Calpain Gene Research Articles

Characterization of the Goose CAPN3 Gene and its Expression Pattern in Muscle Tissues of Sichuan White Geese at Different Growth Stages.

Calpain 3 (CAPN3), also known as p94, is associated with multiple production traits in domestic animals. However, the molecular characteristics of the CAPN3 gene and its expression profile in goose tissues have not been reported. In this study, CAPN3 cDNA of the Sichuan white goose was cloned, sequenced, and characterized. The CAPN3 full-length cDNA sequence consists of a 2,316-bp coding sequence (CDS) that encodes 771 amino acids with a molecular mass of 89,019 kDa. The protein was predicted to have no signal peptide, but several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structure of CAPN3 was predicted to be 38.65% α-helical. Sequence alignment showed that CAPN3 of Sichuan white goose shared more than 90% amino acid sequence similarity with those of Japanese quail, turkey, helmeted guineafowl, duck, pigeon, and chicken. Phylogenetic tree analysis showed that goose CAPN3 has a close genetic relationship and small evolutionary distance with those of the birds. qRT-PCR analysis showed that in 15-day-old animals, the expression level of CAPN3 was significantly higher in breast muscle than in thigh tissues. These results serve as a foundation for further investigations of the function of the goose CAPN3 gene.

Impaired regeneration in calpain-3 null muscle is associated with perturbations in mTORC1 signaling and defective mitochondrial biogenesis

BackgroundPrevious studies in patients with limb-girdle muscular dystrophy type 2A (LGMD2A) have suggested that calpain-3 (CAPN3) mutations result in aberrant regeneration in muscle.MethodsTo gain insight into pathogenesis of aberrant muscle regeneration in LGMD2A, we used a paradigm of cardiotoxin (CTX)-induced cycles of muscle necrosis and regeneration in the CAPN3-KO mice to simulate the early features of the dystrophic process in LGMD2A. The temporal evolution of the regeneration process was followed by assessing the oxidative state, size, and the number of metabolic fiber types at 4 and 12 weeks after last CTX injection. Muscles isolated at these time points were further investigated for the key regulators of the pathways involved in various cellular processes such as protein synthesis, cellular energy status, metabolism, and cell stress to include Akt/mTORC1 signaling, mitochondrial biogenesis, and AMPK signaling. TGF-β and microRNA (miR-1, miR-206, miR-133a) regulation were also assessed. Additional studies included in vitro assays for quantifying fusion index of myoblasts from CAPN3-KO mice and development of an in vivo gene therapy paradigm for restoration of impaired regeneration using the adeno-associated virus vector carrying CAPN3 gene in the muscle.ResultsAt 4 and 12 weeks after last CTX injection, we found impaired regeneration in CAPN3-KO muscle characterized by excessive numbers of small lobulated fibers belonging to oxidative metabolic type (slow twitch) and increased connective tissue. TGF-β transcription levels in the regenerating CAPN3-KO muscles were significantly increased along with microRNA dysregulation compared to wild type (WT), and the attenuated radial growth of muscle fibers was accompanied by perturbed Akt/mTORC1 signaling, uncoupled from protein synthesis, through activation of AMPK pathway, thought to be triggered by energy shortage in the CAPN3-KO muscle. This was associated with failure to increase mitochondria content, PGC-1α, and ATP5D transcripts in the regenerating CAPN3-KO muscles compared to WT. In vitro studies showed defective myotube fusion in CAPN3-KO myoblast cultures. Replacement of CAPN3 by gene therapy in vivo increased the fiber size and decreased the number of small oxidative fibers.ConclusionOur findings provide insights into understanding of the impaired radial growth phase of regeneration in calpainopathy.

Autosomal dominant calpainopathy due to heterozygous CAPN3 C.643_663del21.

A calpain-3 (CAPN3) gene heterozygous deletion (c.643_663del21) was recently linked to autosomal dominant (AD) limb-girdle muscular dystrophy. However, the possibility of digenic disease was raised. We describe 3 families with AD calpainopathy carrying this isolated mutation. Probands heterozygous for CAPN3 c.643_663del21 were identified by targeted next generation or whole exome sequencing. Clinical findings were collected for probands and families. Calpain-3 muscle Western blots were performed in 3 unrelated individuals. Probands reported variable weakness in their 40s or 50s, with myalgia, back pain, or hyperlordosis. Pelvic girdle muscles were affected with adductor and hamstring sparing. Creatine kinase was normal to 1,800 U/L, independent of weakness severity. Imaging demonstrated lumbar paraspinal muscle atrophy. Electromyographic findings and muscle biopsies were normal to mildly myopathic. Muscle calpain-3 expression was reduced. This study provides further evidence for AD calpainopathy associated with CAPN3 c.643_663del21. No pathogenic variants in other genes known to cause myopathy were detected. Muscle Nerve 57: 679-683, 2018.

P 49 A slight change with a major impact – a report on a patient with limb girdle muscular dystrophy

Objectives Limb girdle muscular dystrophy is characterized by weakness of the proximal arm and leg muscles. Both autosomal dominant and autosomal recessive inheritance patterns are known. Up to 50 genes are identified to cause autosomal recessive muscular dystrophy, the most prevalent mutations affect the Calpain 3 (CAPN3) gene. Case report We report on a 53-year-old woman, suffering from muscle weakness since her adolescence. At the age of 15, she detected problems climbing stairs; she could attend sport lessons in school but failed to acquire good results. The symptoms worsened over time and she is wheel-chair bound since the age of 32. The family history was uneventful. Neurological examinations as well as the CK analysis were normal in the patient”s parents and her brother and sister. The clinical investigation revealed proximal paresis of the lower extremities as well as bilateral scapula alata. Muscle tendon reflexes were diminished and she reported no sensory disturbances. The laboratory work-up showed elevated levels of CK (maximum 500 U/l). Electromyography and a muscle biopsy of the thigh confirmed a myopathy, but no complementary details emerged. In 2016, genetic analysis was supplemented. We found two heterozygote mutations of the CAPN3 gene (c.550delA and c.2105C > T). Both mutations are rated as pathogenic, the compound heterozygote presentation was confirmed by investigation of the parents which each carried one of the above-mentioned mutations. Conclusion We report on a patient with a severe limb girdle muscular dystrophy with an uneventful family history. Genetic analysis revealed a compound heterozygote mutation of the CAPN3 gene in our index patient.

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.

Integrative variants, haplotypes and diplotypes of the CAPN3 and FRMD5 genes and several environmental exposures associate with serum lipid variables

To determine whether the integrative variants, haplotypes and diplotypes of the calpain 3 (CAPN3) and the FERM domain containing 5 genes (FRMD5) and several environmental exposures are associated with an implication in lipid homeostasis, which are associated with cardiovascular risk. Genotyping of the CAPN3 rs4344713 and FRMD5 rs524908 was performed by Sanger sequencing in 1,640 subjects (Jing, 819 and Han, 821). Multivariate analyses of covariance models that adjusted by age, gender, body mass index (BMI), blood pressure and lifestyle (smoking and drinking), were constructed using variants, haplotypes and diplotypes of the CAPN3 rs4344713 and FRMD5 rs524908 as predictors and changes in lipid variables. Significant associations with low-density lipoprotein cholesterol and apolipoprotein (Apo) B were found. Linkage disequilibrium with each other showed the haplotype-phenotype associations with triglyceride and ApoA1. This study also suggested pleiotropic associations of the CAPN3-FRMD5 diplotypes with lipid variables. As potential confounders, diastolic blood pressure (DBP) and BMI were significantly associated with lipid variables. We conclude that integrative variants, haplotypes and diplotypes of the CAPN3 rs4344713 and FRMD5 rs524908, as well as DBP and BMI are associated with serum lipid variables in the Jing and Han populations.

Redox-Dependent Calpain Signaling in Airway and Pulmonary Vascular Remodeling in COPD.

The calcium-dependent cytosolic, neutral, thiol endopeptidases, calpains, perform limited cleavage of their substrates thereby irreversibly changing their functions. Calpains have been shown to be involved in several physiological processes such as cell motility, proliferation, cell cycle, signal transduction, and apoptosis. Overactivation of calpain or mutations in the calpain genes contribute to a number of pathological conditions including neurodegenerative disorders, rheumatoid arthritis, cancer, and lung diseases. High concentrations of reactive oxygen and nitrogen species (RONS) originated from cigarette smoke or released by numerous cell types such as activated inflammatory cells and other respiratory cells cause oxidative and nitrosative stress contributing to the pathogenesis of COPD. RONS and calpain play important roles in the development of airway and pulmonary vascular remodeling in COPD. Published data show that increased RONS production is associated with increased calpain activation and/or elevated calpain protein level, leading to epithelial or endothelial barrier dysfunction, neovascularization, lung inflammation, increased smooth muscle cell proliferation, and deposition of extracellular matrix protein. Further investigation of the redox-dependent calpain signaling may provide future targets for the prevention and treatment of COPD.

Association between single nucleotide polymorphism in ovine Calpain gene and growth performance in three Egyptian sheep breeds.

The aim of the present study was to assess the association of single nucleotide polymorphisms (SNPs) of Calpain (CAPN) gene with birth weight (BW), final weight (FW) and average daily gain (ADG) in three Egyptian sheep breeds: Barki, Rahmani and Ossimi. Blood samples were collected from 108 animals representing the three breeds. DNA was isolated using salting out procedure and then the quality and quantity of DNA extracted were measured. A 190 bp of CAPN was amplified by PCR using specific primers. The allele and genotype frequencies for all the identified SNPs were calculated. The PCR products corresponding to each genotype were sequenced to identify SNPs associated with the traits in question. Two SNPs (C→T) were detected in the nucleotides 44 and 154. For each SNP, the two mentioned alleles were named C and T, respectively. The sequenced CAPN segments were subjected to nucleotide blast at NCBI, which revealed 99% identity with that reported for sheep in Genbank. The TT was the least common genotype, whereas frequencies of CT and CC genotypes were fluctuated in the three sheep breeds under study. Animal carrier TT genotype had higher BW, FW and ADG than those with CT genotype, while the lowest values were associated with CC genotype. For the three traits under study, Rahmani had the highest estimates followed by Ossimi and Barki. Males exhibited heavier BW and FW as well as higher ADG compared with females. The results generated provide preliminary indication of the functional diversity present in Barki, Rahmani and Ossimi sheep and the possibility of using this polymorphism in Egyptian sheep genetic improvement.

Association analysis of g.68G → A SNP in CAPN1 gene with carcass and meat quality traits in goose raised in organic <i>dehesa</i>

Abstract. Meat quality is an important concern for the poultry industry. Domestic geese products obtained from free-range systems usually have rather tough meat, and it is necessary to select them to improve meat tenderness. The relation of the calpain 1 (CAPN1) gene with the post-mortem tenderness process of meat has been demonstrated in several species. Thus, the objective of the present study was to identify polymorphisms in this gene and to perform an association analysis between these polymorphisms and related economic traits in goose raised in the dehesa ecosystem. For the analysis, 50 geese of 3 different subpopulations (20 Embden Anser anser; 20 Toulouse Anser anser; 10 F1 cross) were studied. The experimental protocols were followed complying with principles of animal welfare. A novel SNP was found in the CAPN1 gene, g.68G → A. This polymorphism was statistically associated with different carcass and meat quality traits such as thigh muscle width (P = 0.020) and the b* 10-day meat colour parameter (P = 0.024) for the global goose population. The association of this gene with meat tenderness (Warner–Bratzler shear force) was confirmed in the case of female individuals of the Toulouse breed (P = 0.043). The results suggest the possibility of using molecular markers in CAPN1 gene as a potential tool for improving carcass and meat quality traits in goose breeding programmes.

NOVEL SNP OF CALPAIN-1 (CAPN1) GENE AND ITS ASSOCIATION WITH CARCASS AND MEAT CHARACTERISTICS TRAITS IN BALI CATTLE

Calpain-1 gene (CAPN1) produces an calpain enzyme controlling structure of meat protein and tenderness. The aims of this study were to identify Single Nucleotide Polymorphism (SNP) in exon 5 and 6 of CAPN1 gene and its associate with carcass and meat characteristic traits in bali cattle. A total of 48 bali cattles from BPTU-HMT Bali Cattle, Bali Province were used in the research. SNP in exon 5 and 6 of CAPN1 gene were identify with direct sequencing using MEGA 5 program. Analysis of polymorphism was conducted by PopGen 1.32 software to identify frequencies of genotype, allele and Hardy-Weinberg equilibrium. The association of CAPN1 gene genotype with carcass and meat characteristic traits was analyzed using Generalized Linear Model (GLM) procedure of SAS. Sequencing analysis at exon 5-6 of CAPN1 gene in Bali cattle resulted in eight polymorphic SNPs. They are c.3669T>C, c.3854G>A, c.3881T>C, c.3899C>T, c.3908C>G, c.4002C>A, c.4021G>T and c.4037A>C. The SNPs c.3669T>C, c.3854G>A and c.3899C>T were significantly (P<0.05) associated with rump thickness (RT), rump fat thickness (RFT) and marbling score (MS), while SNP c.4037A>C was not significantly associated with carcass and meat characteristic traits. The SNPs were significantly associated with carcass and meat characteristic traits namely c.3669T>C, c.3854G>A and c.3899C>T. Those SNPs may be used as candidate marker for Marker Assisted Selection (MAS) in bali cattle.

Association of genetic polymorphisms of Calpain and Calpastatin genes with growth traits in Bandur sheep

The study was undertaken to determine genetic polymorphisms of Calpain and Calpastatin genes and to study their association with growth traits (birth weight, weaning weight, 6-month body weight and ADG) in Bandur ram lambs (100) distributed over core area of the home tract as well as from Livestock Research and Information Centre (Sheep), KVAFSU, Nagamangala. Miller’s high salt method was used for isolation of genomic DNA from venous blood. A 192 bp of CAPN (exons 5 and 6) and 620 bp of CAST (exons 1C/1D) genes were amplified by PCR using published primers. The PCR-SSCP analysis of CAPN gene revealed genotype frequency for AA (0.672) and AB (0.295) and allele frequency for A (0.820) and B (0.180), respectively. Alignment of A and B allele of Calpain by CLC Main Workbench 6.8.1 showed addition/insertion of one nucleotide ‘A’ in B allele. The PCR-RFLP analysis of CAST gene revealed frequency for genotypes MM (0.24), MN (0.59) and NN (0.17), and for alleles M (0.535) and N (0.465), respectively. The Chi-square test indicated that the studied population was in Hardy-Weinberg equilibrium. Five SNPs were identified in the intron between exon 1C and 1D of CAST. The association of CAPN and CAST genotypes with growth traits was analysed by ANOVA using GLM procedures of SAS 9.3. No significant associations were observed between polymorphisms of CAPN and CAST loci and growth traits.

Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies.

BackgroundLimb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis.MethodsWe investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes.ResultsAnalysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound – c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene – c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present.ConclusionsGenetic diagnosis was possible in 12 of 60 patients (20 %). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-1058-z) contains supplementary material, which is available to authorized users.

312. Gene Correction of LGMD2A Patient-Specific iPS Cells for Targeted Autologous Cell Therapy

Limb girdle muscular dystrophy type 2A (LGMD2A), an incurable autosomal recessive disorder and the most common form of LGMD, is characterized by symmetric and progressive weakness of limb-girdle muscles leading to loss of ambulation. LGMD2A is caused by mutations in the Calpain 3 (CAPN3) gene which encodes a muscle-specific intracellular calcium-sensitive cysteine protease, hypothesized to act as a gate keeper for proper assembly and maintenance of the sarcomere. A cell therapy based on induced pluripotent stem (iPS) cells has the potential for treating muscular dystrophy. We have demonstrated that transplantation of skeletal muscle progenitors obtained from disease-free human iPS cells can restore muscle contractility in a mouse model for Duchenne Muscular Dystrophy. The goal of this study is to apply gene editing tools to correct the CAPN3 mutation in LGMD2A iPS cells, and to determine the feasibility of an iPS-cell based autologous cell therapy for LGMD2A. Here we investigated two approaches to genetically correct a LGMD2A patient-specific iPS cell line with homozygous deletion of exon 17 to 24 in the CAPN3 gene. The first approach consisted of knocking in the coding region of CAPN3 cDNA with SV40 polyadenylation signal sequence downstream in exon 1 of the endogenous CAPN3 gene by CRISPR-CAS9 induced double stranded break (DSB) mediated homology directed DNA repair from exogenous donor template, allowing for universal correction of all types of CAPN3 mutations. PCR, sequencing and Southern blot analysis on iPS colonies post- genome editing confirmed the integration of the cDNA in exon 1 of the CAPN3 gene. The second approach involved inserting the missing exons 17-24 with SV40 polyadenylation signal sequence downstream contiguous with the exon 16 of the endogenous CAPN3 gene. PCR and sequencing analysis on iPS colonies post-genome editing confirmed proper integration in this approach as well. LGMD2A iPS cell clones, genetically corrected using these two gene editing methods will be differentiated into skeletal muscle progenitors using conditional expression of Pax7, as previously described by our group, and evaluated in vitro and in vivo for restoration of full length CAPN3. For the latter, we have generated an immunodeficient LGMD2A mouse model by combining the CAPN3-KO mouse model with NOD/SCID and IL2Rg mutations. This mouse is ideal for studies on the transplantation of human cells. In summary, we have corrected the CAPN3 mutation in LGMD2A iPS cells for the first time, and ongoing transplantation studies with corrected iPS cells will provide proof-of-principal for a potential new cell therapy that can permanently restore CAPN3 function in all LGMD2A patients.

Association between Single Nucleotide Polymorphism of Calpain1 Gene and Meat Tenderness Traits in Different Genotypes of Chicken: Malaysian Native and Commercial Broiler Line

Association between Single Nucleotide Polymorphism of Calpain1 Gene and Meat Tenderness Traits in Different Genotypes of Chicken: Malaysian Native and Commercial Broiler Line

Loci asociados con enfermedades genéticas y calidad de carne en bovinos Charolais mexicanos

Se determinaron las frecuencias alélicas y genotípicas de ocho marcadores localizados en los genes calpaína (CAPN, 4751 y 316), calpastatina (CASTT1) y tiroglobulina (TG5), asociados a calidad de carne, y en los genes, miostatina (MSTN, Q204X), arginino succinato sintasa (ASS), monofosfato sintasa (UMPS) y miofosforilasa (PYGM), asociados a enfermedades genéticas de ganado bovino. Se muestrearon 493 animales Charolais de registro de dos hatos ubicados en Sonora (n=157) y tres en Nuevo León (n=336). No se encontraron portadores de los alelos T-ASS y T-UMPS, pero sí portadores del alelo Q204X del gen MSTN en frecuencias de ≤ 1 % en las poblaciones de Sonora y de 8.6 a 14.4 % en las de Nuevo León. Además, se identificaron portadores del marcador del gen PYGM, en frecuencias del 6.5 y de 1.0 % para un hato de Sonora y otro de Nuevo León, respectivamente. El análisis de diferenciación génica pareado entre las poblaciones y con los cuatro loci mostró que hay diferencias altamente significativas dentro de poblaciones del noroeste (P<0.0001) y entre éstas y las del noreste (P<0.001), la cual es explicada principalmente por los loci CAPN-316 y TG5. De acuerdo a los resultados obtenidos se recomienda el monitoreo del marcador del gen PYGM y del alelo Q204X del gen MSTN, así como también implementar estrategias para confirmar la utilidad de los marcadores asociados a calidad y productividad como herramienta para complementar los programas de mejoramiento genético.

Investigation of biochemical changes of the ovine calpain 3 exon-10 polymorphism

Calpain 3 (CAPN3) is a tissue specific calpain, and its mRNA is the most expressed calpain isoform in skeletal muscles. Many mutations and polymorphisms within the human CAPN3 gene have been reported and related to limb-girdle muscular dystrophy. Several reports link CAPN3 polymorphisms and meat quality. An association between three allele variants in exon-10 of ovine CAPN3 and the yield of fat trimmed meat cuts has been reported. This research investigated the biochemical significance of polymorphic variation in CAPN3. CAPN3 mRNA sequences were obtained from muscle samples collected from lambs which were homozygous for each of the three alleles. Four single base substitutions were found besides those in exon-10, but none of them, including the variations within exon-10, caused a change in amino acid sequence. The expression of CAPN3 mRNA and the amounts of CAPN3 protein were also compared among genotypes, and no significant differences were found. These results suggest that the reported association of specific allele variants within CAPN3 exon-10 to phenotype variations were not direct effects of CAPN3 polymorphisms. Interspecies analyses of the CAPN3 sequences indicated that the sequence reported here is more likely to be the correct common ovine CAPN3 sequence than the reference sequence.

Association of Calpain (CAPN) 10 (UCSNP-43, rs3792267) gene polymorphism with elevated serum androgens in young women with the most severe phenotype of polycystic ovary syndrome (PCOS)

Objectives: To highlight a possible association of Calpain (CAPN 10) gene UCSNP-43 polymorphism with hormonal and metabolic traits of young women with different phenotypes of polycystic ovary syndrome (PCOS).Design: PCOS women were genotyped for the CAPN 10 gene UCSNP-43 polymorphism. A comparison of clinical and biochemical features of women with PCOS stratified on the basis of the CAPN 10 gene UCSNP-43 variants was assessed.Methods: Anthropometric, hormonal and biochemical measurements were carried out in 668 PCOS women and 200 healthy controls. Subjects were also genotyped for the CAPN 10 gene UCSNP-43 polymorphism. The genotype frequency distributions between groups and controls were compared using the chi-square test. The association of the polymorphism with the clinical and biochemical features of the study cohort was estimated as well.Results: No association of the frequency of CAPN 10 gene UCSNP-43 polymorphism with PCOS was detected. No association of the polymorphism with the anthropometric, biochemical and hormonal features was detected both in PCOS and control women. The polymorphism was associated with serum Δ4 androstenedione (p = 0.018), as well as with 17-OH progesterone (17-hydroxyprogesterone) among women with PCOS phenotype A (p = 0.012).Conclusions: CAPN 10 gene polymorphism UCSNP-43 is deprived of a metabolic contribution to cardiovascular disease (CVD). However, due to its association with androgen excess in phenotype A, CAPN 10 gene polymorphism UCSNP-43 could be used as a genetic marker for CVD in young PCOS women.

The normalisation of CAPN gene expression in M. pectoralis superficialis in broiler lines differing in growth rate and their relationship to breast muscle tenderness

The aim of this study was to assess mRNA abundance of calpain 1 (CAPN1) and calpain 3 (CAPN3) in breast muscle of 80 fast-growing (FG) and slow-growing broilers (SG) and relate gene expression in relation to growth and Warner Bratzler (WB) shear force of breast muscle.The expression of CAPN1 and CAPN3 genes was higher in the FG compared to the SG line, but significant results were obtained only for CAPN1. The CAPN1 mRNA level was strongly dependent on line and gender interaction.Lower values of shear force were observed in the FG line, where a higher level of calpain expression was shown.A new panel of housekeeping genes (RPL4 and SDHA) for normalisation of gene expression in muscle tissues could be used in other studies of gene expression in chicken.

Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells.

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.

Differential Diagnosis between Duchenne Muscular Dystrophy and Limb Girdle Muscular Dystrophy 2a.

Limb Girdle Muscular Dystrophy 2A (LGMD 2A) is the most common form of limb girdle muscular dystrophies caused by mutations in the calpain-3 gene (CAPN-3). The pattern of LGMD 2A can be clinically indistinguishable from that of Duchenne Muscular Dystrophy (DMD). We report a case of a 14-year-old boy which has the initial diagnosed as DMD at 6 years old, based on clinical features and very elevated serum creatine kinase levels. A muscle biopsy at the age of 10 showed atypical features which suggested a histiocytosis or neural damage. An MRI conducted 2 years later revealed fatty degeneration predominantly in the posterior region of the thigh and led the diagnosis to LGMD 2A, as well as the necessity to repeat the biopsy. Immunohistochemical analysis was normal for dystrophin, but the Western Blott showed a normal/borderline amount of calpain-3 in the muscle. We also performed a molecular analysis that identified a compound heterozygous mutation of the calpain 3 gene (CAPN 3). LGMD 2A was often misdiagnosed as DMD due to the similarities in clinical manifestations and technique limitations; the immunohistochemical examination, the magnetic resonance imaging examination and the molecular analysis are an essential tool for establishing a right diagnosis.