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Analysis of Somatostatin Receptors 2 and 5 Polymorphisms in Patients with Acromegaly

Institute of Endocrine Sciences (M.F., C.R., E.B., S.B., A.G.L., P.B.-P., A.S.), University of Milan, Ospedale Maggiore Policlinico Mangiagalli e Regina Elena, Istituto di Ricovero e Cura a Curattere Scientifico, 20122 Milan, Italy; Pituitary Unit (M.L.), Department of Neurosurgery, Ospedale San Raffaele, Istituto di Ricovero e Cura a Curattere Scientifico, 20132 Milan, Italy; and Clinical Biochemistry (S.G., C.O.) and Endocrine Units (A.P.), Department of Clinical Physiopathology, University of Florence, 50121 Florence, Italy

Address all correspondence and requests for reprints to: Anna Spada, M.D., Istituto di Scienze Endocrine Ospedale Maggiore, Istituto di Ricovero e Cura a Curattere Scientifico, Via Francesco Sforza 35, 20122 Milano, Italy. E-mail: anna.spada{at}unimi.it.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Objective: The aim of the study was to investigate the possible correlation of single nucleotide polymorphisms in somatostatin receptor (SSTR)2 and SSTR5 genes with the responsiveness to somatostatin analogs in a cohort of acromegalic patients.

Study Design: Three single nucleotide polymorphisms (a-83 g, c-57 g, and t80c) of SSTR2 and three (t-461c, c325t, and c1004t) of SSTR5 were analyzed in 66 acromegalic patients with different responsiveness to somatostatin analogs and 66 healthy controls.

Results: Allele frequencies in patients and controls were similar. No association between SSTR2 genotypes and GH and IGF-I levels was found. When considering SSTR5 variants, patients homozygous or heterozygous for the substitution c1004 (P+) showed basal IGF-I levels significantly lower than patients homozygous for 1004t (P–). Moreover, serum GH levels were lower in patients with P+/T– haplotype (having c1004 allele and no t-461 allele) than in those with P–/T+. No correlation between SSTR2 and SSTR5 genotypes, responsiveness to somatostatin therapy, and mRNA expression in the removed adenomas (n = 10) was found.

Conclusions: These data suggest a role for SSTR5 t–461c and c1004t alleles in influencing GH and IGF-I levels in patients with acromegaly, whereas SSTR2 and SSTR5 variants seem to have a minor role in determining the responsiveness to somatostatin analogs.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
SOMATOSTATIN EXERTS ANTISECRETIVE and antiproliferative effects on different endocrine cells by acting through a family of G protein-coupled receptors that includes 5 subtypes (SSTR1–5). Normal human pituitary and pituitary adenomas have been shown to express almost all SSTR subtypes, with the exception of SSTR4 (1, 2, 3). Consistent with the observation that octreotide and other somatostatin analogs bind to SSTR2 and SSTR5 with high affinity (4, 5, 6), these genes have been screened for quantitative/qualitative abnormalities in tumors removed from patients with poor responsiveness to somatostatin analogs treatment (7, 8, 9, 10). Data obtained in GH-secreting adenomas suggested that resistance to octreotide was frequently associated with low expression of SSTR2 mRNA (8), although other authors failed to confirm this finding (10). To date, the only mutational change involving SSTR2 and SSTR5 is the Arg to Trp substitution in codon 240 of the SSTR5 gene that was found in one acromegalic patient resistant to octreotide (11).

In recent years, molecular studies investigated the possible association of gene polymorphisms and susceptibility to diseases and/or resistance to drugs. As far as polymorphic variants of SSTR genes are concerned, a recent study reported the association of polymorphisms in SSTR5 gene and bipolar affective disorder in the Danish and British population, consistent with the hypothesis that reduced somatostatinergic tone or altered SSTR5 interaction with dopamine D2 receptor might be involved in the pathogenesis of this disorder (12). Moreover, a polymorphism identified on human SSTR2 promoter responsible for reduced SSTR2 transcription has been identified in human pancreatic cancer cells (13).

In the present study, we searched for single nucleotide polymorphisms in coding and in 5'-upstream regions of SSTR2 and SSTR5 in a cohort of acromegalic patients and investigated the possible correlation of these variants with the clinical and hormonal characteristics as well as the responsiveness to somatostatin analog therapy.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

The study was carried out on 66 patients (31 men and 35 women, aged 43.3 ± 12.7 yr, 66.1% macroadenomas) with active acromegaly who received the somatostatin analogs lanreotide-SR at the dose of 60 mg im every 21–28 d (n = 28) and octreotide-LAR at the dose of 10–30 mg im every 28 d (n = 38). The levels of GH and IGF-I were evaluated before and after at least 6 months of therapy. According to the current criteria, serum IGF-I levels within the age-adjusted normal range and basal GH values (mean of at least three consecutive samples, collected every 30 min during saline infusion) less than 2.5 µg/liter were considered indicative of disease control. Twenty-one patients underwent adenomectomy by the transphenoidal route, and tumoral tissue samples from 10 patients were collected and frozen for mRNA extraction. No patients had previously undergone pituitary irradiation. The study included 66 sex- and age-matched healthy subjects as controls.

The study protocol was approved by the Ospedale Maggiore Istituto di Ricovero e Cura a Curattere Scientifico Ethics Committee. Informed consent was obtained from all subjects

DNA extraction and genotyping

Genomic DNA was extracted from peripheral blood nucleated cells according to standard procedures, as previously described (11). PCRs were carried out in a 50-µl reaction mix [0.5 µg of DNA sample, 50 nmol/liter KCl, 50 nmol/liter Tris-KCl (pH 8.3), 1 nmol/liter MgCl₂, 25 pmol of each primer, and 2.5 U Taq DNA polymerase AmpliTaq (PerkinElmer Corp., Foster City, CA)] and subjected to denaturation at 94 C for 3 min, followed by 35 cycles of 94 C for 60 sec, specific couple primers melting temperature for 60 sec, and 72 C for 1 min. A final cycle at 72 C for 10 min was carried out to allow complete extension of the amplified fragments. PCR amplification was carried out using primers pairs shown in Table 1. Before denaturing HPLC (DHPLC) analysis, heteroduplex formation was induced by heat denaturation of PCR products at 98 C for 5 min, followed by a reannealing at 62 C for 30 min. Five microliters of these PCR products were automatically loaded on the column (Varian, Inc.), Palo Alto, CA) and were eluted on a linear acetonitrile gradient in a 0.1 M triethylamine acetate buffer (pH 7.0), with a constant flow rate of 0.45 ml/min. The start and end point of the gradient were adjusted according to the size of the PCR products. The denaturing run temperatures predicted by the Stanford DHPLC Melt program (http://insertion.stanford.edu/melt.html) were tested by running a wild-type PCR product for each polymorphism at the calculated run temperature ± 2 C. Experimental run temperatures were as follows: 55 C for a-83 g, 57 C for c-57 g, 60 C for t80c SSTR2 polymorphisms, 64.5 C for t-461c, 63 C for c325t, and 64 C for c1004t SSTR5 polymorphisms. Data were acquired using a UV detector at 260 nm, and heterozygous profiles were identified by visual inspection of superimposed chromatograms. Homozygous samples were then pooled with known homozygous DNA, reannealed and reanalyzed. Subsequently heterozygous sample were interpreted as homozygous for the allele absent in reference DNA. Heterozygous samples, when available, were used as internal positive controls. Standard restriction digestion (pUC 18 HaeIII digest) was used at 50 C to test the DHPLC system and column.

SSTR5 mRNA quantification was performed by real-time RT-PCR, based on TaqMan technologies, as previously reviewed (14). The amount of product was measured by interpolation from a standard curve with RNA extracted from K-Ras-activated colon HCT 116 cell line, which overexpresses SSTR5 mRNA. HCT 116 RNA (1 µg) and neuroblastoma cell line CHP404, which overexpresses SSTR2 mRNA was reverse transcribed, and cDNA was then serially diluted to obtain five standard solutions to be used in the PCR to generate the reference curve, as previously described (15). SSTR2 mRNA measurements were carried out as previously reported (14). SSTR5 mRNA upstream PCR primer corresponds to the region from base 872, 5'-TCCTCTCCTACGCGAACAGC-3', the reverse primer corresponds to the region from base 946, 5'-GGAAGCTCTGGCGGAAGTT-3'. The internal oligonucleotide probe was labeled with the fluorescent dye 5-carboxyfluorescein on the 5-end and N,N,N,N-tetramethyl-6-carboxyrhodamine on the 3-end. The internal probe hybridizes within the region amplified by the PCR primers and has the sequence 5-carboxyfluorescein-CCCGTCCTCTACGGCTTCCTCTCTGA-N,N,N,N-tetramethyl-6-carboxyrhodamine-3'.

Hormone assay

Serum GH levels were measured in acromegalic patients before and after somatostatin treatment by a highly sensitive two-site monoclonal immunofluorimetric assay method supplied by AutoDelfia kit (Wallac OY, Turku, Finland), with a sensitivity of 0.01 µg/liter and intra- and interassay coefficients of variation of 2 and 1.7%, respectively. Serum IGF-I levels were measured in acromegalic patients and healthy controls using the commercial RIA kits from Mediagnost (Tübingen, Germany). The intra- and interassay coefficients of variation were 3.2 and 8.9%, respectively, and IGF-II cross-reactivity less than 0.05%.

Statistical analysis

Quantitative variables were reported in the text as mean ± SD. Differences of the means were tested by t test or one-way ANOVA with Bonferroni posttest, according to normal distribution. Nongaussian variables were analyzed with Mann-Whitney U test or Kruskal-Wallis test with Dunn’s posttest. ² analysis was used to test the hypothesis of no difference between patients and control alleles frequency. Fisher’s exact test was applied whenever expected values were less than 5. Two-tailed P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Analysis of SSTR2 polymorphisms

Analysis of three polymorphisms (a-83 g, c-57 g, and t80c) of SSTR2 by DHPLC showed comparable proportions of various alleles in acromegalic patients and controls (Table 2). In particular, SSTR2 t80 (L27S) allele was detected in homozygosis in all the samples and thus excluded from further considerations, whereas the other polymorphisms had largest allelic frequencies. Due to the small number of patients with some genotypes (e.g. –83 g homozygotes), possible associations between polymorphisms and quantitative variables were evaluated by grouping heterozygotes and homozygotes for the minor allele. Accordingly, –83 g homozygotes or a/g heterozygotes was defined as G+ and a –83 homozygotes as G–, whereas c-57 homozygotes or c/g heterozygotes were defined as C+ and –57 g homozygotes as C–.

Analysis of three polymorphisms (t-461c, c325t, and c1004t) in SSTR5 gene showed no differences in the percent of patients heterozygous or homozygous for the SSTR5 polymorphisms, compared with those found in controls (Table 2). In particular, 325t (P109S) was not found in our series, whereas a previously unreported variation (g994a, A332T) was detected in one healthy subject. As assessed for SSTR2 polymorphisms, subjects homozygous for less common allele were grouped with the corresponding heterozygotes. Accordingly, c1004 (P335) homozygotes or c/t heterozygotes were defined as P+ and 1004t (335L) homozygotes as P–, whereas t-461 homozygotes or t/c heterozygotes were defined as T+ and –461c homozygotes as T–.

No significant difference in tumor size between P+ and T+ genotypes was recorded (Table 3). By contrast, the different genotypes were associated with different hormonal levels.

In particular, P+ patients (n = 39, aged 41.2 ± 12.6 yr) had IGF-I levels significantly lower than P– (n = 27, aged 42.9 ± 9.9 yr) [772.3 ± 241.0 µg/liter (100.3 ± 31.3 nmol/liter)] vs. 1018.7 ± 348.0 µg/liter [132.3 ± 45.2 nmol/liter), P = 0.02 by Student’s t test], whereas the reduction of GH levels did not reach the statistical significance (Table 3). This association was not present in healthy controls [P+: 139.4 ± 27.0 µg/liter (18.1 ± 3.5 nmol/liter) vs. P–: 157.8 ± 45.4 µg/liter (20.5 ± 5.9 nmol/liter, Student’s t test, P > 0.05].

The study of c1004t and t-461c SSTR5 polymorphisms combined in P/T haplotypes revealed GH levels lower in P+/T– (n = 4) than in P–/T+ (n = 6) patients (6.2 ± 2.5 vs. 43.2 ± 27.7 µg/liter, P = 0.04 by Kruskal-Wallis test, P < 0.05 by Dunn’s posttest, Fig. 1B). Basal IGF-I levels in P/T haplotypes showed a trend similar to that of GH levels, although the difference between the two groups did not reach the statistical significance (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Basal circulating levels of GH (A) (mean of at least three consecutive samples during saline infusion) and IGF-I (B) in 66 acromegalic patients categorized by SSTR2 and SSTR5 genotypes and haplotypes as follow: P+ subjects were c1004 homozygotes and heterozygotes; P– subjects were t1004 homozygotes; T+ subjects were t-461 homozygotes and heterozygotes; T– subjects were c-461 homozygotes; P+/T+ subjects had c1004 and t-461 alleles; P–/T– did not have both alleles; and P+/T– had only c1004 allele and P–/T+ conversely. a, Kruskal-Wallis test, P < 0.05, Dunn’s posttest on P+/T– and P–/T groups; b, Student’s t test, P = 0.02. Mean ± SEM.

SSTR2 and SSTR5 mRNA expression

SSTR2 and SSTR5 mRNA levels were detected in 10 adenomatous tissue samples, obtained by surgery, using real-time PCR technique. SSTR5 was expressed at higher levels than SSTR2, although both SSTR2 and SSTR5 mRNA were highly variable and overlapping (SSTR2: 8.26 ± 0.76 Log n° molecules/µg RNA, SSTR5: 8.81 ± 1.00 Log n° molecules/µg RNA, where Log n° molecules means common logarithm of the number of SSTR2 or SSTR5 RNA molecules). No clear differences in mRNA expression of both receptors in patients with polymorphisms in the coding and 5' upstream regions of SSTR2 and SSRT5 genes were observed (data not shown).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
This is the first report of a systematic search for single nucleotide polymorphisms in SSTR2 and SSTR5 genes in acromegalic patients. Although it cannot be excluded that minor changes in the promoter or coding regions may affect the expression or function of the receptor, we focused our analysis on the reported single nucleotide polymorphisms located in promoter element-binding sites or causing nonsynonymous amino acid substitutions. By this approach, we failed to detect significant differences in SSTR2 and SSTR5 allele frequencies between acromegalic patients and controls. In particular, considering SSTR2 t80c (the only missense substitution reported in National Center for Biotechnology Information single nucleotide polymorphisms database, http://www.ncbi.nlm.nih.gov/SNP/), all subjects showed t80 allele suggesting a low, if any, frequency of the 80c variant. Similarly, by analyzing the SSTR5 polymorphisms, we did not find the 325t allele in any subject, consistent with the low frequency of this allele (0.04) reported by Nyegaard et al. (12) in British and Danish populations. The remaining single nucleotide polymorphisms in SSTR2 and SSTR5 genes analyzed showed a variable prevalence, which was, however, comparable in the affected and control populations.

We then investigated the possible associations of the different genotypes with the clinical and hormonal characteristics of the patients as well as the responsiveness to somatostatin analogs therapy. With regard to SSTR2 polymorphisms, the two single nucleotide substitutions detected in the promoter sequence at positions –83 and –57 from the major transcription initiation site were not associated with differences in clinical characteristics, hormonal profile, or responsiveness to somatostatin analog therapy. This is at least in part in contrast with the functional phenotypes of these variants that have been previously identified and characterized in human pancreatic cancers and pancreatic cell lines (13). In fact, –83 g substitution reduced SSTR2 transcription by 60–70%, the repression being due to the specific fixation of nuclear factor I, whereas –57 g variant was without effect (13). The results of our study indicated that, although the poor responsiveness to somatostatin analogs in acromegalic patients have been correlated to the reduced SSTR2 expression by the corresponding tumor tissues (8, 16, 17), this phenomenon was not due to polymorphic variants in SSTR2 gene promoter. Taken together, these data suggest a minor, if any, impact of SSTR2 polymorphisms on GH secretion and responsiveness to medical treatment.

As far as single nucleotide polymorphisms in SSTR5 were concerned, patients with SSTR5 c1004 allele (P335) showed IGF-I levels significantly lower than patients homozygous for 1004t (335L) and a tendency to lower GH levels, although this observation was obtained in a rather limited cohort of patients due to the rarity of the disease. This substitution is a common polymorphism causing a nonconservative amino acid change, i.e. proline to leucine. In fact, proline is structurally important because it has a rigid cyclic structure, it is often present in turns between helices, and it has no structural similar counterpart. The c1004t is located in the C-terminal intracellular tail of the receptor, and mutation analysis of this domain has shown that it is involved in the interaction with adenylyl cyclase and in receptor desensitization and internalization (18). Moreover, this variant has been recently associated with bipolar affective disorder in the British population, consistent with the hypothesis that reduced somatostatinergic tone or altered SSTR5 interaction with dopamine D2 receptor might be involved in the pathogenesis of this disorder (12, 19). Therefore, it is tempting to speculate that the variant receptor may confer a low sensibility to the hypothalamic somatostatin tone, resulting in an impaired negative feedback and higher IGF-I levels in acromegalic patients. The finding that this variant was associated with increased IGF-I levels in acromegalic patients but not in controls might be related to the increased somatostatinergic tone reported in acromegalic patients as well as in animals transgenic for GH (20, 21). Conversely, the lack of differences in the responsiveness to somatostatin analogs between the different genotypes was likely related to the supraphysiological doses of peptide reached during octreotide or lanreotide administration. Taking together these data suggest a possible role of SSTR5 in influencing GH and IGF-I levels in patients with acromegaly. On this line, loss of heterozygosity at the SSTR5 gene locus and retention of the 335L variant have been recently observed in one aggressive TSH-secreting adenoma removed from a patient resistant to octreotide therapy (22).

Recently, a polymorphism (192 bp AC repeat) in the IGF-I gene that influences the age-related serum IGF-I decline has been reported by Rietveld et al. (23). In this study, carried out on healthy subjects aged between 55 and 75 yr (mean 64.3 yr), subjects homozygous for the 192-bp polymorphism showed a significant age-related decline of IGF-I levels, whereas the other genotypes (heterozygotes and noncarriers of the 192-bp polymorphism) did not show this trend. Although we cannot exclude the presence of this association also in patients with acromegaly, it is unlikely that the reduction in IGF-I levels observed in patients with SSTR5 c1004 allele may be related to IGF-I gene variants because patients included in the present study were relatively young (mean 43.3 yr ± 12.7 yr), and the age distribution was similar in the various subgroups considered.

When considering the SSTR5 c1004t and t-461c haplotypes (P/T), patients with P+/T– haplotype showed GH levels significantly lower than P–/T+. This association suggested opposite actions of SSTR5 c1004 and t-461 alleles on GH secretion, as also indicated by the observation that in patients having them both present or absent (P+/T+ or P–/T– haplotypes) GH level were superimposable. Accordingly, taking into account that t-461c polymorphism is included in a sequence recognized by Hmx1, a transcription factor that belongs to a family of homeodomain proteins (24), it is tempting to hypothesize that this polymorphism might affect SSTR5 transcription. Admittedly, the low number of patients in P+/T– and P–/T+ groups and the low number of corresponding tumors did not allow definitive conclusions on the impact of t-461c polymorphism on SSTR5 expression.

In conclusion, these findings suggest a possible role of SSTR5 c1004t and t-461c alleles in influencing GH and IGF-I levels in patients with acromegaly, although further studies on large populations are required to confirm these data. Moreover, polymorphic variants in SSTR2 and SSTR5 genes seem to have a minor, if any, role in determining the different responsiveness to somatostatin analog in patients with acromegaly.

	Footnotes

 
First Published Online May 24, 2005

Abbreviations: DHPLC, Denaturing HPLC; SSTR, somatostatin receptor.

Received January 24, 2005.

Accepted May 18, 2005.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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