Co-segregation of the malignant hyperthermia and the Arg615-Cys615 mutation in the skeletal muscle calcium release channel protein in five European Landrace and Pietrain pig breeds

A total of 392 pigs of European Landrace and Pietrain origin segregating for malignant hyperthermia (MH) were genotyped using a polymerase chain reaction (PCR)/restriction endonuclease test for the C—T mutation at nucleotide (nt) 1843 in the skeletal muscle ryanodine receptor (RYR1) gene, earlier identified as the causal mutation for MH. All pigs had been halothane tested and genotyped at linked polymorphic marker loci. There was complete correlation between MH status of the 392 animals, as diagnosed by a combination of the halothane challenge test with S, GPI, H, A1BG, PGD haplotyping, and the DNA-based test. DNA-based detection of the MH status in 238 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 2% diagnostic error that is associated with the halothane challenge test. The mutation was also associated with an allele of a polymorphic microsatellite (ETH5 001) at the RYR1 locus.     

The IGF2-intron3-G3072A substitution explains a major imprinted QTL effect on backfat thickness in a Meishan x European white pig intercross

A paternally expressed QTL for muscle growth and backfat thickness (BFT) has previously been identified near the IGF2 locus on the distal tip of pig chromosome 2 (SSC2p) in three experimental F2 populations. Recently, a mutation in a regulatory element of the IGF2 gene was identified as the quantitative trait nucleotide (QTN) underlying the major QTL effect on muscle growth and BFT in crosses between Large White and Wild Boar or Pietrain. This study demonstrates that the IGF2 mutation also controls the paternally expressed QTL for backfat thickness in a cross between Meishan and European Whites. In addition, a comparison of QTL of backfat thickness measured by Hennessy grading probe (HGP) and by ultrasound measurement (USM) was made. In the USM analyses, the IFG2 mutation explains the entire QTL effect on SSC2p, whereas in the HGP analysis the presence of a second minor QTL can not be excluded. Finally, this study shows that this particular IGF2 mutation does not cause the paternally expressed QTL for teat number mapping to the same region of SSC2p as the BFT QTL.     

The Ile2453Thr mutation in the ryanodine receptor gene 1 is associated with facilitated calcium release from sarcoplasmic reticulum by 4-chloro-m-cresol in human myotubes

Central core disease (CCD) is a congenital disorder of skeletal muscle that is characterised histologically by typical central cores in type 1 skeletal muscle fibres. This disease is associated with malignant hyperthermia susceptibility and has been linked to the gene of skeletal muscle ryanodine receptor RYR1. In this study, we present a family with the spontaneous occurrence of the RYR1 Ile2453Thr mutation. Affected individuals were diagnosed as susceptible to malignant hyperthermia in the in vitro contracture test (IVCT) and showed histological signs of CCD. Myotubes were derived from the index patient. The calcium homeostasis in response to the ryanodine receptor agonist 4-chloro-m-cresol (4CmC) was investigated by calcium imaging using the Ca(2+)-sensitive fluorescent probe FURA 2. In the myotubes derived from the mutation carrier, the EC(50) of 4CmC was reduced to 94 micro as compared to 201 microM in a control group of 16 individuals non-susceptible to malignant hyperthermia. In the myotubes of the non-affected family members, the EC(50) was found within the same range as that of the control group. The reduction of EC(50) indicates a facilitated calcium release from sarcoplasmic reticulum in the myotubes of the index patient suggesting that the RYR1 Ile2453Thr mutation is pathogenic for the malignant hyperthermia susceptibility and CCD of the two affected individuals.     

Indirect effect of IGF2 intron3 g.3072G>A mutation on prolificacy in sows

A QTL located in the paternally expressed insulin-like growth factor 2 (IGF2) gene is known to increase muscle growth and reduce fat deposition in pigs. This makes the QTL in IGF2 a good marker for use in pig breeding programmes. However, care has to be taken as it is postulated that increased leanness and lowered fat deposition may have a negative effect on the prolificacy and longevity of sows. Selection of sire and dam lines for different alleles of the mutation in the paternally imprinted IGF2 gene could actually provide a solution to this problem. Therefore, in this study, the effect of the IGF2 QTL on prolificacy-related traits in sows was investigated. It was found that the paternal IGF2 wild-type allele was associated with higher reproduction performance in the sow. Moreover, it was also examined whether the difference in prolificacy in sows could be a consequence of differential IGF2 expression in the ovarian follicles of the sow or whether it is mainly a secondary effect caused by differences in fatness traits. Therefore, IGF2 expression was measured in follicles of different sizes from sows with different genotypes for the paternal IGF2 allele. It was observed that, however, while the size of the follicles was associated with follicular IGF2 expression level, the IGF2 genotype was not. It could be concluded that the difference in prolificacy of sows with a different paternal IGF2 genotype could be a secondary effect, resulting from differences in fat deposition.     

Associations of the IGF2 gene with growth and meat efficiency in Large White pigs

The insulin-like growth factor 2 gene (IGF2) has been described in several studies as a candidate gene for meat efficiency in pigs. IGF2 is a member of the growth factors family and has an effect on development of muscle tissue. The effect of IGF2 gene polymorphism on meat efficiency was analysed in a population of 121 Large White pigs. A PCR-based test and RFLP methods were used for detection of genotypes. Allele A, lacking the restriction site, was characterised by the presence of a 0.9-kb fragment. In allele B, the amplimer was cut into a 0.8-kb fragment and some barely detectable fragments. The statistical analysis was carried out according to the General Linear Model procedure. The genotype frequencies observed were: 1.65%, 33.88%, 64.46% for AA, AB and BB genotypes, respectively. There was a significant difference (P < or = 0.05) between the AB and BB genotypes in live weight before the test. A significant association between AB and BB genotypes and body weight before the test was found. No significant difference in other traits of growth and meat efficiency was observed (P > 0.05).     

Discordance and cosegregation of swine halothane susceptibility and 1843 C-T mutation of their RYR1 locus responsible for the ryanodine receptor

In two Siberian swine populations, the halothane test and PCR were used to determine the halothane susceptibility of the animals and to reveal a point mutation in their RYR1 gene, respectively. No correlations were found between the halothane susceptibility of an animal and the presence or absence of a point mutation at its RYR1 locus. However, the population changes in halothane susceptibility and the frequency of the mutation proved to be unidirectional. In the studied swine populations, the halothane-susceptible animals had no hyperthermia and the frequencies of their Hal and RYR1 genes changed similarly. These phenomena along with the phylogenesis of malignant hyperthermia and the porcine stress syndromes (PSS) in different breeds are discussed. In the populations studied, PSS is suggested to be under the polygenic control.     

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.     

A domesticated transposon mediates the effects of a single‐nucleotide polymorphism responsible for enhanced muscle growth

Single‐nucleotide polymorphisms (SNPs) in the regulatory regions of the genome can have a profound impact on phenotype. The G3072A polymorphism in intron 3 of insulin‐like growth factor 2 (IGF2) is implicated in higher muscle content and reduced fat in European pigs and is bound by a putative repressor. Here, we identify this repressor—which we call muscle growth regulator (MGR)—by using a DNA protein interaction screen based on quantitative mass spectrometry. MGR has a bipartite nuclear localization signal, two BED‐type zinc fingers and is highly conserved between placental mammals. Surprisingly, the gene is located in an intron and belongs to the hobo‐Ac‐Tam3 transposase superfamily, suggesting regulatory use of a formerly parasitic element. In transactivation assays, MGR differentially represses the expression of the two SNP variants. Knockdown of MGR in C2C12 myoblast cells upregulates Igf2 expression and mild overexpression retards growth. Thus, MGR is the repressor responsible for enhanced muscle growth in the IGF2 G3072A polymorphism in commercially bred pigs.     

The gene structure of the insulin-like growth factor family

The insulin-like growth factors (IGF) constitute a family of proteins with insulin-like and growth-stimulating properties. The best characterized members of this family are IGF-I, a protein of 70 amino acids which plays an important role in post-natal growth, and IGF-II, a 67 amino acid protein which is most likely involved in fetal development.The gene structure of IGF-II has been elucidated for the human and the rat and shows extensive interspecies homologies. The gene structure of IGF-I has only partially been established. A striking feature of the IGF genes is that they are controlled by multiple promoters which are expressed in a tissue-specific and development-dependent way.     

Discordance, in a Malignant Hyperthermia Pedigree, between in Vitro Contracture-Test Phenotypes and Haplotypes for the MHS1 Region on Chromosome 19q12–13.2, Comprising the C1840T Transition in the RYR1 Gene

A point mutation in the gene encoding the skeletal muscle calcium release channel (RYR1) has been proposed as the probable cause of malignant hyperthermia (MH) in swine, where it segregates with the disease in all MH–prone strains investigated. The same C-to-T exchange in nucleotide position 1840 of the human RYR1 cDNA sequence was found in a few human MH pedigrees. We report a German MH pedigree where in vitro contracture test (IVCT) results and haplotypes of markers for the MHS1/RYR1 region including this base transition have yielded several discrepancies. The MH-susceptible phenotype was defined by IVCT performed according to the European standard protocol. Haplotypes were constructed for markers for the MHS1/RYR1 region on chromosome 19 and include the C1840T base exchange. Discussing the probabilities for a number of hypotheses to explain these data, we suggest that our results may challenge the causative role of this mutation—and possibly the role of the RYR1 gene itself—in human MH susceptibility, at least in some cases.     

Does the A3333G mutation in the CACNL1A3 gene, detected in malignant hyperthermia, also occur in central core disease?

Malignant hyperthermia (MH) and central core disease (CCD) are two conditions associated with susceptibility to volatile anesthetics and depolarizing muscle relaxants. The gene RYR1, encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum, is responsible for about 50% of the cases of MH and some cases of CCD. However, genetic heterogeneity occurs in MH and a mutation in a second gene (CACLN1A3), encoding the alpha1-subunit of the dihydropyridine (DHP) channel, has recently been found in a large MH French family. The presence of this mutation in patients with CCD has not yet been reported. In this study, we analyzed the A3333G mutation in 5 unrelated patients affected by CCD and 31 MH-susceptible relatives (from 19 MH families) and did not find this mutation in any of them. Nevertheless, the report of data on newly described mutations in different populations is important to estimate the contributions of each gene mutation to the phenotype of MH and CCD.     

Evidence for genetic heterogeneity of malignant hyperthermia susceptibility

A locus for malignant hyperthermia susceptibility (MHS) has been localized on chromosome 19q12-13.2, while at the same time the gene encoding the skeletal muscle ryanodine receptor (RYR1) also has been mapped to this region and has been found to be tightly linked to MHS. RYR1 was consequently postulated as the candidate for the molecular defect causing MHS, and a point mutation in the gene has now been identified and is thought to be the cause of MH in at least some MHS patients. Here we report the results of a linkage study done with 19q12-13.2 markers, including the RYR1 cDNA, in two Bavarian families with MHS. In one of the families, three unambiguous recombination events between MHS and the RYR1 locus were found. In the second family only one informative meiosis was seen with RYR1. However, segregation analysis with markers for D19S75, D19S28, D19S47, CYP2A, BCL3, and APOC2 shows that the crossovers in the first family involve the entire haplotype defined by these markers flanking RYR1 and, furthermore, reveals multiple crossovers between these haplotypes and MHS in the second family. In these families, pairwise and multipoint lod scores below -2 exclude MHS from an interval spanning more than 26 cM and comprising the RYR1 and the previously described MHS locus. Our findings thus strongly suggest genetic heterogeneity of the MHS trait and prompt the search for another MHS locus.     

Effects of a novel SNP of IGF2R gene on growth traits and expression rate of IGF2R and IGF2 genes in gluteus medius muscle of Egyptian buffalo

Insulin-like growth factor 2 receptor (IGF2R) is responsible for degradation of the muscle development initiator, IGF2, and thus it can be used as a marker for selection strategies in the farm animals. The aim of this study was to search for polymorphisms in three coding loci of IGF2R, and to analyze their effect on the growth traits and on the expression levels of IGF2R and IGF2 genes in the gluteus medius muscle of Egyptian buffaloes. A novel A266C SNP was detected in the coding sequences of the third IGF2R locus (at nucleotide number 51 of exon 23) among Egyptian water buffaloes. This SNP was non-synonymous mutation and led to replacement of Y (tyrosine) amino acid (aa) by D (aspartic acid) aa. Three different single-strand conformation polymorphism patterns were observed in the third IGF2R locus: AA, AC, and CC with frequencies of 0.555, 0.195, and 0.250, respectively. Statistical analysis showed that the homozygous AA genotype significantly associated with the average daily gain than AC and CC genotypes from birth to 9 mo of age. Expression analysis showed that the A266C SNP was correlated with IGF2, but not with IGF2R, mRNA levels in the gluteus medius muscle of Egyptian buffaloes. The highest IGF2 mRNA level was estimated in the muscle of animals with the AA homozygous genotype as compared to the AC heterozygotes and CC homozygotes. We conclude that A266C SNP at nucleotide number 51 of exon 23 of the IGF2R gene is associated with the ADG during the early stages of life (from birth to 9 mo of age) and this effect is accompanied by, and may be caused by, increased expression levels of the IGF2 gene.