CAPN1 Research Articles

A new SNP in the 3′UTR region of the bovine calpain small subunit (CAPNS1) gene

Calpains are a ubiquitous cytoplasmic cysteine protease, the activity of which is absolutely dependent on calcium. This proteolytic system degrades myofibrillar protein under post-mortem conditions and appears to be the primary enzyme in the postmortem tenderization process. In the present study a new single nucleotide polymorphism was found in the bovine CAPNS1 gene exon 11 coding for the 3'UTR. Transition C --> T at position 6536 was detected and identified using PCR-SSCP and DNA sequencing techniques, and then analysed with PCR-RFLP using MboII nuclease. The genotype frequencies and alleles distribution were studied in 190 bulls including, Charolaise, Hereford, Limousine, Simmental, Polish Red and Fresian breeds.

Evaluation of biochemical parameters and genetic markers for association with meat tenderness in South African feedlot cattle

A large proportion of South African feedlot cattle are crossbreds of Brahman (BrX, Bos indicus), and Simmental (SiX, Bos taurus). A sample of 20 grain fed bulls from each of these crossbreeds was used to compare meat quality with that of the small frame indigenous Nguni (NgX, Sanga) by evaluating a variety of biochemical and genetic parameters previously shown to be associated with meat tenderness. Shear force values were generally high (5.6 kg average at 14 days post mortem), with SiX animals higher than BrX or NgX ( P = 0.051) despite higher calpastatin:calpain ratio in BrX ( P < 0.05). Calpain activity and cold shortening were both correlated with tenderness for all classes. The sample size was too small to accurately estimate genotypic effects of previously published markers in the CAST and CAPN1 genes, but the allele frequencies suggest that only modest progress would be possible in these South African crossbreds using these markers.

New Single Nucleotide Polymorphisms in the µ-Calpain Gene in Spanish Maternal Beef Breeds

Calpains play an important role in the postmortem tenderization process of meat and several SNP in the μ-calpain gene (CAPN1) have been reported to be associated with tenderness in beef cattle. Our objectives were to identify the previously reported CAPN1 331G>C SNP and to detect new polymorphisms in this gene in Spanish maternal beef breeds. A fragment (exon 8 to 10) of the bovine CAPN1 gene was sequenced and genotyped in a sample of the main Spanish maternal beef breeds including Retinta, Morucha, and Avileña Negra-Ibérica. These breeds are characterized for their high meat quality, their adaptation to adverse environmental conditions, and their good maternal aptitude. This adaptation makes it possible to rear these breeds in the south and west of Spain, where drought and feed shortages occur frequently. Six SNP in the μ-calpain gene were found, five of which (CAPN1 80C>T, 302C>G, 310G>A, 445C>T, 524A>C) have not been reported previously. Sequences obtained for these five newly found SNP were submitted to GenBank (Accessions EU386166 to EU386183).

Association of selected SNP with carcass and taste panel assessed meat quality traits in a commercial population of Aberdeen Angus-sired beef cattle

BackgroundThe purpose of this study was to evaluate the effects of eight single nucleotide polymorphisms (SNP), previously associated with meat and milk quality traits in cattle, in a population of 443 commercial Aberdeen Angus-cross beef cattle. The eight SNP, which were located within five genes: μ-calpain (CAPN1), calpastatin (CAST), leptin (LEP), growth hormone receptor (GHR) and acylCoA:diacylglycerol acyltransferase 1 (DGAT1), are included in various commercial tests for tenderness, fatness, carcass composition and milk yield/quality.MethodsA total of 27 traits were examined, 19 relating to carcass quality, such as carcass weight and fatness, one mechanical measure of tenderness, and the remaining seven were sensory traits, such as flavour and tenderness, assessed by a taste panel.ResultsAn SNP in the CAPN1 gene, CAPN316, was significantly associated with tenderness measured by both the tenderometer and the taste panel as well as the weight of the hindquarter, where animals inheriting the CC genotype had more tender meat and heavier hindquarters. An SNP in the leptin gene, UASMS2, significantly affected overall liking, where animals with the TT genotype were assigned higher scores by the panellists. The SNP in the GHR gene was significantly associated with odour, where animals inheriting the AA genotype produced steaks with an intense odour when compared with the other genotypes. Finally, the SNP in the DGAT1 gene was associated with sirloin weight after maturation and fat depth surrounding the sirloin, with animals inheriting the AA genotype having heavier sirloins and more fat.ConclusionThe results of this study confirm some previously documented associations. Furthermore, novel associations have been identified which, following validation in other populations, could be incorporated into breeding programmes to improve meat quality.

How to tackle the diagnosis of limb-girdle muscular dystrophy 2A

Limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy) is the most frequent form of LGMD in many European countries. The increasing demand for a molecular diagnosis makes the identification of strategies to improve gene mutation detection crucial. We conducted both a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified LGMD, unclassified myopathy and hyperCKemia, and a functional assay of calpain-3 autolytic activity in 108 cases with LGMD and normal protein quantity. Subsequently, screening of CAPN3 gene mutations was performed using allele-specific tests and simplified SSCP analysis. We diagnosed a total of 94 LGMD2A patients, carrying 66 different mutations (six are newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). Our data show a high predictive value for reduced-absent calpain-3 or lost autolytic activity. These biochemical assays are powerful tools for otherwise laborious genetic screening of cases with a high probability of being primary calpainopathy. Our multistep diagnostic approach is rational and highly effective. This strategy has improved the detection rate of the disease and our extension of screening to presymptomatic phenotypes (hyperCKemia) has allowed us to obtain early diagnoses, which has important consequences for patient care and genetic counseling.

The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration.

Cell migration involves the dynamic formation and release of cell-substrate adhesions, where the exertion and detection of mechanical forces take place. Members of the calpain family of calcium-dependent proteases are believed to have a central role in these processes, possibly through the regulation of focal adhesion dynamics. The ubiquitous calpains, calpain 1 (mu-calpain) and calpain 2 (m-calpain), are heterodimers consisting of large catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and the small regulatory subunit encoded by Capn4. We have examined the role of the calpain regulatory small subunit in traction force production and mechanosensing during cell migration. Capn4-deficient or rescued cells were plated on flexible polyacrylamide substrates, for both the detection of traction forces and the application of mechanical stimuli. The total force output of Capn4-deficient cells was approximately 75% lower than that of rescued cells and the forces were more randomly distributed and less dynamic in Capn4-deficient cells than in rescued cells. Furthermore, Capn4-deficient cells were less adhesive than wild-type cells and they also failed to respond to mechanical stimulations by pushing or pulling the flexible substrate, or by engaging dorsal receptors to the extracellular matrix. Surprisingly, fibroblasts deficient in calpain 1 or calpain 2 upon siRNA-mediated knockdown of Capn1 or Capn2, respectively, did not show the same defects in force production or adhesion, although they also failed to respond to mechanical stimulation. Interestingly, stress fibers were aberrant and also contained fewer colocalised vinculin-containing adhesions in Capn4-deficient cells than Capn1- and Capn2-knockdown cells. Together, these results suggest that the calpain small subunit plays an important role in the production of mechanical forces and in mediating mechanosensing during fibroblast migration. Furthermore, the Capn4 gene product might perform functions secondary to, or independent of, its role as a regulatory subunit for calpain 1 and calpain 2.

G.P.14.03 Quantitative analysis of CAPN3 and DMD transcripts: Involvement of nonsense-mediated mRNA decay

Duchenne and Becker muscular dystrophy (DMD/BMD) are associated with mutations in the DMD gene (Xp21); limb girdle muscular dystrophy type 2A (LGMD2A) with mutations in the CAPN3 gene (15q15). Pathological mutations connected with these phenotypes frequently disrupt the translational reading frame, giving rise to truncated proteins. Nonsense-mediated mRNA decay (NMD) is a type of quality control mechanism that selectively degrades mRNAs with premature termination codons (PTCs). Using real-time PCR, we performed quantitative analysis of DMD and CAPN3 transcripts carrying various mutated alleles and examined mRNA degradation by reason of NMD.

G.P.14.02 MLPA analysis of the CAPN3 gene detects large deletions in LGMD2A patients

G.P.14.02 MLPA analysis of the CAPN3 gene detects large deletions in LGMD2A patients

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras V12 oncogenic cells: Modulation of cell survival and migration

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras V12). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras V12 cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras V12 cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras V12 cells. In contrast, RasGAP knockdown in PC3Ras V12 cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

Analysis of SNPs in partial exons and 3'UTR of&lt;I&gt;CAPN1&lt;/I&gt;in porcine

In this study, Wild boar, Min pig and Yorkshire were used to investigate the effect of CAPN1 gene on lean meat percentage and tenderness. Seven pairs of primers for exons and 3'untranslated region of CAPN1 gene were designed according to pig sequences from GenBank. Polymorphisms within exons were detected by PCR-SSCP method. SNPs in 3'untranslated region were detected by DNA sequencing, and PCR-RFLP method was then developed to screen the population. Totally, 11 SNPs, of which 5 from exons including one missense mutation resulting in amino acid substitution of M/V at the position of 260, 4 from introns and 2 from 3'untranslated region, were identified. Population genetics analysis showed that the distribution of genotypes among Yorkshire, Min pig and Wild boar were extremely significant difference (P<0.01), while there were no significant difference between Min pig and Wild boar (P>0.05), and extremely significant difference existed between Min pig and Yorkshire (P<0.01). Association of polymorphisms with breed characteristics indicated that the mutations detected by P4, P6 primers and that resulting in the change of Hinf 1 recognition site may be related to lean meat percentage.

CDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were suspected to have LGMD2A, based on western blot results. Four of these patients were shown to have LGMD2I upon molecular analysis, whereas 16 of the remaining 42 patients harbored mutations in CAPN3 by both direct genomic sequencing and cDNA analyses. In 10 patients, we identified both mutant alleles. In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish origin, indicating a five- to sixfold lower prevalence in Denmark compared to other European countries. A total of 16 different CAPN3 mutations were identified, of which 5 were novel. The present study demonstrates the value of cDNA analysis for CAPN3 in LGMD2A patients and indicates that calpainopathy is an uncommon cause of LGMD in the Denmark.

G.P.4.12 Analysis of the diagnostic strategy for limb girdle muscular dystrophy 2A in the UK

Diagnosis of limb girdle muscular dystrophy type 2A can be complex due to phenotypic variability, accuracy of protein analysis in muscle biopsies, and lack of mutational hot spots in the CAPN3 gene. The aim of the study was to review clinical and biopsy data from a group of patients with known CAPN3 genetic status to validate and refine our current diagnostic strategy, which combines clinical information and protein analysis to direct gene testing. We analysed 85 patients in whom CAPN3 gene sequencing had been performed.

G.P.4.13 Mutation screening of CAPN3 gene in 13 Turkish LGMD2A patients

G.P.4.13 Mutation screening of CAPN3 gene in 13 Turkish LGMD2A patients

Frequency of calpain-3 c.550delA mutation in limb girdle muscular dystrophy type 2 and isolated hyperCKemia in German patients

Calpain-3 deficiency is the most common cause of autosomal-recessive limb girdle muscular dystrophy (LGMD2). The c.550delA mutation in the CAPN3 gene was frequently identified in LGMD2A patients from Eastern Europe and is considered a Slavic founder mutation. We screened for the c.550delA mutation in unrelated German patients with LGMD2 (n = 98) and in patients with asymptomatic or minimally symptomatic (myalgia or fatigue) hyperCKemia of unknown origin (n = 102). Results of Western blot analysis were available in 75 patients with LGMD2 and 65 patients with hyperCKemia. In samples that were heterozygous for the c.550delA mutation, the whole CAPN3 gene was analyzed by sequencing in order to detect the second mutation. The c.550delA mutation was found in 8.1% of LGMD2 (n = 1 homozygous, n = 7 heterozygous) and 1.9% of hyperCKemia patients (n = 2 heterozygous). In 8 of the 9 hetrozygous patients, a second CAPN3 mutation was identified by direct sequencing. Two mutations (Val509Phe and Gln565Stop) have not been reported before. Absent or deficient calpain-3 protein in Western blot analysis was found in 22.5% of the LGMD2 patients and 11% of the patients with hyperCKemia. Western blot results were available in 9 out of the 10 patients with genetically confirmed LGMD2A and were clearly abnormal in 6 patients, suspicious in 2 and entirely normal in 1. Two LGMD2 patients with the c.550delA mutation and onset within the first 2 decades had joint contractures. Muscle biopsy revealed inflammatory changes in three patients. The CAPN3 gene mutation c.550delA is rather frequently observed in German patients with LGMD2, but also occasionally in cases with isolated hyperCKemia.

A unique case of limb‐girdle muscular dystrophy type 2A carrying novel compound heterozygous mutations in the human CAPN3 gene

A unique sib pair afflicted by limb girdle muscular dystrophy type 2A (LGMD2A) is described showing a slowly progressive autosomal recessive type of muscular dystrophy with onset in the third and fourth decades. The patients had early asymmetric muscle involvement characterized by prominent biceps brachii atrophy with sparing of the knee extensors. Additional findings included elevation of serum creatine kinase level, myopathic EMG changes and dystrophic type of pathology on muscle biopsy. Asymmetrical wasting of muscles in the extremities exhibited uniform and highly selective CT imaging patterns. RNA and DNA analyses confirmed novel compound heterozygous mutations (R147X/L212F) in the human CAPN3 gene.

Correlation Analysis on Single Nucleotide Polymorphism of CAPN1 Gene and Meat Quality and Carcass Traits in Chickens

Abstract The selection of meat quality has received considerable focus in chicken breeding. This study was aimed at investigating the effect of CAPN1 gene on meat quality traits in chicken populations. Primer pairs for 3′UTR in CAPN1 were designed from database of chicken genomic sequence. Polymorphisms were detected using PCR-SSCP and DNA sequencing. A mutation at position 9950 nt (G/A, locus A) was found among individuals in each population. The allele and genotype frequencies significantly differed among eight lines with higher frequencies of allele A2 and genotype A1A2 ( P<0.01). The least square analysis showed that there was significant difference ( P<0.05) in muscle fiber density and some carcass traits among genotypes and that the breast muscle fiber density (BFD) of birds of A1A1 genotype was significantly higher ( P<0.05) than that of birds of A2A2 genotype. It was concluded that the CAPN1 gene was the major gene affecting the muscle fiber traits of chicken or was linked with the major gene. These results were useful for studying the molecular mechanism that influences meat traits and were used as the base of molecular-assisted selection to meat quality traits. So, this site may be a potential marker affecting the muscle traits of chickens.

Age-Related Cataracts in α3Cx46-Knockout Mice Are Dependent on a Calpain 3 Isoform

Previous studies have demonstrated that in 129alpha3Cx46-/- mice, age-related nuclear cataract is formed. In the present study, a more in vivo-relevant model was generated to test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice. To test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice, 129alpha3Cx46-/- and CAPN3-/- mice were mated to generate homozygous double-knockout (dKO) mice. Lenses from the mice were examined by visual observation, laser scan analysis, and histologic and biochemical methods. In the absence of the CAPN3 gene, the formation of a cataract was delayed, and its appearance was changed to a more diffuse, pulverulent type. Unlike in the 129alpha3Cx46-/- mouse, cleavage of gamma-crystallin was not detected in the dKO mouse. In both 129alpha3Cx46-/- and dKO mice, total Ca2+ increased. The present study shows for the first time that calpain 3 is necessary for the formation of age-dependent nuclear cataracts in alpha3Cx46-/- mice. Evidence that the calpain 3 gene is directly involved in, or part of the pathway that leads to, gamma-crystallin cleavage is presented. These results are consistent with the hypothesis that the loss of alpha3Cx46 leads to increased levels of Ca2+ ions, and this increase activates the CAPN3 isoform, Lp82/85, which results in the formation of a nuclear cataract.

Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy 2A

Diagnosis of limb girdle muscular dystrophy type 2A can be complex due to phenotypic variability, lack of precision of protein analysis in muscle biopsies, and absence of mutational hot spots in the CAPN3 gene. The aim of this study was to review clinical and biopsy data from a group of patients with known CAPN3 genetic status to validate and refine our current diagnostic strategy, which combines clinical information and protein analysis to direct gene testing. We analysed 85 patients in whom CAPN3 gene sequencing had been performed. Forty-two patients had two mutations, 15 a single mutation and in 28 no mutation was found. We identified clinical features that clearly discriminated the LGMD2A patients. These were: presence of scapular winging, contractures and normal respiratory function. In addition, a typical pattern of muscle weakness on manual muscle testing could be confirmed. Interpretation of protein expression obtained by Western blot was complex and involved the analysis of a number of bands detected by two antibodies for calpain 3. Loss of all calpain 3 bands was 100% specific for LGMD2A, but this pattern was found in only 23%. Absence or reduction of the approximately 60 kDa bands was also highly specific for LGMD2A, while increased abundance was highly predictive of no mutations being found even where other bands were reduced, suggesting that this is the most sensitive marker of artefactual protein degradation. Twenty-three percent of the patients with two mutations had normal full-sized calpain 3 protein, consistent with the finding of mutations localized in parts of the gene likely or proven to be involved in autolytic activity. Clinical and biochemical findings in patients with only one mutation were similar to patients with two mutations, indicating that other gene analysis techniques should be used before excluding the diagnosis. Our analysis confirms that our strategy is still valid to prioritize genetic testing in this complex group of patients, provided patients with normal protein but a suggestive clinical phenotype are not excluded from genetic testing.

Association of CAPN1 and CAST gene polymorphisms with meat tenderness in Bos taurus beef cattle from Argentina

The activity of the calpains/calpastatin proteolytic system is closely related to the postmortem tenderization of meat. We investigated the association between beef tenderness and single nucleotide polymorphism (SNP) markers on the CAPN1 gene (SNP316, alleles C/G; SNP530 alleles A/G) and the CAST gene 3' untranslated region (SNP2870, alleles A/G). We sampled nine slaughter groups comprising 313 steers which had been reared in beef production systems in Argentina between 2002 and 2004 from crosses between Angus, Hereford and Limousin cattle. Minor allele frequencies for the markers were 0.27 to 0.46 (C), 0.02 to 0.18 (A), and 0.24 to 0.53 (A), respectively. The presence of CAPN1 markers had significant effects on meat shear force but no detectable effects were demonstrated for the CAST marker. The shear force of meat from steers with the SNP316 CC genotype was 11% lower than for the SNP316 CG genotype and 17% lower than for the SNP316 GG genotype. There were very few steers with the SNP530 AA genotype and, contrary to previous studies, meat from steers with the SNP530 GG genotype showed an 11.5% higher shear force than that from steers with the SNP530 GA genotype. Final body weight, carcass weight and rib eye area were not affected by any of the markers. These results support the concept that CAPN1 variants are associated with tenderness across a wide range of beef production systems.