CAPN1 Research Articles

Polymorphism of the CAPN1 and GH genes and its relationship with the productivity of cattle of the Kazakh white-headed breed

The improvement of beef cattle is associated with the use of molecular genetic testing for genes associated with signs of meat productivity. The article presents the results of studies of gene polymorphism of calpain (CAPN1) and growth hormone (GH) in different sex and age groups of the Kazakh white-headed breed and its effect on the dynamics of live weight of young animals. It was found that polymorphism of the CAPN1 and GH genes is represented by three genotypes of CC, CG, GG and VV, LV, LL, respectively. The most common in the CAPN1 gene are the G allele and the GG genotype, the frequency of which ranged between 0.83-0.89 and 0.72-0.81, in the GH gene, the L allele and the LL genotype, the occurrence of which, respectively, was within 0.60-0.81 and 0.51-0.66. The carriers of the desired alleles, both in the homozygous and heterozygous state, were insignificant: in the CAPN1 gene, in the range of 0.03-0.22, in the GH gene, 0.03-0.31. In the CAPN1 gene, a lack of heterozygotes was observed in all groups, while in repair young animals it was higher than in animals of the parent herd. In the GH gene, a slight excess of heterozygotes was found in manufacturing bulls and repair bulls, while among cows and repair heifers they were deficient. Significant superiority in live weight at 8 and 12 months of age of gobies and heifers carriers of genotypes CAPN1CC and GHVV over peers CAPN1GG and GHLL was established by an average of 13.7 (P <0.05) and 6.8 %%; 12.1 (P <0.05) and 6.7 %%.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.63 AAV.CAPN3 gene therapy for Limb-girdle muscular dystrophy type 2A (LGMD2A): Phenotypic improvements in the calpain3 (CAPN3)-null mouse

LGMD2A is caused by mutations in CAPN3 gene. CAPN3 loss of function is shown to play a role in disease pathogenicity, which might be improved by CAPN3 gene therapy. rAAVrh74.tMCK.CAPN3 vector was systemically delivered to young (YC, 6-10 week-old) and old cohorts (OC, 21-23 week-old) of CAPN3-KO mice. Each cohort included two treatment groups receiving low dose (LD; 3E12 vg, n=12) and high dose (HD; 6E12 vg, n=12) of the vector and age-matched untreated (UT) control groups (UT-YC, n=13;UT-OC, n=15). Outcome measures were performed at 20 weeks post-injection. AAV.CAPN3 improved treadmill performance in both cohorts, at both LD and HD subgroups (p<0.0001). There was no significant age-related effect on treadmill activity and the average performance of the treatment groups, HD vs. LD did not differ significantly. In the OC however, females (n=5) from the HD group run longer distance than males from same group (n=7; p<0.01). In the LD and HD treatment groups from OC, in vivo muscle contractility assay showed an improvement in both Twitch (p=0.001) and Tetanic Max responses (p=0.047) at the endpoint. The average of percent change between baseline and endpoint tetanic responses increased in the treated-YC while there was a decline in the UT cohort (p=0.002). Histological analyses of the skeletal muscle (gastroc, tibialis anterior, quadriceps and triceps) showed remodeling of muscle, a switch to fatigue resistant oxidative fibers in both cohorts along with fiber size increase. In-house toxicology studies in the HD cohort revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. AAV.CAPN3 gene therapy improved the LGMD2A-related phenotype in the CAPN3-KO mouse model in both low and high doses tested, independent of age at the time of vector-administration. Improvements shown were supported with the absence of cardiotoxicity, enhancing the efficacy and safety of AAV.CAPN3 vector as a potential gene therapy for LGMD2A.

Heterozygous CAPN3 missense variants causing autosomal-dominant calpainopathy in seven unrelated families.

Recessive variants in CAPN3 gene are the cause of the commonest form of autosomal recessive limb girdle muscle dystrophy. However, two distinct in-frame deletions in CAPN3 (NM_000070.3:c.643_663del21 and c.598_621del15) and more recently, Gly445Arg and Arg572Pro substitutions have been linked to autosomal dominant (AD) forms of calpainopathy. We report 21 affected individuals from seven unrelated families presenting with an autosomal dominant form of muscular dystrophy associated with five different heterozygous missense variants in CAPN. We have used massively parallel gene sequencing (MPS) to determine the genetic basis of a dominant form of limb girdle muscular dystrophy in affected individuals from seven unrelated families. The c.700G>A, [p.(Gly234Arg)], c.1327T>C [p.(Ser443Pro], c.1333G>A [p.(Gly445Arg)], c.1661A>C [p.(Tyr554Ser)] and c.1706T>C [p.(Phe569Ser)] CAPN3 variants were identified. Affected individuals presented in young adulthood with progressive proximal and axial weakness, waddling walking and scapular winging or with isolated hyperCKaemia. Muscle imaging showed fatty replacement of paraspinal muscles, variable degrees of involvement of the gluteal muscles, and the posterior compartment of the thigh and minor changes at the mid-leg level. Muscle biopsies revealed mild myopathic changes. Western blot analysis revealed a clear reduction in calpain 3 in skeletal muscle relative to controls. Protein modelling of these variants on the predicted structure of calpain 3 revealed that all variants are located in proximity to the calmodulin-binding site and are predicted to interfere with proteolytic activation. We expand the genotypic spectrum of CAPN3-associated muscular dystrophy due to autosomal dominant missense variants.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.145 Prevalence of CAPN3 mutations in Limb-girdle muscular dystrophy type 2A: From a multi-center analysis in China to a worldwide perspective

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a rare autosomal recessive limb-girdle muscular dystrophy caused by mutations in CAPN3 gene. To date, no systemic evaluation was performed to investigate the mutational distribution and the hot spot regions. We retrospectively reviewed CAPN3 gene mutations from 124 Chinese patients recruited from four diagnostic centers (2012 January to 2019 September). Then we integrated the multicenter analysis with open-source databases (Clinvar, LOVD, gnomAD) and annotated the combined dataset (Ref: NM_000070.3). Functional evaluating tools (SIFT, PolyPhen, dbscSNV and MaxEntScan) were used to analyze the pathogenicity of the mutations. We evaluated the frequency and distribution of allelic CAPN3 mutation in LGMD2A patients. In the allelic mutational dataset from Chinese cohort (n=112), 62 novel mutations (55.4%) were identified according to ClinVar. The most prevalent mutation in China was c.2120A>G (18.9%) while the previous reported hotspot in LOVD was c.550del. Functional analysis with Polyphen and dbscSNV revealed a higher deleterious effect in Chinese cohort in comparison to the reported data (PolyPhen: g= 0.52, p < 0.0001; dbscSNV: g=0.49, p = 0.0036). After integrating the Chinese data with reported alleles (n= 467 in total), mutations in exon 1 (8.9%) and exon 10 (8.0%) of CAPN3 had the highest frequencies, while missense and frameshift mutations accounted for the majority of mutations. CAPN3 mutations had a high frequency in Exon 1 and 10 among LGMD2A patients. Exon 1 encodes the muscle-specific NS region of Calpain-3 protein while Exon 10 corresponds to Protease Core subdomain 2 (PC2). The frequent mutational regions revealed in the LGMD2A patients.

Dystrophin Is Required for the Proper Timing in Retinal Histogenesis: A Thorough Investigation on the mdx Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked muscular disease caused by defective expression of the cytoskeletal protein dystrophin (Dp427). Selected autonomic and central neurons, including retinal neurons, express Dp427 and/or dystrophin shorter isoforms. Because of this, DMD patients may also experience different forms of cognitive impairment, neurological and autonomic disorders, and specific visual defects. DMD-related damages to the nervous system are established during development, suggesting a role for all dystrophin isoforms in neural circuit development and differentiation; however, to date, their function in retinogenesis has never been investigated. In this large-scale study, we analyzed whether the lack of Dp427 affects late retinogenesis in the mdx mouse, the most well studied animal model of DMD. Retinal gene expression and layer maturation, as well as neural cell proliferation, apoptosis, and differentiation, were evaluated in E18 and/or P0, P5, P10, and adult mice. In mdx mice, expression of Capn3, Id3 (E18-P5), and Dtnb (P5) genes, encoding proteins involved in different aspects of retina development and synaptogenesis (e.g., Calpain 3, DNA-binding protein inhibitor-3, and β-dystrobrevin, respectively), was transiently reduced compared to age-matched wild type mice. Concomitantly, a difference in the time required for the retinal ganglion cell layer to reach appropriate thickness was observed (P0–P5). Immunolabeling for specific cell markers also evidenced a significant dysregulation in the number of GABAergic amacrine cells (P5–P10), a transient decrease in the area immunopositive for the Vesicular Glutamate Transporter 1 (VGluT1) during ribbon synapse maturation (P10) and a reduction in the number of calretinin+ retinal ganglion cells (RGCs) (adults). Finally, the number of proliferating retinal progenitor cells (P5–P10) and apoptotic cells (P10) was reduced. These results support the hypothesis of a role for Dp427 during late retinogenesis different from those proposed in consolidated neural circuits. In particular, Dp427 may be involved in shaping specific steps of retina differentiation. Notably, although most of the above described quantitative alterations recover over time, the number of calretinin+ RGCs is reduced only in the mature retina. This suggests that alterations subtler than the timing of retinal maturation may occur, a hypothesis that demands further in-depth functional studies.

Novel CAPN1 mutations extend the phenotypic heterogeneity in combined spastic paraplegia and ataxia

ObjectiveRecessive mutations in the CAPN1 gene have recently been identified in spastic paraplegia 76 (SPG76), a complex hereditary spastic paraplegia (HSP) that is combined with cerebellar ataxia, resulting in an ataxia‐spasticity disease spectrum. This study aims to assess the influence of CAPN1 variants on the occurrence of SPG76 and identify factors potentially contributing to phenotypic heterogeneity.MethodsWe screened a cohort of 240 unrelated HSP families for variants in CAPN1 using high‐throughput sequencing analysis. We described in detail the clinical and genetic features of the SPG76 patients in our cohort and summarized all reported cases.ResultsSix unreported CAPN1‐associated families containing eight patients with or without cerebellar ataxia were found in our cohort of HSP cases. These patients carried three previously reported homozygous truncating mutations (p.V64Gfs*103, c.759+1G>A, and p.R285*), and three additional novel compound heterozygous missense mutations (p.R481Q, p.P498L, and p.R618W). Lower limbs spasticity, hyperreflexia, and Babinski signs developed in about 94% of patients, with ataxia developing in 63% of cases. In total, 33 pathogenic mutations were distributed along the three reported functional domains of calpain‐1 protein, encoded by CAPN1, with no hotspot region. A comparison of gender distribution between the two groups indicated that female SPG76 patients were significantly more likely to present with complicated HSP than male patients (P = 0.015).InterpretationOur study supports the clinically heterogeneous inter‐ and intra‐family variability of SPG76 patients, and demonstrates that gender and calpain‐1 linker structure may contribute to clinical heterogeneity in SPG76 cases.

Genetic diversity, population structure of seven Indian domestic goat breeds based SNPs identified in CAPN1 gene

The micromolar calcium-activated neutral protease (CAPN1) gene encodes μ-calpain that degrades myofibril proteins under the post-mortem conditions which appears to be the primary enzyme in the postmortem tenderization process, hence affecting the meat quality. Objective of this study was to analyze genetic polymorphism of the CAPN1 gene in seven diverse Indian goat breeds. Direct DNA sequencing was carried out to identify the genetic polymorphisms in 84 unrelated goats representing seven different goat breeds (Barbari, Beetal, Ganjam, Sirohi, Black Bengal, Osmanabadi and Malabari). Six single nucleotide polymorphisms (SNPs) were identified in CAPN1 gene intron 17. The observed heterozygosity (Ho) was highest (0.50) in Sirohi and Osmanabadi (0.42) breeds and lowest in Black Bengal (0.06) breed. The expected heterozygosity (He) was highest (0.45) in Sirohi followed by Osmanabadi (0.37), Barbari (0.34) and Beetal (0.37). The allele frequencies ranged from 0.083 to 0.917 and the major allele frequencies ranged from 0.595 (locus 6499 T/C) to 0.917 (locus 6529 T/C). In Black Bengal, polymorphism at all theloci was fixed with the exception of 6417 T/G and 6533C/G. The identity values oscillated from 0.7866 to 0.9593. The Nei's genetic distances were below 0.0294. Genetic diversity among different goat meat breeds indicates possible role in meat quality trait. Allelic diversity at this locus could be used in further for the association between gene polymorphism and enzyme activity as well as caprine meat quality traits.

Biochemical and histological indicators of blood and m. longissimus dorsi of young bulls of Kazakh white-headed breed of different genotypes by the CAPN1 and GH genes

The use of molecular genetic markers for productivity is one of the modern approaches in breeding beef cattle. The article presents the results of the studies on the influence of genotypes by the calpain (CAPN1) and growth hormone (GH) genes on live weight, fatty acid composition of blood plasma, quantitative and qualitative indicators of meat of young cattle of Kazakh white-headed breed. It was established that animals of the homozygous geno-types СС–CAPN1 and VV-GH had a larger live weight and its average daily gain on the 240th and 365th days. Their superiority over the GG and LL genotypes was 13.8% (P&lt;0.05) and 6.9%; 14.6% (P&lt;0.05) and 6.9%, respectively. The carriers of the desirable alleles in the homozygous state had the sum of unsaturated fatty acids higher by 6.3% and 7.1%, respectively, than the young bulls, in which genotypes they were absent. The predominance of unsaturated fatty acids over saturated fatty acids also determined lower values of lipid me-tabolism direction index (0.92 and 1.00) in the animals with the desirable genotype. Muscle tissue of animals of the СС and VV genotypes was characterized by a higher level of protein, fat by 0.28–2.13 abs. per cent, energy value, and the quantity of muscle fibers with a smaller diameter per unit area compared to the GG and LL genotypes by on average 8.7% and 25.5% (P&lt;0.01). Due to the greater number of interfiber fat inclusions, muscle tissue of the desirable genotypes received higher marbling scores. The obtained data testify to the prospects of replication of animals, which genotype has the desirable alleles for further improvement of traits of meat productivity of Kazakh white-headed breed.

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay.

Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often-overlooked splicing effect of missense variants, we developed the functional assay ("minigene") for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon-intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning-based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the "minigene" functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics.

Myostatin inhibition promotes fast fibre hypertrophy but causes loss of AMP-activated protein kinase signalling and poor exercise tolerance in a model of limb-girdle muscular dystrophy R1/2A.

Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. The effect of genetic and pharmacological inhibition of myostatin signalling on the disease phenotype in a mouse model of LGMD R1 (CAPN3 knockout mouse-C3KO) was studied. Inhibition of myostatin signalling in C3KO muscles resulted in significant muscle hypertrophy; however, there were no improvements in muscle strength and exacerbation of exercise intolerance concomitant with further reduction of muscle oxidative capacity was observed. Inhibition of myostatin signalling is unlikely to be a valid therapeutic strategy for LGMD R1. Limb-girdle muscular dystrophy R1 (LGMD R1) is caused by mutations in the CAPN3 gene and is characterized by progressive muscle loss, impaired mitochondrial function and reductions in the slow oxidative gene expression programme. There are currently no therapies available to patients. We sought to determine if induction of muscle growth, through myostatin inhibition, represents a viable therapeutic strategy for this disease. Myostatin is a negative regulator of muscle growth, and its inhibition improves the phenotype in several muscle wasting disorders. However, the effect of myostatin depends on the genetic and pathophysiological context and may not be efficacious in all contexts. We found that genetic inhibition of myostatin through overexpression of follistatin (an endogenous inhibitor of myostatin) in our LGMD R1 model (C3KO) resulted in 1.5- to 2-fold increase of muscle mass for the majority of limb muscles. However, muscle strength was not improved and exercise intolerance was exacerbated. Pharmacological inhibition of myostatin, using an anti-myostatin antibody, resulted in statistically significant increases in muscle mass; however, functional testing did not reveal changes in muscle strength nor endurance in treated C3KO mice. Histochemical and biochemical evaluation of follistatin overexpressing mice revealed a reduction in the percentage of oxidative fibres and decreased activation of AMP-activated protein kinase signalling in transgenics compared to C3KO muscles. Our data suggest that muscle hypertrophy, induced by myostatin inhibition, leads to loss of oxidative capacity, which further compromises metabolically impaired C3KO muscles and thus is unlikely to be a valid strategy for treatment of LGMD R1.

Associations of CAST, CAPN1 and MSTN Genes Polymorphism with Slaughter Value and Beef Quality – A Review

Abstract The slaughter value of cattle and beef quality are influenced by many factors, which can generally be divided into antemortem (breed, sex, age, housing system, diet, pre-slaughter handling) and postmortem (post-slaughter processing, chilling temperature, packaging). Studies of many authors have shown that meat quality traits can be also influenced by the individual genetic background of an animal. Numerous studies have been conducted worldwide to determine the functions of various genes as well as polymorphisms with potential effects on fattening and slaughter value of cattle and on beef quality. This study reviews the most important research done on the associations of polymorphisms in the calpain, calpastatin and myostatin genes with carcass traits and beef quality. Knowledge about the genes and chromosome regions associated with desired meat quality characteristics may prove very helpful when selecting pairs for mating and estimating the breeding value of offspring, mainly because it is difficult to improve meat quality traits based on conventional selection methods due to their low heritability and polygenic regulation. Furthermore, meat quality evaluation is expensive and can only be carried out after slaughter.

Novel CAPN3 variant associated with an autosomal dominant calpainopathy.

The most common autosomal recessive limb girdle muscular dystrophy is associated with the CAPN3 gene. The exclusively recessive inheritance of this disorder has been recently challenged by the description of the recurrent variants, c.643_663del21 [p.(Ser215_Gly221del)] and c.598_612del15 [p.(Phe200_Leu204del)], associated with autosomal dominant inheritance. Our objective was to confirm the existence of autosomal dominant calpainopathies. Through our activity as one of the reference centres for genetic diagnosis of calpainopathies in France and the resulting collaborations through the French National Network for Rare Neuromuscular Diseases (FILNEMUS), we identified four families harbouring the same CAPN3 heterozygous variant with supposedly autosomal dominant inheritance. We identified a novel dominantly inherited CAPN3 variant, c.1333G>A [p.(Gly445Arg)] in 14 affected patients from four unrelated families. The complementary phenotypic, functional and genetic findings correlate with an autosomal dominant inheritance in these families, emphasizing the existence of this novel transmission mode for calpainopathies. The mild phenotype associated with these autosomal dominant cases widens the phenotypic spectrum of calpainopathies and should therefore be considered in clinical practice. We confirm the existence of autosomal dominant calpainopathies as an entity beyond the cases related to the in-frame deletions c.643_663del21 and c.598_612del15, with the identification of a novel dominantly inherited and well-documented CAPN3 missense variant, c.1333G>A [p.(Gly445Arg)]. In addition to the consequences for genetic counselling, the confirmation of an autosomal dominant transmission mode for calpainopathies underlines the importance of re-assessing other myopathies for which the inheritance is considered as strictly autosomal recessive.

Analysis of polymorphism of CAPN1 and GH genes in cattle of Kazakh White-Headed breed in association with the peculiarities of beef productivity

The intensive development of genetics at the molecular level has now opened up the possibility for breeders to identify and study genes that are directly or indirectly related to desirable economically useful traits. The purpose of the research was to identify desirable genotypes in the breeding of Kazakh White-Headed cattle based on the genes-markers of meat productivity of calpain (CAPN1) and somatotropin (GH) for use in breeding to increase meat productivity and beef quality. Experimental researches have been carried out on steers of the Kazakh White-Headed breed on the basis of the APC collective farm “Gigant” in the Blagodarnensky district in the Stavropol Territory. Analysis of growth and development indicators of calves has shown that the value of live weight and average daily gains was higher in calves carrying the C allele at calpain gene locus and V allele at somatotropin gene locus. Dominant alleles C and V in the homozygous state characterize the desired genotype of calves. During weaning the studied indicators were higher with a diff erence on average up to 13,0 % in young animals of homozygous genotypes (CC and VV). The largest number of muscle fi bers has been observed in steers with the desired CC and VV genotypes. They outperformed their herdmates with GG genotype by 36,82 pcs/мм² or 19,7 % in calpain gene group (CAPN1) by 55,7 pcs/мм² or 31,4 % in somatotropin gene group (GH), respectively. The diameter of muscle fibers in carcasses with the desired genotype was less by 4,23 microns or 11,9 %, and by 6,82 microns or 16,8 %. The meat of steers with homozygous CC and VV alleles according to the studied genes differed from the meat of animals with GG and LL genotypes by 12,6 % in calpain gene (CAPN1) and 15,1 % in somatotropin gene (GH). The connective tissue content was higher in animals with the undesirable type of G and L alleles than in animals of the desired genotype, by 2,13 and 3,34 abs. percent’s. The area of the eye of loin in animals of desired VV genotype of somatotropin cluster (GH) was higher than in animals of CC genotype of calpain cluster (CAPN1) by 4,84 cм² or 10,1 %.

Frizzled related protein deficiency impairs muscle strength, gait and calpain 3 levels

BackgroundLimb-girdle muscular dystrophy recessive 1 calpain3-related (LGMDR1), previously known as LGMD2A, is a disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness and muscle degeneration. Frizzled related protein (FRZB), upregulated in LGMDR1, was identified as a key regulator of the crosstalk between Wnt and integrin signalling pathways. FRZB gene silencing showed a recovery in the expression of some of the costamere protein levels in myotubes.ResultsHere, we performed a comprehensive characterization of Frzb−/− mice muscles to study the absence of Frzb in skeletal muscle and eventual links with the molecular characteristics of LGMDR1 patient muscles. Frzb−/− mice showed reduced muscle size and strength. Gait analysis showed that Frzb−/− mice moved more slowly but no impaired regeneration capacity was observed after muscle injury. Additionally, Frzb−/− mice muscle showed an increased number of mesoangioblasts. Lack of Frzb gene in Frzb−/− mice and its increased expression in LGMDR1 patients, showed contrary regulation of Rora, Slc16a1, Tfrc and Capn3 genes. The reciprocal regulation of Frzb and Capn3 genes further supports this axis as a potential target for LGMDR1 patients.ConclusionsOur data confirm a role for Frzb in the regulation of Rora, Slc16a1, Tfrc, and Capn3 genes in muscle cells. In vivo, reduced muscle strength and gait in the Frzb−/− mice are intriguing features. The reciprocal relationship between FRZB and CAPN3 further supports a key role for this axis in patients with LGMDR1.

CAPN1 and hereditary spastic paraplegia: a novel variant in an Iranian family and overview of the genotype-phenotype correlation

Purpose SPG76 is one of the rare forms of hereditary spastic paraplegia (HSP) which causes by mutations in the CAPN1 gene. The mode of inheritance of SPG76 is autosomal recessive (AR) and so far, only 24 families and 25 mutations in this gene have been reported worldwide. These mutations have been associated with a spectrum of disorders from pure HSP to spastic ataxia. HSP genetically is one of the most heterogeneous neurological disorders and to date, 79 types of HSP (SPG1-SPG79) have been identified, however, it has been suggested that many HSP-genes, particularly in AR-HSPs, remained unknown. AR-HSPs clinically overlap with other neurodegenerative disorders, making an accurate diagnosis of the disease difficult. Therefore, in addition to clinical examination, a high throughout genetic method like whole exome sequencing (WES) may be necessary for the diagnosis of this type of neurodegenerative disorders. Methods and Results Herein, we present the clinical features and results of WES in the first Iranian family with a novel CAPN1 variant, c.C853T:p.R285* and pure HSP. Conclusion Some of the previous studies have mentioned that the “spasticity-ataxia phenotype might be conducted to the diagnosis of SPG76” but recently the number of pure HSP patients with CAPN1 mutation is increasing. The present study also expands the mutation spectrum of pure CAPN1-related SPG76; emphasizing that CAPN1 screening is required in both pure HSP and spasticity-ataxia phenotypes. As noted in some other literature, we suggest the clinical spectrum of this disorder to be considered as “CAPN1-associated neurodegeneration”.

Deletion of the Capn1 Gene Results in Alterations in Signaling Pathways Related to Alzheimer's Disease, Protein Quality Control and Synaptic Plasticity in Mouse Brain.

Calpains represent a family of calcium-dependent proteases participating in a multitude of functions under physiological or pathological conditions. Calpain-1 is one of the most studied members of the family, is ubiquitously distributed in organs and tissues, and has been shown to be involved in synaptic plasticity and neuroprotection in mammalian brain. Calpain-1 deletion results in a number of phenotypic alterations. While some of these alterations can be explained by the acute functions of calpain-1, the present study was directed at studying alterations in gene expression that could also account for these phenotypic modifications. RNA-seq analysis identified 354 differentially expressed genes (DEGs) in brain of calpain-1 knock-out mice, as compared to their wild-type strain. Most DEGs were classified in 10 KEGG pathways, with the highest representations in Protein Processing in Endoplasmic Reticulum, MAP kinase and Alzheimer’s disease pathways. Most DEGs were down-regulated and validation of a number of these genes indicated a corresponding decreased expression of their encoded proteins. The results indicate that calpain-1 is involved in the regulation of a significant number of genes affecting multiple brain functions. They also indicate that mutations in calpain-1 are likely to be involved in a number of brain disorders.

Single Loci and Haplotypes in CAPN1 and CAST Genes are Associated with Growth, Biometrics, and in Vivo Carcass Traits in Santa Inês Sheep

Abstract µ-calpain (CAPN1) and calpastatin (CAST) genes play key roles in protein turnover. The present study aimed to identify the variants in these genes associated with growth and ultrasound carcass traits in Santa Inês sheep. A sample of 192 no full sibling Santa Inês lambs was used. Fragments of the CAST and CAPN1 genes were amplified and next-generation sequencing was performed in the MiSeq platform. Variants in the CAPN1 and CAST sequences were then detected using bioinformatic tools. Withers and croup heights, body length, thoracic and croup widths, thoracic and leg girths, body depth, carcass fat score, rib eye area, fat thickness, body weights were recorded at weaning and at 140 days post-weaning, and average daily gain post-weaning was calculated. Both single-locus and haplotype association analyses were performed with the model as follows: farm (2 levels), year (4 levels), the month of birth (12 levels), and the covariate age of the animal. The fragments amplified included 4,514 bp between the 20th and 23rd exons of CAST as well as 3,927 bp between the 12th and 21st exons of CAPN1. In these regions, 58 (CAST) and 45 (CAPN1) variants were identified. In the CAST gene, the single-locus analysis revealed 22 suggestive additive effects (P&lt;0.05) on several growth and carcass traits. Moreover, haplotype substitutions were associated with rib eye area (–0.689±0.290), average daily gain (–23.6±10.4), thoracic girth (–2.72±1.27), body length (–3.38±1.49), and leg girth (–2.84±1.37). Regarding the CAPN1 gene, the single-locus analysis identified seven suggestive additive effects, while only one haplotype replacement effect on fat thickness (–0.0143±0.0053) was detected. The results of the present study suggest that variants in the CAPN1 and CAST genes are associated with growth and ultrasound carcass traits in Santa Inês sheep, which may be a source of information to improve knowledge regarding the genetic control of these traits.

Mutational spectrum of autosomal recessive limb-girdle muscular dystrophies in a cohort of 112 Iranian patients and reporting of a possible founder effect

BackgroundLimb-girdle muscular dystrophies are a group of genetically heterogeneous diseases that are inherited in both autosomal dominant (LGMDD) and autosomal recessive forms (LGMDR), the latter is more common especially in populations with high consanguineous marriages like Iran. In the present study, we aimed to investigate the genetic basis of patients who are suspicious of being affected by LGMDR.DNA samples of 60 families suspected of LGMD were extracted from their whole blood. Four short tandem repeat (STR) markers for each candidate genes related to LGMD R1 (calpain3 related)- R6 (δ-sarcoglycan-related) were selected, and all these 24 STRs were applied in two sets of multiplex PCR. After autozygosity mapping, Sanger sequencing and variant analysis were done. Predicting identified variants’ effect was performed using in-silico tools, and they were interpreted according to the American College of Medical Genomics and Genetics (ACMG) guideline. MLPA was used for those patients who had large deletions.Fresh muscle specimens were taken from subjects and were evaluated using the conventional panel of histochemical stains.Resultsforty out of sixty families showed homozygote haplotypes in CAPN3, DYSF, SGCA, and SGCB genes. The exons and intron-exon boundaries of the relevant genes were sequenced and totally 38 mutations including CAPN3 (n = 15), DYSF (n = 9), SGCB (n = 11), and SGCA (n = 3) were identified. Five out of them were novel. The most prevalent form of LGMDs in our study was calpainopathy followed by sarcoglycanopathy in which beta-sarcoglycanopathy was the most common form amongst them. Exon 2 deletion in the SGCB gene was the most frequent mutation in this study.We also reported evidence of a possible founder effect in families with mutations in DYSF and SGCB genes. We also detected a large consanguineous family suffered from calpainopathy who showed allelic heterogeneity.ConclusionsThis study can expand our knowledge about the genetic spectrum of LGMD in Iran, and also suggest the probable founder effects in some Iranian subpopulations which confirming it with more sample size can facilitate our genetic diagnosis and genetic counseling.

Association of CAPN1 and GH genes with productivity traits in Kazakh Whiteheaded cattle

The Kazakh Whiteheaded cattle was developed by crossing local breeds with the Hereford breed in Kazakhstan in the 30s of the last century. This breed is adapted to arid conditions of extreme continental climate. The goal of the investi-gations was the assessment of the genetic structure of the Kazakh Whiteheaded breed based on the genes of calpain (CAPN1) and somatotropic hormone (GH) and analysis of the association of genotypes for these genes with productivity traits. The main argument for this aim is research works illustrated that both genes could affect on meat productivity traits in other breeds of cattle. 352 blood samples from representatives of Kazakh Whiteheaded animals of different sex and age groups were genotyped for the CAPN1 and GH loci. As a result, it was firstly proved that Kazakh Whiteheaded animals, who are CC homozygosis at the CAPN1 locus and VV homozygosis at the GH locus, reliably exceed animals without C and V alleles on the productivity traits such as milk production, average daily body weight gain, and pre-slaughter body weight, slaughter weight, carcass weight, flesh weight, chemical composition and histological characteristics of meat. It was also shown that the frequencies of the desired alleles and genotypes of these loci are rather low the frequency of the C allele was 0.11–0.17, and the frequency of the CC genotype was 0.03–0.06 in different age and sex groups; the frequency of the V allele was 0.19-0.40, and the frequency of the VV genotype was 0.03-0.31. Thus, planning selection for Kazakh Whiteheaded cattle, it would be useful to take into account the assessment of ani-mal genotypes at the CAPN1 and GH loci to increase the proportion of animals with the desired genotypes.

Polymorphism and Association of 5’UTR CAPN1 Gene with Growth Traits in Bali Cattle by PCR-RFLP

The aim of this study was to identify the variation of 5’UTR CAPN1 gene and its association to growth traits in Bali cattle. DNA samples were obtained from 80 heads of Bali cattle originated from BPTU-HPT Denpasar. The average of Bali cattle age was 784 days (631 days-1098 days). Bali cattle were divided into 3 age groups namely, the first group (1.5 years to 2 years), the second group (2 years to 2.5 years), and the third group (2.5 years to 3 years). The observed growth traits were birth weight (kg), live weights (kg), average daily gain (kg), body length (cm), chest depth (cm), withers height (cm), hip height (cm), and heart girth (cm). Polymorphism identification of 5’UTR CAPN1 gene was conducted by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) with BglII as the restriction enzyme. Growth traits data association were analyzed using the General Linear Model (GLM) analysis. The 5’UTR CAPN1 gene|BglII was polymorphic in Bali cattle (GG, GT, and TT). Genotype frequencies for Bali cattle were 0.30 (GG), 0.66 (GT), and 0.04 (TT). The allele frequencies of G and T allele were 0.63 and 0.37, respectively. The G allele was the most frequent allele and GT genotype was the most frequent genotype among the cattle. The CAPN1|BglII had a significant effect (p<0.05) on growth traits in Bali cattle. Animal carrier of GG genotype had higher live weight and average daily gain than those with GT genotype, while the lowest values were associated with TT genotype.