Variation in the ovine PRKAG3 gene

Variation in the ovine PRKAG3 gene

GENE-40516; No. of pages: 4; 4C: Gene xxx (2015) xxx–xxx Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/ge...

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GENE-40516; No. of pages: 4; 4C: Gene xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Short communication

Variation in the ovine PRKAG3 gene Guo Yang a,b, Huitong Zhou b, Ruoyu Wang a,⁎,1, Jon Hickford b,⁎,1 a b

Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China Gene-Marker Laboratory, Faculty of Agriculture and Life Sciences, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand

a r t i c l e

i n f o

Article history: Received 19 August 2014 Received in revised form 8 April 2015 Accepted 2 May 2015 Available online xxxx Keywords: PRKAG3 Genetic variation PCR-SSCP Sheep Nucleotide substitution Amino acid substitution

a b s t r a c t The 5′AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that controls cellular energy homeostasis in response to environmental or nutritional stress. The PRKAG3 gene (PRKAG3) encodes the γ3 subunit of the AMPK. Variation in this gene has been found to be associated with meat quality traits in pigs. In this study, we used polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) to investigate variation in exon 3 and exons 4–6 of ovine PRKAG3. In 160 New Zealand Suffolk sheep, two variant sequences (named a and b) were identified in the exon 3 region of the gene and three variant sequences (named A, B and C) were identified in the exon 4–6 region of the gene, respectively. A total of three nucleotide substitutions were revealed and these were located in intron 4, exon 4 and intron 5, respectively. The nucleotide substitution identified in the exon 4 (g.2656 C N T) could nominally lead to an amino acid substitution of tryptophan to arginine at position 230 (R230W) in ovine PRKAG3. In comparison with the PRKAG3 amino acid sequences in other species, this R230W substitution appeared to occur only in sheep. This is the first report of genetic variation in ovine PRKAG3, and the variation found in this study could be functionally important for AMPK activity, which in turn may affect meat quality traits in sheep. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The 5′AMP-activated protein kinase (AMPK) is an important enzyme regulating cellular energy flux and protects cells from depletion of ATP in response to cellular metabolic stresses by enabling the metabolism of energy reverse (Winder and Hardie, 1999). It is involved in the metabolism of fatty acid and carbohydrate in the liver, adipose tissue, pancreatic beta cells and skeletal muscle (Winder and Hardie, 1999). Mammalian AMPK is heterotrimeric, and is composed of one activating α domain (composed of α1 and α2 subunits), one regulatory β domain (composed of β1 and β2 subunits) and one binding domain γ (composed of γ1, γ2 and γ3 subunits). The γ3 subunit of AMPK (5′-AMP-activated protein kinase subunit γ3, PRKAG3) is encoded by PRKAG3 and is predominantly expressed in skeletal muscle (Cheung et al., 2000). It has been reported that the PRKAG3 subunit is important for the function of AMPK (Cheung et al., 2000). In PRKAG3 knockout mice, impaired glucose and lipid metabolism has been reported (Barnes et al., 2005). In humans, variation in PRKAG3 has been associated with impaired fatty acid oxidation and glucose uptake (Weyrich et al., 2007). In pure bred Hampshire Rendement Napole (RN) Abbreviations: PCR, polymerase chain reaction; SSCP, single-stranded conformational polymorphism; PRKAG3, 5′AMP-activated protein kinase subunit gamma-3; AMPK, 5′ AMP-activated protein kinase. ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Wang), [email protected] (J. Hickford). 1 These authors contributed equally to this work.

pigs, a natural mutation of arginine to glutamine at position 200 of PRKAG3 (R200Q) has been described (Milan et al., 2000) and the 200Q allele has been associated with a dramatically increased muscle glycogen content, decreased protein content and decreased meat pH (Milan et al., 2000). Recently, variation in the porcine PRKAG3 has been reported to be associated with glycogen content in skeletal muscle (Ryan et al., 2012). The bovine PRKAG3 has been reported to have a total length of 8048 bp, contains 13 coding exons as well as two alternative splicing sites (Roux et al., 2006). Recently, variation in bovine PRKAG3 has been found to be associated with carcass and meat quality traits in beef cattle (Li et al., 2012). To date, the ovine PRKAG3 has been sequenced and reported to contain 11 exons (GenBank accession number: FJ685774). However, the variation in the ovine PRKAG3 is still unclear. Given the important role that PRKAG3 appears to play in the animal glycogen metabolism, it could be hypothesized that variation in this gene may be important for meat quality traits. Accordingly, in this study we investigated variation in ovine PRKAG3 using polymerase chain reaction-single stranded conformational polymorphism (PCRSSCP).

2. Materials and methods 2.1. Sheep analyzed and DNA collection Blood samples were collected from the 160 NZ Suffolk sheep onto FTA cards (Whatman, Middlesex, UK). A two-step DNA extraction

http://dx.doi.org/10.1016/j.gene.2015.05.021 0378-1119/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Yang, G., et al., Variation in the ovine PRKAG3 gene, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.021

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Table 1 PCR primers used for amplification of ovine PRKAG3. Primer

Coordinateb

Primer sequence (5′–3′)

Expected amplicon size

Tm (°C)

Exon 3-up Exon 3-down Exons 4–6 upa Exons 4–6 downa

g.2313 g.2432 g.2623 g.3124

GCCTTCTTTGCCCTGGTG GGAACACCCCGCTAAG ACAGACTTCATCTTGGTT AGGAACGGGACGTGTCT

120 bp

59

502 bp

59

a b

The upstream primer for exons 4–6 is located at exon 4 of ovine PRKAG3, and the downstream primer is located at intron 6 of the gene. The positions of nucleotide substitutions are given related to the first nucleotide of ovine PRKAG3 (GenBank accession number: FJ685774).

method was used to get purified genomic DNA from the blood samples on the FTA cards (Zhou et al., 2006).

found in heterozygous sheep, a band corresponding to the rare allele was excised as a gel slice from the first SSCP gel, macerated, and then used as a template for reamplification with the original primers to produce a SSCP gel pattern equivalent to a sheep homozygous for that rare allele. This second amplicon was then sequenced. Sequence alignments, translations and comparisons were carried out using DNAMAN (version 5.2.10, Lynnon Biosoft, Canada). The BLAST algorithm was used to search the NCBI GenBank (http://blast.ncbi.nlm.nih.gov/) databases for homologous sequences. The genetic indices including Hardy–Weinberg Equilibrium (HWE), gene homozygosity (Ho), gene heterozygosity (He), effective allele numbers (Ne), and Polymorphism Information Content (PIC) in NZ Suffolk sheep population, were calculated using POPGENE version 1.32 (Molecular Biology and Biotechnology Centre, University of Alberta, Canada). Linkage Disequilibrium (LD) measures between pairs of SNPs were calculated using measures of Lewontin's coefficient (D′) and squared correlation coefficient (r2) using the Haploview (Version 4.2, Broad Institute of MIT and Harvard, USA). The amino acid sequence alignment of PRKAG3 was performed using DNAMAN (version 5.2.10, Lynnon Biosoft, Canada). These sequences included bovine (GenBank accession number: NP_001155893), pig (GenBank accession number: XP_005672270), rat (GenBank accession number: NM_001106921), human (GenBank accession number: BAG61882) and horse (GenBank accession number: NP_001075384) PRKAG3 sequences, together with the ovine PRKAG3 variant sequences identified in this study.

2.2. PCR amplification and SSCP analysis Two regions of ovine PRKAG3 were investigated in this study. Region 1 is approximately 120 bp in size and covers the partial of exon 3, and region 2 is approximately 502 bp in size and covers partial of exon 4, whole of exon 5 and exon 6. Two pairs of primers (Table 1) were designed to amplify these two regions based on the published ovine PRKAG3 sequence (GenBank accession number: FJ685774). Amplifications were performed in a 15 μL reaction containing the genomic DNA on one 1.2 mm punch of FTA paper, 0.25 μM of each primer, 150 μM of dATP, dCTP, dGTP and dTTP (Eppendorf, Hamburg, Germany), 2.5 mM Mg2+, 0.5 U of Taq DNA polymerase (Qiagen, Hilden, Germany), and 1× the reaction buffer supplied. The thermal profiles for amplification of the two fragments consisted of 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 59 °C and 30 s at 72 °C, with a final extension step of 5 min at 72 °C. Amplification was carried out in an iCycler (Bio-Rad, Hercules, CA, USA). Amplicons were visualized by electrophoresis in 1% agarose (Quantum Scientific, Queensland, Australia) gels, using 1 × TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA) containing 200 ng/mL of ethidium bromide. An aliquot of 0.7 μL of each amplicon was mixed with 7 μL of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene-cyanol). After denaturation at 95 °C for 5 min, samples were cooled rapidly on wet ice and then loaded onto 16 × 18 cm, 14% acrylamide/bisacrylamide (37.5:1) (Bio-Rad) gels. Electrophoresis was performed using Protean II xi cells (Bio-Rad), The amplicons of exon 3 and exons 4–6 were run at 230 V, 4.2 °C and 280 V, 22 °C, respectively, for 18 h in 0.5 × TBE buffer. Gels were silver-stained according to the method of Byun et al. (2009).

3. Results PCR amplicons were obtained of approximately 120 bp in size in region 1 and 485 bp in size in the region 2 of ovine PRKAG3, using the methods described above. Two and three different SSCP banding patterns were observed respectively from amplicons of regions 1 and 2 (Fig. 1). After sequencing, these patterns were confirmed as novel sequence variants and were named variants a and b for exon 3 and variants A–C for exons 4–6. The sequences of variants A–C were deposited into GenBank with accession numbers: JF508174–JF508176. GenBank will no longer accept sequences shorter than 200 bp, so our 120 bp

2.3. Sequencing of the allelic variants and sequence analysis For the 160 NZ Suffolk sheep investigated, PCR amplicons representative of different SSCP patterns from sheep that appeared to be homozygous in the amplified region for ovine PRKAG3 were directly sequenced at the Lincoln University DNA Sequencing Facility. For rare alleles only

Exon 4-6

Exon 3

aa

bb

ab

AC

AB

AB

BB

AB

Fig. 1. SSCP banding patterns for the amplicons of ovine PRKAG3 exon 3 and exons 4–6.

Please cite this article as: Yang, G., et al., Variation in the ovine PRKAG3 gene, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.021

G. Yang et al. / Gene xxx (2015) xxx–xxx

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Table 2 Frequencies and genetic indices of SNPs found in ovine PRKAG3 in NZ Suffolk sheep using PCR-SSCP. SNPa

g.2399 A N G

SSCP variants Variant a

Variant b

A

G

Variant A

Variant B

g.2656 C N T

C

C

g.2690 C N T

C

T

Genotypic frequency

Allelic frequency

AA 0.575 CC 0.574 CC 0.669

A 0.738 C 0.743 C 0.807

Variant C

T (R230Wb) C

AG 0.325 CT 0.338 CT 0.275

GG 0.100 TT 0.088 TT 0.056

G 0.262 T 0.257 T 0.193

χ2 (HWE)

Ho

He

Ne

PIC

P b 0.050

0.613

0.387

1.631

0.312

P b 0.050

0.619

0.381

1.618

0.309

P b 0.050

0.688

0.312

1.453

0.264

HWE: Hardy–Weinberg Equilibrium; Ho: gene homozygosity; He: gene heterozygosity; Ne: effective allele numbers; PIC: Polymorphism Information Content. a The positions of nucleotide substitutions are given related to the first nucleotide of ovine PRKAG3 (GenBank accession number: FJ685774). b The position of mutation in the amino acid sequence of ovine PRKAG3 was referred from the published ovine PRKAG3 amino acid sequence (GenBank accession number: ACM91654).

Table 3 Linkage Disequilibrium (LD) measures between the pairs of SNPs found in ovine PRKAG3 gene in NZ Suffolk sheep. Locus 1

Locus 2

D′

r2

g.2399 A N G g.2399 A N G g.2656 C N T

g.2656 C N T g.2690 C N T g.2690 C N T

0.846 0.324 1.000

0.694 0.009 0.083

D′: Lewontin's coefficient; r2: squared correlation coefficient.

exon 3 sequences a and b are available in fasta format in the Supplementary file. One SNP (g.2399 G N A) in intron 4, a SNP (g.2656 C N T) in exon 4, and a SNP (g.2690 C N T) in intron 4 were revealed (Table 2). The SNP g.2656 C N T would notionally result in a non-synonymous amino acid substitution of arginine to tryptophan at position 230 (R230W) of the ovine PRKAG3 amino acid sequence (GenBank accession number: ACM91654). The frequencies and the genetic indices of Ho, He, Ne and PIC of each SNPs in NZ Suffolk sheep are shown in Table 2. Each of the SNPs was checked for HWE using the standard χ2 test with significance level of 0.001 (Table 2). The measures of LD (r2 and D′) between pairs of SNPs in ovine PRKAG3 are shown in Table 3. 4. Discussion This is the first report identifying the variation in ovine PRKAG3. According to the classification of PIC (PIC value ≤ 0.250, low polymorphism; 0.250 b PIC value b 0.500, intermediate polymorphism; and PIC value ≥ 0.500, high polymorphism), NZ Suffolk sheep showed intermediate genetic diversity in these three SNPs loci of PRKAG3, suggesting that this gene is naturally polymorphic in sheep. Given the variation in PRKAG3 reported in other species such as cattle (Roux et al., 2006), pigs (Uimari and Sironen, 2014) and humans (Hoffman et al.,

2013), it could be concluded that more variants of ovine PRKAG3 may exist when extended gene regions are analyzed, or more sheep of more breeds around the world are investigated. The SNP g.2656 C N T and SNP g.2690 C N T are in complete LD (D′ = 1.0, r2 = 0.083, Table 3), indicating that there is no recombination between these two SNPs. It is notable that r2 between these two SNPs is low (0.083). This may be due to minor allelic frequencies of g.2656 T (8.8%, Table 2) and g.2690 T (5.6%, Table 2) presented in this two SNPs which affects r2 value (Wray, 2005). The χ2-test showed that the genotype distributions do not comply with Hardy–Weinberg Equilibrium (P b 0.050) at all of three SNP loci in the NZ Suffolk sheep population. This discrepancy between observed and expected PRKAG3 genotype frequencies might be due to a small sample size or artificial selection pressure on NZ Suffolk sheep. It is notable that the SNP g.2656 C N T would notionally encode a putative tryptophanat residue at position 230 of PRKAG3 and this non-synonymous substitution appears to occur exclusively in sheep (Fig. 2). The amino acid substitution R230W is possibly located in a potential cystathionine beta-synthase (CBS) pair region of PRKAG3 (CBS domain: GenBank COG0517; CBS pair: GenBank cl15354). These CBS domains are important for PRKAG3's binding to the adenosine component of AMP (Hardie and Hawley, 2001). The CBS pair coexists with a variety of other functional domains. Although its exact function is unknown, it has been proposed that the CBS domain may play a regulatory role in making proteins sensitive to adenosyl carrying ligands (Shan et al., 2001). Mutations of conserved residues within these CBS domains have been associated with a variety of human hereditary diseases, including congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, classic Bartter syndrome, Wolff–Parkinson– White syndrome, retinitis pigmentosa, and homocystinuria (Scott et al., 2004). Studies in pigs revealed that an amino acid substitution in the CBS domain could cause variation in muscle glycogen content and protein content, which decreases meat value and affects yields of cooked meat, respectively (Barnes et al., 2004; Ciobanu et al., 2001; Enfält et al., 1997; Gariépy et al., 1999; Lundström and Enfält, 1997). However, further

Fig. 2. Alignment of the ovine PRKAG3 amino acid sequence obtained in this study with various species' PRKAG3 amino acid sequences. These sequences include bovine (GenBank accession number: NP_001155893), pig (GenBank accession number: XP_005672270), rat (GenBank accession number: NM_001106921), human (GenBank accession number: BAG61882) and horse (GenBank accession number: NP_001075384) PRKAG3 sequences.

Please cite this article as: Yang, G., et al., Variation in the ovine PRKAG3 gene, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.021

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works are needed to clarify the association between the variation found in PRKAG3 and meat traits in sheep, by investigating more sheep with phenotypic data. Acknowledgments This study was financially supported by the Gene-Marker Laboratory of Lincoln University, National Natural Science Foundation of China (No. 31370447) and Hundred Talents Program (No. 27Y127L41002) of the Chinese Academy of Sciences, China. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.05.021. References Barnes, B.R., Marklund, S., Steiler, T.L., Walter, M., Hjalm, G., Amarger, V., et al., 2004. The 5′-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J. Biol. Chem. 279, 38441–38447. Barnes, B.R., Long, Y.C., Steiler, T.L., Leng, Y., Galuska, D., Wojtaszewski, J.F., et al., 2005. Changes in exercise-induced gene expression in 5′-AMP-activated protein kinase gamma3-null and gamma3 R225Q transgenic mice. Diabetes 54, 3484–3489. Byun, S.O., Fang, Q., Zhou, H., Hickford, J.G., 2009. An effective method for silver-staining DNA in large numbers of polyacrylamide gels. Anal. Biochem. 385, 174–175. Cheung, P.C., Salt, I.P., Davies, S.P., Hardie, D.G., Carling, D., 2000. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem. J. 346, 659–669. Ciobanu, D., Bastiaansen, J., Malek, M., Helm, J., Woollard, J., Plastow, G., et al., 2001. Evidence for new alleles in the protein kinase adenosine monophosphate-activated gamma(3)-subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality. Genetics 159, 1151–1162. Enfält, A.C., Lundström, K., Hansson, I., Johansen, S., Nyström, P.E., 1997. Comparison of non-carriers and heterozygous carriers of the RN-allele for carcass composition, muscle distribution and technological meat quality in Hampshire-sired pigs. Livest. Prod. Sci. 47, 221–229.

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Please cite this article as: Yang, G., et al., Variation in the ovine PRKAG3 gene, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.05.021