J Appl Genet 46(4), 2005, pp. 403-406
Short communication
Single nucleotide polymorphisms in exon 10
of the chinchilla growth hormone receptor (GHR) gene
Daniel Polasik1, Marek Kmieć1, Silvia Liefers2, Arkadiusz Terman1
1
Department of Genetics and Animal Breeding, Agricultural University in Szczecin, Poland
2
Wageningen University, Animal Breeding and Genetics Group, Netherlands
Abstract. The aim of this study was to detect SNPs in exon 10 of the chinchilla growth hormone receptor gene
(GHR) by comparative sequencing. Sixty females of the same breed (Standard) were analysed. Four new SNPs
were identified, which cause 3 amino acid substitutions in the intracellular domain of the receptor: G/C at position
135 bp (in relation to the total sequence of exon 10) (gln/his), CAG/AAA at 352 bp and 354 bp (gln/lys), and C/A
at 641 bp (thr/asn).
Key words: chinchilla, GHR gene, SNP.
Chinchilla is a South American rodent of The full-length membrane form of the receptor is
the suborder Hystricognathi and family composed of 620 amino acids (aa). The first exon
Chinchillidae. There are three species of chinchil- is noncoding. Exon 2 codes for 18 signal peptides
las: Chinchilla lanigera, C. brevicaudata and exons 3 to 7 code for the 246-aa extracellular
and C. real. Only C. lanigera is bred as a fur ani- domain. Exon 8 codes for the 24-aa
mal. The most important elements of the value transmembrane segment, and exons 9 and 10 for
of all fur animals are their size and the quality the 350-aa cytoplasmic domain. The mouse GHR
of their coat. gene consists of 11 coding exons. Nine of them are
Growth hormone (GH) is responsible for many similar in size and sequence to the human GHR
metabolic transformations, such a growth, repro- exons, while exons 4B and 8A do not have any ho-
duction, aging, immune responsiveness (Feng mologous equivalents in the human GHR gene.
et al. 1998) and skin homeostasis (Edmontson Exon 4A encodes an 8-aa segment, which is
et al. 2003), and acts through its receptor (GHR) unique to the mouse GHBP domain, and exon 8A
on target cells. The GHR protein belongs to encodes the 3 terminus of GHBP mRNA (Moffat
the cytokine/haematopoietin superfamily due et al. 1999). Exon 10 of the GHR gene in chinchilla
to its partial amino acid homology. This family is most homologous to the pacarana (94%), Amer-
consists of hormones like prolactin, erythropoie- ican porcupine (93%), paca (93%) and guinea pig
tin, interferons, and interleukins 3 to 7 (Herrington (90%). The same exon in the human, rat and mouse,
and Carter-Su 2001; Schwartzbauer and Menon shows 75 81% similarity to the exon chinchilla (as-
1998). GHRs were also found in a soluble form sessed by BLAST software on the NCBI website,
(GHBP). The human GHR gene consists http://www.ncbi.nlm.nih.gov/). Defects in the GHR
of 10 exons (Godowski et al. 1989). gene often cause insensitivity to GH, which in hu-
Received: May 11, 2005. Revised: July 13, 2005. Accepted: July 20, 2005.
Correspondence: D. Polasik, Department of Genetics and Animal Breeding, Agricultural University in Szczecin, Judyma 6,
71-466 Szczecin, e-mail: [e-mail]
404 D. Polasik et al.
mans causes an extremely short stature called the sequence, with forward and backward primers
Laron-type dwarfism or Laron syndrome separately, was performed by using the DYEnamic
(Godowski et al. 1989; Freeth et al. 1997). Besides ET Dye Terminator Kit MegaBACE (Amersham
short stature, patients also demonstrate metabolic Biosciences) in a GeneAmp PCR System 9600
changes like: delayed puberty, hypoglycaemia, thermocycler (Perkin Elmer Cetus). The following
obesity, prematurely aged skin, normal or higher PCR conditions were applied: 30 cycles of 95°C
biologically active GH concentrations in serum for 20 s, 55°C for 50 s, and 60°C for 60 s.
(Rosenfeld et al. 1994). GHR is an important can- To remove contaminating salts and unincorporated
didate gene for improving growth, carcass dye terminators before sequencing, The Montage
and milk traits in livestock (Ge et al. 2003). SEQ96 Sequencing Reaction Cleanup Kit
The aim of this study was to detect polymorphism (Millipore) was used. Sequencing was performed
within exon 10 of the chinchilla GHR gene. by using a capillary sequencer (Abi Prism 3100).
Sixty female chinchillas of the same breed Pregap4 and Gap4 software were used to visualise
(Standard) were kept under the same conditions in and analyse the sequence of GHR polymorphisms.
a Polish chinchilla farm. DNA was isolated by Four single nucleotide polymorphisms (SNPs)
using a standard blood isolation kit (Masterpure, were found in exon 10 of the chinchilla growth
Epicentre). Lysis of two red cells was needed to hormone receptor gene (NCBI, AY701337).
obtain a sufficient quantity and quality of DNA. The first one is a missense mutation (G/C) at posi-
Primers CHINGHR1 and CHINGHR2 were based tion 135 bp and causes a change of glutamine to
on chinchilla exon 10 of the GHR gene (NCBI, histidine. Frequency of genotypes with this SNP is
AF520660). The primers, with sequences as follows: GG 0.78, GC 0.18 and CC 0.04.
CHINGHR1 5 -AACGATGACTCTTGGGTTG The second and third SNPs were found in the same
Figure 1. Sequence of exon 10 of the chinchilla GHR gene with SNPs and amino acid substitutions
AA-3 and CHINGHR2 5 -GGCAGCTGCATT codon at positions 352 bp and 354 bp
GAGTATGA-3 , were designed by using Primer3 (CAG/AAA), with frequencies: CAG/CAG 0.38,
software. To estimate optimal annealing CAG/AAA 0.31 and AAA/AAA 0.31. These 2 nu-
temperature, a gradient thermocycler was used cleotide substitutions cause a change from
(T-Gradient, Biometra). A 807-bp PCR product glutamine to lysine. The fourth SNP was found at
was obtained by applying the following position 641 bp (C/A). It is also a missense muta-
parameters of PCR reaction: 95°C for 5 min, tion, leading to a threonine to asparagine substitu-
35 cycles of 95°C for 45 s, 60°C for 60 s, 72°C for tion. The following frequencies of genotypes were
50 s and the last extension 72°C for 5 min. observed: CC 0.29, AC 0.49, and AA 0.22. Posi-
To detect polymorphism in this fragment, tions of all SNPs are given in relation to the total
comparative sequencing was used. PCR products sequence of exon 10 and are presented in Figure 1.
were purified from primers by using The Montage Most of the mutations within the GHR gene
PCR96 Filter Plates (Millipore). The PCR of
have been reported in humans with Laron syn-
SNPs in exon 10 of the chinchilla GHR gene 405
Acknowledgements. This work was supported by
drome (Godowski et al. 1989; Kou et al. 1993;
the Animal Breeding and Genetics Group,
Sobrier et al. 1997; Sanchez et al. 1998; Gastier
Wageningen University, Netherlands.
et al. 1999). Amino acid changes within
the extracellular region of GHR can lead to defects
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