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A polymorphism in the human GALR3 galanin receptor gene (GALNR3)
Molecular and Cellular Probes (1999) 13, 325–327 Article No. mcpr.1999.0254, available online at http://www.idealibrary.com. on
Polymorphism Report
A polymorphism in the human GALR3 galanin receptor gene (GALNR3 ) N. M. Lapsys, S. M. Furler, N. K. Henderson, J. L. Dutton, Y. J. Hort, J. A. Eisman, J. Shine and T. P. Iismaa∗ The Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney NSW 2010, Australia (Received 6 May 1999, Accepted 11 June 1999)
KEYWORDS: galanin receptor, neuropeptide, polymorphism, obesity, bone.
INTRODUCTION The neuropeptide galanin is widely expressed in the central and peripheral nervous system and has many physiological effects, including modulation of pituitary hormone secretion and stimulation of appetite. It has mitogenic effects during development and in certain types of cancer, and is expressed in developing bone tissue.1–3 Three galanin receptor subtypes, which are members of the seven transmembrane G proteincoupled receptor superfamily, have been cloned to date.4–6 The GALR3 galanin receptor (Online Mendelian Inheritance in Man, NCBI, #603692) is expressed in hypothalamic nuclei that regulate feeding and appetite, and in the pituitary, spinal cord and a number of peripheral tissues.2,6,7 The gene encoding human GALR3 (GALNR3 ) is located at 22q13.1 and comprises two exons, with exon 1 encoding the N-terminal extracellular region and the first three transmembrane domains, and exon 2 encoding the remainder of the receptor.8 We report here identification of a polymorphism within GALNR3 and
lack of association of this polymorphism with adiposity and bone mineral density.
SOURCE/DESCRIPTION A 255 bp fragment of exon 1 of GALNR3, amplified by polymerase chain reaction (PCR) from a genomic clone (HGW131)9 and corresponding to nucleotides 18–272 of the coding sequence of human GALR3, was used to isolate three distinct clones from a human
Fig. 1. PstI-digested polymerase chain reaction (PCR) products of genomic DNA, analysed on a 2·5% agarose gel. Lane 1: homozygote (TT genotype); Lane 2: homozygote (tt genotype); Lane 3: heterozygote (Tt genotype). Fragment sizes are given in bp.
The nucleotide sequence described in this paper has been submitted to the GenBank/EMBL Data Base under Accession No. AF129513 and AF129514. ∗ Address to whom all correspondence should be addressed at: Neurobiology Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney NSW 2010, Australia. Tel: +61 2 9295 8293; Fax: +61 2 9295 8281; E-mail: [email protected]
0890–8508/99/040325+03 $30.00/0
1999 Academic Press
N. M. Lapsys et al.
326
Table 1. Human GALNR3 PstI polymorphism allele frequency Genotype tt tT TT
Number
Percent
130 132 55
41·0 41·6 17·4
digestion was performed on one-half of the reaction product as recommended (Promega Corp. Madison, WI, USA), digest products were separated on an agarose gel and were visualized by ethidium bromide staining. Individuals homozygous for absence of the PstI site (TT) exhibited a single fragment of 413 bp, individuals homozygous for presence of the PstI site (tt) exhibited bands of 255 and 158 bp, and heterozygous individuals (Tt) exhibited all three bands (Fig. 1).
T: absence of PstI site. t: presence of PstI site.
P1 genomic DNA library (Genome Systems, St Louis, MO, USA). All three clones contained both coding exons of GALNR3. Restriction endonuclease analysis and sequencing of these clones identified a PstI site within intron 1 that was polymorphic, with the sequence [5′-TTGCAG-3′] occurring instead of [5′CTGCAG-3′] in one of the three clones.
FREQUENCY A total of 438 individuals, including 121 twin pairs (59 dizygotic, 62 monozygotic), were genotyped for the GALNR3 polymorphism. Observed genotype frequencies, which were calculated by including data from only one twin (chosen at random) from each twin pair, are shown in Table 1. The genotype and allele distributions were not in Hardy-Weinberg equilibrium (v2=4·4, df=1, p=0·04), but this marginally significant result requires confirmation in other populations.
PRIMER SEQUENCES intF: 5′-CCTGGCTGGGCAGGGCTG-3′ intR: 5′-GCTGTGGCAGGAGAATCGCTGG-3′
PCR CONDITIONS ASSOCIATION ANALYSIS A 413 bp fragment spanning the polymorphic PstI site was amplified by PCR using AmpliTaq Gold (PerkinElmer Cetus), oligonucleotide primers [intF and intR; 200 n in 100 ll reaction volume] and cycling conditions as follows: 95°C for 6 min, followed by 30 cycles of 94°C for 45 s, 60°C for 1 min and 72°C for 1 min, then incubation at 72°C for 10 min. PstI
In a total of 201 females and 73 males, including individuals from the twin cohort and a small subset (n=32) of unrelated individuals, direct measures of adiposity and bone mineral density (BMD) were available from dual energy X-ray absorptiometry (DXA).10 Subjects in this group ranged in age from 20 to 83
Table 2. Lack of association of genotype at GALNR3 with measures of adiposity and bone mineral density Physical trait
Significance level of association analysis (P-value) Males
Females
Adiposity Weight Body Mass Index Whole-Body Fat (%) Central Fat (%)
0·68 0·83 0·32 0·67
0·58 0·82 0·72 0·82
Bone Mineral Density Whole-Body Lumbar Spine Femoral Neck Trochanter Ward’s Triangle
0·68 0·46 0·89 0·79 0·34
0·88 0·75 0·20 0·21 0·81
The analysis for adiposity-related traits included a correction for age. Bone mineral density data were corrected for both age and weight. Non-significant results were also obtained when age and weight corrections were applied separately.
A polymorphism in the GALR3 galanin receptor
years (mean=51 years). They were not specifically selected for a particular phenotype; the sample distributions of body weight, adiposity and BMD were typical of the general population. An association analysis by ANOVA was undertaken to investigate possible co-variation in fat depot size or BMD with genotype at the GALNR3 locus. As well as wholebody measures, specific traits considered were central fat content, and BMD determined at the lumbar spine, femoral neck, trochanter and Ward’s triangle. A random-effect nested ANOVA design was used to model the twin relationship. Data for males and females were analysed separately after adjusting for age. For the BMD data only, additional analyses were undertaken after adjusting for body weight alone, or weight and age.11 Irrespective of analytical approach, no association was apparent between marker genotype and measures of adiposity or BMD (Table 2). These data are consistent with an earlier report of lack of association or linkage of galanin with body fat deposition12 and suggest further that any effects of galanin on bone physiology3 are not mediated by the GALR3 galanin receptor subtype.
3.
4.
5.
6.
7.
8.
9.
ACKNOWLEDGEMENTS This work was supported by the National Health and Medical Research Council (Australia) and a grant from Aza Research Pty Ltd, a joint venture of the Garvan Institute of Medical Research and Eli Lilly Australia Pty Ltd.
10.
11.
REFERENCES 1. Crawley, J. N. (1995). Biological actions of galanin. Regulatory Peptides 59, 1–16. 2. Iismaa, T. P. & Shine, J. (1999). Galanin and galanin receptors. In Results and Problems in Cell Differentiation, Vol 26: Regulatory Peptides and Cognate
12.
327
Receptors (Richter, D., ed.) Pp 257–292. Heidelberg, Germany: Springer-Verlag. Xu, Z.-Q., Shi, T.-J. & Ho¨kfelt, T. (1996). Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos. Proceedings of the National Academy of Sciences USA 93, 14901– 905. Habert-Ortoli, E., Amiranoff, B., Loquet, I., Laburthe, M. & Mayaux, J.-F. (1994). Molecular cloning of a functional human galanin receptor. Proceedings of the National Academy of Sciences USA 91, 9780–783. Howard, A. D., Tan, C., Shiao, L.-L. et al. (1997). Molecular cloning and characterization of a new receptor for galanin. FEBS Letters 405, 285–290. Wang, S. K., He, C. G., Hashami, T. & Bayne, M. (1997). Cloning and expressional characterization of a novel galanin receptor–identification of different pharmacophores within galanin for the three galanin receptor subtypes. Journal of Biological Chemistry 272, 31949–52. Kolakowski, L. F. Jr, O’Neill, G. P., Howard, A. D. et al. (1998). Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. Journal of Neurochemistry 71, 2239–51. Iismaa, T. P., Fathi, Z., Hort, Y. J., Iben, L. G., Dutton, J. L., Baker, E., Sutherland, G. R. & Shine, J. (1998). Structural organization and chromosomal localization of three human galanin receptor genes. Annals of the New York Academy of Sciences 863, 56–63. Fathi, Z., Cunningham, A. M., Iben, L. G. et al. (1997). Cloning, pharmacological characterization and distribution of a novel galanin receptor. Molecular Brain Research 51, 49–59. Carey, D. G., Jenkins, A. B., Campbell, L. V., Freund, J. & Chisholm, D. J. (1996). Abdominal fat and insulin resistance in normal and overweight women: Direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM. Diabetes 45, 633–8. Pocock, N., Noakes, K., Griffiths, M., Bhalerao, N., Sambrook, P., Eisman, J. & Freund, J. (1997). A comparison of longitudinal measurements in the spine and proximal femur using lunar and hologic instruments. Journal of Bone and Mineral Research 12, 2113–18. Kofler, B., Lapsys, N., Furler, S. M., Klaus, C., Shine, J. & Iismaa, T. P. (1998). A polymorphism in the 3′ region of the human preprogalanin gene. Molecular and Cellular Probes 12, 431–32.
Polymorphism Report
A polymorphism in the human GALR3 galanin receptor gene (GALNR3 ) N. M. Lapsys, S. M. Furler, N. K. Henderson, J. L. Dutton, Y. J. Hort, J. A. Eisman, J. Shine and T. P. Iismaa∗ The Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney NSW 2010, Australia (Received 6 May 1999, Accepted 11 June 1999)
KEYWORDS: galanin receptor, neuropeptide, polymorphism, obesity, bone.
INTRODUCTION The neuropeptide galanin is widely expressed in the central and peripheral nervous system and has many physiological effects, including modulation of pituitary hormone secretion and stimulation of appetite. It has mitogenic effects during development and in certain types of cancer, and is expressed in developing bone tissue.1–3 Three galanin receptor subtypes, which are members of the seven transmembrane G proteincoupled receptor superfamily, have been cloned to date.4–6 The GALR3 galanin receptor (Online Mendelian Inheritance in Man, NCBI, #603692) is expressed in hypothalamic nuclei that regulate feeding and appetite, and in the pituitary, spinal cord and a number of peripheral tissues.2,6,7 The gene encoding human GALR3 (GALNR3 ) is located at 22q13.1 and comprises two exons, with exon 1 encoding the N-terminal extracellular region and the first three transmembrane domains, and exon 2 encoding the remainder of the receptor.8 We report here identification of a polymorphism within GALNR3 and
lack of association of this polymorphism with adiposity and bone mineral density.
SOURCE/DESCRIPTION A 255 bp fragment of exon 1 of GALNR3, amplified by polymerase chain reaction (PCR) from a genomic clone (HGW131)9 and corresponding to nucleotides 18–272 of the coding sequence of human GALR3, was used to isolate three distinct clones from a human
Fig. 1. PstI-digested polymerase chain reaction (PCR) products of genomic DNA, analysed on a 2·5% agarose gel. Lane 1: homozygote (TT genotype); Lane 2: homozygote (tt genotype); Lane 3: heterozygote (Tt genotype). Fragment sizes are given in bp.
The nucleotide sequence described in this paper has been submitted to the GenBank/EMBL Data Base under Accession No. AF129513 and AF129514. ∗ Address to whom all correspondence should be addressed at: Neurobiology Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney NSW 2010, Australia. Tel: +61 2 9295 8293; Fax: +61 2 9295 8281; E-mail: [email protected]
0890–8508/99/040325+03 $30.00/0
1999 Academic Press
N. M. Lapsys et al.
326
Table 1. Human GALNR3 PstI polymorphism allele frequency Genotype tt tT TT
Number
Percent
130 132 55
41·0 41·6 17·4
digestion was performed on one-half of the reaction product as recommended (Promega Corp. Madison, WI, USA), digest products were separated on an agarose gel and were visualized by ethidium bromide staining. Individuals homozygous for absence of the PstI site (TT) exhibited a single fragment of 413 bp, individuals homozygous for presence of the PstI site (tt) exhibited bands of 255 and 158 bp, and heterozygous individuals (Tt) exhibited all three bands (Fig. 1).
T: absence of PstI site. t: presence of PstI site.
P1 genomic DNA library (Genome Systems, St Louis, MO, USA). All three clones contained both coding exons of GALNR3. Restriction endonuclease analysis and sequencing of these clones identified a PstI site within intron 1 that was polymorphic, with the sequence [5′-TTGCAG-3′] occurring instead of [5′CTGCAG-3′] in one of the three clones.
FREQUENCY A total of 438 individuals, including 121 twin pairs (59 dizygotic, 62 monozygotic), were genotyped for the GALNR3 polymorphism. Observed genotype frequencies, which were calculated by including data from only one twin (chosen at random) from each twin pair, are shown in Table 1. The genotype and allele distributions were not in Hardy-Weinberg equilibrium (v2=4·4, df=1, p=0·04), but this marginally significant result requires confirmation in other populations.
PRIMER SEQUENCES intF: 5′-CCTGGCTGGGCAGGGCTG-3′ intR: 5′-GCTGTGGCAGGAGAATCGCTGG-3′
PCR CONDITIONS ASSOCIATION ANALYSIS A 413 bp fragment spanning the polymorphic PstI site was amplified by PCR using AmpliTaq Gold (PerkinElmer Cetus), oligonucleotide primers [intF and intR; 200 n in 100 ll reaction volume] and cycling conditions as follows: 95°C for 6 min, followed by 30 cycles of 94°C for 45 s, 60°C for 1 min and 72°C for 1 min, then incubation at 72°C for 10 min. PstI
In a total of 201 females and 73 males, including individuals from the twin cohort and a small subset (n=32) of unrelated individuals, direct measures of adiposity and bone mineral density (BMD) were available from dual energy X-ray absorptiometry (DXA).10 Subjects in this group ranged in age from 20 to 83
Table 2. Lack of association of genotype at GALNR3 with measures of adiposity and bone mineral density Physical trait
Significance level of association analysis (P-value) Males
Females
Adiposity Weight Body Mass Index Whole-Body Fat (%) Central Fat (%)
0·68 0·83 0·32 0·67
0·58 0·82 0·72 0·82
Bone Mineral Density Whole-Body Lumbar Spine Femoral Neck Trochanter Ward’s Triangle
0·68 0·46 0·89 0·79 0·34
0·88 0·75 0·20 0·21 0·81
The analysis for adiposity-related traits included a correction for age. Bone mineral density data were corrected for both age and weight. Non-significant results were also obtained when age and weight corrections were applied separately.
A polymorphism in the GALR3 galanin receptor
years (mean=51 years). They were not specifically selected for a particular phenotype; the sample distributions of body weight, adiposity and BMD were typical of the general population. An association analysis by ANOVA was undertaken to investigate possible co-variation in fat depot size or BMD with genotype at the GALNR3 locus. As well as wholebody measures, specific traits considered were central fat content, and BMD determined at the lumbar spine, femoral neck, trochanter and Ward’s triangle. A random-effect nested ANOVA design was used to model the twin relationship. Data for males and females were analysed separately after adjusting for age. For the BMD data only, additional analyses were undertaken after adjusting for body weight alone, or weight and age.11 Irrespective of analytical approach, no association was apparent between marker genotype and measures of adiposity or BMD (Table 2). These data are consistent with an earlier report of lack of association or linkage of galanin with body fat deposition12 and suggest further that any effects of galanin on bone physiology3 are not mediated by the GALR3 galanin receptor subtype.
3.
4.
5.
6.
7.
8.
9.
ACKNOWLEDGEMENTS This work was supported by the National Health and Medical Research Council (Australia) and a grant from Aza Research Pty Ltd, a joint venture of the Garvan Institute of Medical Research and Eli Lilly Australia Pty Ltd.
10.
11.
REFERENCES 1. Crawley, J. N. (1995). Biological actions of galanin. Regulatory Peptides 59, 1–16. 2. Iismaa, T. P. & Shine, J. (1999). Galanin and galanin receptors. In Results and Problems in Cell Differentiation, Vol 26: Regulatory Peptides and Cognate
12.
327
Receptors (Richter, D., ed.) Pp 257–292. Heidelberg, Germany: Springer-Verlag. Xu, Z.-Q., Shi, T.-J. & Ho¨kfelt, T. (1996). Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos. Proceedings of the National Academy of Sciences USA 93, 14901– 905. Habert-Ortoli, E., Amiranoff, B., Loquet, I., Laburthe, M. & Mayaux, J.-F. (1994). Molecular cloning of a functional human galanin receptor. Proceedings of the National Academy of Sciences USA 91, 9780–783. Howard, A. D., Tan, C., Shiao, L.-L. et al. (1997). Molecular cloning and characterization of a new receptor for galanin. FEBS Letters 405, 285–290. Wang, S. K., He, C. G., Hashami, T. & Bayne, M. (1997). Cloning and expressional characterization of a novel galanin receptor–identification of different pharmacophores within galanin for the three galanin receptor subtypes. Journal of Biological Chemistry 272, 31949–52. Kolakowski, L. F. Jr, O’Neill, G. P., Howard, A. D. et al. (1998). Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. Journal of Neurochemistry 71, 2239–51. Iismaa, T. P., Fathi, Z., Hort, Y. J., Iben, L. G., Dutton, J. L., Baker, E., Sutherland, G. R. & Shine, J. (1998). Structural organization and chromosomal localization of three human galanin receptor genes. Annals of the New York Academy of Sciences 863, 56–63. Fathi, Z., Cunningham, A. M., Iben, L. G. et al. (1997). Cloning, pharmacological characterization and distribution of a novel galanin receptor. Molecular Brain Research 51, 49–59. Carey, D. G., Jenkins, A. B., Campbell, L. V., Freund, J. & Chisholm, D. J. (1996). Abdominal fat and insulin resistance in normal and overweight women: Direct measurements reveal a strong relationship in subjects at both low and high risk of NIDDM. Diabetes 45, 633–8. Pocock, N., Noakes, K., Griffiths, M., Bhalerao, N., Sambrook, P., Eisman, J. & Freund, J. (1997). A comparison of longitudinal measurements in the spine and proximal femur using lunar and hologic instruments. Journal of Bone and Mineral Research 12, 2113–18. Kofler, B., Lapsys, N., Furler, S. M., Klaus, C., Shine, J. & Iismaa, T. P. (1998). A polymorphism in the 3′ region of the human preprogalanin gene. Molecular and Cellular Probes 12, 431–32.