Six novel coding SNPs of the nucleophosmin 1 ( NPM 1 ) gene and their associations with growth traits in bovine ( Brief Report )

The Nucleophosmin 1 (NPM1) gene encodes a multifunctional nucleolar phosphoprotein that has crucial roles in the control of different aspects of cell growth and homeostasis, such as ribosome biogenesis, centrosome duplication, cell cycle progression, apoptosis and cell differentiation (GRISENDI et al. 2006, NAOE et al. 2006). As mutants of NPM1 gene impact protein synthesis, NPM1 is an essential protein in mouse development and cell growth (MAGGI et al. 2008). The bovine NPM1 gene contains one exon and locates at chromosome 9. In previous work, the 12-bp deletion was detected in bovine NPM1 gene coding region. (HUANG et al. 2010). In this study, the coding region of bovine NPM1 gene has been scanned by PCR-SSCP, DNA sequencing and forced PCR-RFLP methods for SNPs in 1 032 individuals belonging to four Chinese cattle breeds. Association of six mutations of NPM1 gene with growth traits was analyzed.


Background
The Nucleophosmin 1 (NPM1) gene encodes a multifunctional nucleolar phosphoprotein that has crucial roles in the control of different aspects of cell growth and homeostasis, such as ribosome biogenesis, centrosome duplication, cell cycle progression, apoptosis and cell differentiation (GRISENDI et al. 2006, NAOE et al. 2006).As mutants of NPM1 gene impact protein synthesis, NPM1 is an essential protein in mouse development and cell growth (MAGGI et al. 2008).The bovine NPM1 gene contains one exon and locates at chromosome 9.In previous work, the 12-bp deletion was detected in bovine NPM1 gene coding region.(HUANG et al. 2010).In this study, the coding region of bovine NPM1 gene has been scanned by PCR-SSCP, DNA sequencing and forced PCR-RFLP methods for SNPs in 1 032 individuals belonging to four Chinese cattle breeds.Association of six mutations of NPM1 gene with growth traits was analyzed.
The 15μL PCR amplification contained 30 ng of genomic DNA, 0.20 mM dNTP, 2.5 mM MgCl2, and 0.5 U Taq DNA polymerase (MBI).The PCR was performed using the following program: 94 °C for 3 min followed by 34 cycles of 94 °C for 30 s, annealing at 56.5 °C and 56 °C corresponding two different pairs of primers for 30 s, 72 °C for 30 s and a final extension at 72 °C for 10 min.

PCR-SSCP, DNA sequencing and forced PCR-RFLP
Aliquots of 5 μL PCR products were mixed with 5 μL denaturing solution, heated for 10 min at 98 °C and chilled on ice.Denatured DNA was subjected to 12 % PAGE in constant voltage (200 V) for 2.5~3.0 h.The gel was stained with 0.1 % silver nitrate (WANG et al. 2009).
The 205 bp fragment of the NPM1 gene was amplified by P1-F2 and P1-R1 primers.The naturally-occurring ›T‹ nucleotide at nt232 was substituted by an ›A‹ in the forward primer (underlined) in order to introduce a new recognition site (GAT^ATC) for the EcoR V (MBI, Vilnius, Lithuania) restriction endonuclease (size: 205 bp; primers: P1-F2/P1-R1).Also, the new reverse P2-R2 primer was designed to produce a new Hind III (MBI) restriction site (A^AGCTT) in PCR products (size: 149bp; primers: P2-F1/P2-R2) by the same method.Thus, the amplification contains two recognition sites for the detection of SNPs.Then, aliquots of 10 μL PCR products were digested with 10 U EcoR V and Hind III for 6 h at 37 °C, respectively.The digested products were detected by electrophoresis in 3.0 % agarose gel stained with ethidium bromide, respectively.

Results
SNPs were detected in the exon in 1 032 unrelated cattle from four cattle breeds in China .Comparison between the nucleotide sequences of the bovine NPM1 gene and the above sequences revealed six mutations (GenBank acc no.NC_007307: g.236C>G, 489G>A, 516G>A, 624T>C, 630T>C, 632A>C).Moreover, the SNPs at nt489, nt516, nt624, nt630 and nt632 are in complete linkage disequilibrium: with GGT TA always together and AAC CC always together.The discovered sequence of the first missense mutation (236C>G) was deposited in GenBank (acc no.GQ144334; size: 593 bp; primers: P1-F1/P2-R1), the complete linkage of five SNPs were deposited in GenBank (acc no.FJ794270; size: 972 bp; primers: P1-F1/P1-R2).The variation at two SNP loci caused amino acid mutation 236C>G: Ser to Cys; 632A>C: Glu to Asp, respectively.While variation at the other four SNPs loci were synonymous mutations.Two SNPs (236C>G and 516G>A) were genotyped among four cattle populations by forced PCR-RFLP methods.The genotypic frequencies of two SNP loci in the Chinese Holstein population agreed with Hardy-Weinberg equilibrium (P>0.05)(Table 1).The association of six mutations with body weight and average daily gain of Nanyang and Jiaxian were analyzed together.The statistical model: where Yijk is the trait measured on each of the ijk-th animal, μ is the overall population mean, Ai is the fixed effect due to the i-th age, Gj is the fixed effect associated with j-th genotype and eijk is the random error.The least square means estimates (LSM) with standard errors for three genotypes and growth traits were used.The result indicated that the first missense mutation (236C>G) genotype was significantly associated with body weight and average daily gain.The individuals with GG genotype had higher body weight and average daily gain than the individuals with CG and CC genotypes (P<0.05)(Table 2).However, the five linked mutations (489G>A, 516G>A, 624T>C, 630T>C, 632A>C) were not statistically significantly associated with growth traits (P>0.05).

Table 1
Genotype distribution and allelic frequencies at NPM1 gene in cattle Genotypen-Verteilung und relative Allelehäufigkeit des NPM1-Gens beim Rind NY Nanyang breed, BW body weight, ADG average daily gain, mean least square means, SE standard error of the means, Values with different superscripts within the same line differ significantly at a,b P<0.05.transfergene in China (No. 2009ZX08009-157B, No. 2008ZX08007-002), »13115« Sci-Tech Innovation Program of Shaanxi Province (No. 2008ZDKG-11), Program of National Beef Cattle Industrial Technology System, Basic and Foreland Technology Study Program of Henan Province (No. 072300430160).