in ACVD (-1.16; 95%CI, -2.1 to -0.2; P < .05), AMI (-3.37, 95%CI, -4.4 to -2, 3, P < .05), heart failure (-1.25; 95%CI, -2.3 to -0.1; P < .05) and AIS (-1.78; 95%CI, -2.9 to -0.6; P < .05); unstable angina, upward from 2001 to 2007 (24.73; 95%CI, 14.2-36.2; P < .05). CONCLUSIONS: The ACVD analyzed showed a rising trend in hospitalization rates from 2001 to 2015, which was especially marked for heart failure, and a decreasing trend in hospital mortality rates, which were similar in men and women. These data point to a stabilization and a decline in hospital mortality, attributable to established prevention measures.

O-mannosyl glycans: from yeast to novel associations with human disease.

In yeasts and other fungi, O-mannosyl glycans constitute a major protein modification that is essential for cell viability. For several decades, protein O-mannosylation was considered a yeast-specific modification. Thus, it was especially interesting when it became evident that O-mannosyl glycans in mammals are not as rare as previously thought. O-mannosyl glycans are abundant in the mammalian brain and are also an abundant modification of alpha-dystroglycan, a component of the dystrophin-glycoprotein complex. Recently, mutations in genes that are or might be involved in the glycosylation of alpha-dystroglycan have been identified. Their association with neuromuscular diseases has focused the attention of different research areas on protein O-mannosylation.

Bovine CACNA1A gene and comparative analysis of the CAG repeats associated to human spinocerebellar ataxia type-6.

A small expansion of a CAG repeat domain in exon 47 of the human CACNA1A gene, which codes for the pore-forming alpha1A subunit of P/Q-type Ca2+ channels, causes spinocerebellar ataxia type-6. Only the human alpha1A protein has been demonstrated to contain the poly(Q) tract, although this locus has also recently been detected in ape genomes. To our knowledge, no further information has been published on other mammal species. Here, we have cloned the full-length alpha1A subunit in a non-primate species, the cow. The results have made it possible to explore the exon organization of the bovine CACNA1A gene as well as the splice alpha1A isoforms expressed by bovine chromaffin cells. We found a splice variant of the protein that, as in humans, also contains a polymorphic poly(Q) tract. Based on this result and using data from different Genome Databases, we performed an interspecies comparison of exon 47 and discovered that the poly(Q) tract is present in all the species studied, with the exception of primitive fish and rodents. Our results provide insight into the evolution of the CAG repeat tract at the C-terminus coding region of the CACNA1A gene.

Genomic organization of a novel glycosylphosphatidylinositol MAM gene expressed in human tissues and tumors.

We report the genomic organization of a novel human gene mapped to chromosome 6p21, encoding a putative glycosylphosphatidylinositol (GPI) anchored protein containing a MAM (meprin, A5 antigen, protein tyrosine phosphatase mu) domain, that we have termed as GPIM (GPI and MAM) protein. GPIM gene consists of an 8.9 kb transcript composed of 17 coding exons spanning about 65.5 kb of genomic DNA. The deduced polypeptide consists of 955 amino acids and exhibits structural features found in different types of cell adhesion molecules (CAMs), such as the presence of immunoglobulin domains, the presence of a MAM domain or the capacity to anchor to the cell membrane by a GPI motif. Expression analysis in normal human tissues revealed that this gene is expressed as a 5 kb and 9.5 kb mRNA. Furthermore, the smaller transcript is highly expressed in some human cancer cell lines, as well as in different primary tumors (lung, colon, uterus, stomach and breast). Interestingly, the gene was higher expressed in several tumor tissues analysed as compared to their corresponding normal tissues. Thus, GPIM is a novel gene codifying a protein with structural features characteristics of some CAMs, which might be involved in the tumor progression.

Clinical features and molecular characterization of a patient with muscle-eye-brain disease: a novel mutation in the POMGNT1 gene.

Muscle-eye-brain disease is a congenital muscular dystrophy characterized by structural brain and eye defects. Here, we describe a 12-year-old boy with partial agenesis of corpus callosum, ventriculomegaly, flattened brain stem, diffuse pachygyria, blindness, profound cognitive deficiencies, and generalized muscle weakness, yet without a clear dystrophic pattern on muscle biopsy. There was no glycosylation of α-dystroglycan and the genetic screening revealed a novel truncating mutation, c.1545delC (p.Tyr516Thrfs*21), and a previously identified missense mutation, c.1469G>A (p.Cys490Tyr), in the protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) gene. These findings broaden the clinical spectrum of muscle-eye-brain disease to include pronounced hypotonia with severe brain and eye malformations, yet with mild histopathologic changes in the muscle specimen, despite the absence of glycosylated α-dystroglycan.

Promoter alteration causes transcriptional repression of the POMGNT1 gene in limb-girdle muscular dystrophy type 2O.

Limb-girdle muscular dystrophy type 2O (LGMD2O) belongs to a group of rare muscular dystrophies named dystroglycanopathies, which are characterized molecularly by hypoglycosylation of α-dystroglycan (α-DG). Here, we describe the first dystroglycanopathy patient carrying an alteration in the promoter region of the POMGNT1 gene (protein O-mannose ß-1,2-N-acetylglucosaminyltransferase 1), which involves a homozygous 9-bp duplication (-83_-75dup). Analysis of the downstream effects of this mutation revealed a decrease in the expression of POMGNT1 mRNA and protein because of negative regulation of the POMGNT1 promoter by the transcription factor ZNF202 (zinc-finger protein 202). By functional analysis of various luciferase constructs, we localized a proximal POMGNT1 promoter and we found a 75% decrease in luciferase activity in the mutant construct when compared with the wild type. Electrophoretic mobility shift assay (EMSA) revealed binding sites for the Sp1, Ets1 and GATA transcription factors. Surprisingly, the mutation generated an additional ZNF202 binding site and this transcriptional repressor bound strongly to the mutant promoter while failing to recognize the wild-type promoter. Although the genetic causes of dystroglycanopathies are highly variable, they account for only 50% of the cases described. Our results emphasize the importance of extending the mutational screening outside the gene-coding region in dystroglycanopathy patients of unknown aetiology, because mutations in noncoding regions may be the cause of disease. Our findings also underline the requirement to perform functional studies that may assist the interpretation of the pathogenic potential of promoter alterations.

POMT1 is essential for protein O-mannosylation in mammals.

Over the past decade it has emerged that O-mannosyl glycans are not restricted to yeast and fungi but are also present in higher eukaryotes up to humans. In mammals, the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. In humans, mutations in POMT1 and POMT2 result in hypoglycosylation of alpha-dystroglycan (alpha-DG) thereby abolishing its binding to extracellular matrix ligands such as laminin. As a consequence, POMT mutations cause a heterogeneous group of severe recessive congenital muscular dystrophies in humans. However, little is known about the function of O-mannosyl glycans in mammals apart from its crucial role for the ligand binding abilities of alpha-DG. In this chapter we discuss the methods used to analyze the expression of Pomt1 in adult mouse organs and during embryo development. Further, we describe the generation and immunohistochemical analysis of Pomt1 knockout mice.

Expression of the murine Pomt1 gene in both the developing brain and adult muscle tissues and its relationship with clinical aspects of Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is the most severe of a group of congenital disorders that have in common defects in the O-glycosylation of alpha-dystroglycan. WWS is characterized by congenital muscular dystrophy coupled with severe ocular and brain malformations. Moreover, in at least one-fifth of the reported cases, mutations in the POMT1 gene are responsible for this disease. During embryonic development (E8.5 to E11.5), the mouse Pomt1 gene is expressed in the tissues most severely affected in WWS, the muscle, eye, and brain. In this study, we show that mPomt1 expression is maintained in the muscle and eye in later developmental stages and, notably, that its expression is particularly strong in regions of brain and cerebellum that, when affected, could generate the defects observed in patients with WWS. We show that the Pomt1 protein is localized to the sarcoplasmic reticulum of muscle tissue cells in adult mice, where alpha-dystroglycan is O-glycosylated. Furthermore, the Pomt1 protein is localized to the acrosome of maturing spermatids, where alpha-dystroglycan is not glycosylated, so that Pomt1 might have a different target for O-mannosylation in the testes. This expression pattern in the testes could also be related to the gonadal anomalies observed in some patients with WWS.

Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas.

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1alpha-2, podoplanin), a small mucin-type transmembrane glycoprotein originally identified as a cell-surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E-cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell-surface protrusions and reduced Ca(2+)-dependent cell-cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.