Original Paper Hum Hered 1992;42; 134-139
P. LucarcllP E. Mantuanoa PL. Giorgih E. BartoIottab G. Cardinaleh M. Cappac R. Palmarinoa
D N A Restriction Fragment Analysis of the Somatostatin Gene in Familial Isolated Growth Hormone Deficiency Type I
* Centro di Genética Evoluzionistica del CNR, Dipartimento di Genética e Biología Molecolare, Universitá ‘La Sapienza’, Roma; h Dipartimento di Pediatría, Universitá, Ancona; c Ospedale Pediátrico Bambino Gesü, Roma, Italia
Abstract The somatostatin (SST) gene was analyzed to detect possible molecular variations in subjects with familial isolated growth hormone deficiency type I (IGHD I). No gross alterations in restriction fragments were observed with 18 used enzymes. The association with two RFLPs closely linked to the SST gene was also negative, adding weight to the evidence that the SST gene is not involved in the etiology of IGHD I. The nucleotide var iability of a 23-kb DNA segment containing the SST gene and its flanking sequences was studied by restriction analysis of a sample of 19 Italians. The date suggest that ~1 in 400 bp is var iant in this region.
Introduction The somatostatin (SST) gene is relevant to human growth because it encodes a family of SST-related peptides with many important roles among which the well-recognized influ ence on the pituitary secretion of growth hor
mone (GH). It is now clear that GH secretion is the result of the interaction between the in hibitory effect of SST and the inducing effect of GH-releasing factor (GHRF). Therefore, variants of the SST gene may contribute to the etiology of some forms of dwarfism although, as yet, no association was reported between
Dr. Lucarel ¡ Paola Dipartimento di Genética e Biología Molecolare, Universitá ‘La Sapienza’ P. le Aldo Moro 5 1-00185 Rome (Italy)
© 1992 S. Karger AG, Basel 0001-5652/92/ 0422-0134 $ 2.75/0
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Key Words Somatostatin Isolated growth hormone deficiency type I Restriction analysis DNA polymorphisms
Materials and Methods Patients and Reference Population Eighty-seven patients were selected from the pop ulations attending the University Pediatric Clinics, An cona (57). and the Hospital ‘Bambino GesiV. Roma (30). A diagnosis of familial IGHD I was established us ing the following criteria: (1) stature below 3'd ccntilc for age and sex; (2) significantly delayed bone age: (3) peak GH levels below 10 ng/ml after stimulation by in sulin. clonidinc or /-dopa: (4) no demonstrable ana tomic cause for IGHD: (5) growth velocity below 4 cm/ year that responded to exogenous hGH; (6) all other pituitary functions normal; (7) normal thyroid func
tion; (8) hGH-N gene sequences grossly normal | man uscript in preparation]; (9) both parents within the normal height range. The control population included 611 healthy unre lated volunteers randomly collected from II different areas in 5 Italian provinces. Stature was not recorded. DNA Analysis Total genomic DNA was extracted according to Kunkel et al. [5] from fresh or frozen blood samples collected in EDTA, digested to completion with a va riety of suitable restriction enzymes chosen for gener ating resolvable fragments, fractionated by electropho resis on 0.6-2% agarose gels and blotted onto Hybond-N or Hybond-N+ membranes (Amersham). The probe, a 2,667-bp human DNA fragment subcloned in pBR322 and designed pgHS7-2.7 which includes the SST gene [1] was generously provided by G.I. Bell. Both the original recombinant or the genomic EcoRIH indlll fragment were p :P|-labelcd by nick translation to a specific activity greater than 2 • 10s cpm/pg. Rou tinely, 2.4 • 10'’cpm of the probe were used with 200 enr filters for 24 h. Filter hybridization was performed in 6x SSC, 0.5% SDS. 5x Dcnhardt’s solution al 68°C for 24 h and followed by high-stringency washes al 68 °C (twice in 0.2x SSC, 0.1% SDS for 1 h, twice in 0.1 x SSC. 0.1% SDS for 1 h). Bands were visualized by autoradiogra phy following 1- to 15-day exposure of an X-ray film (Kodak X-Omat super rapid) at -70°C .
Results and Discussion Restriction Site Polymorphisms and Nucleotide Variability Estimation o f the SST Gene Region Wc used the SST probe to search for fur ther polymorphic restriction site variations besides those previously reported by Naylor et al. [1]. Out of 20 tested restriction enzymes, 16 produced excellent digestion patterns and were subsequently applied to population screening. Table 1 shows the used enzymes and the expected fragment sizes calculated using the available sequence data of the SST gene and a computer word processor to search for DNA restriction sites. Obviously, the expected lengths of the external fragments were not
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SST deficiency or mutant SST molecule and human disease. Developments in molecular genetics have made it possible to detect variations between individuals at a single gene locus using cloned gene probes. The human SST gene has been cloned, sequenced and localized to the end of the long arm of human chromosome 3, region q28 [1-3]. Two polymorphic sites are tightly linked to the .SST gene [1] in spite of their proximity (only about 5.4 kb apart), they do not appear to be in linkage disequilibrium. The four resulting haplotypes are frequent enough to be suitable for clinical, anthropo logical and gene linkage studies (PIC = 0.28) [41. Our strategy was to look for relevant alter ations of the SST region by comparing the re striction map in and around the SST locus be tween individuals with familial isolated GH deficiency type I (IGHD I) and normal con trols. In this process, we have searched for fur ther restriction fragment length polymor phisms (RFLPs) near the SST gene and from these data the nucleotide variability was cal culated. We also investigated the frequency distributions of the alleles and haplotypes defined by EcoRI and BamHI restriction enzymes in patients and controls.
Table 1. Restriction enzymes used in the polymorphie survey of the SST locus Restric tion enzymes
No. Fragments, kb of expected bases
Avail BamHI Ban! Band Bell BgHI EcoRI EcoRV H aelll Hindlll MspI Narl Pst I Pvull Rsal Sau 961 TaqI Xhol
5 6 6 6 6 6 6 6 4 6 4 6 6 6 4 5 4 6
* 0.177 ♦ * 1.173 * 0.356 « ♦ 0.069 * * * * 0.131 * * 1.116 * 0.155 *
*
1.03
observed
* * 0.155 * 0.052 1.414 * 1.262 0.049 *
0.566 0.97
*
0.095 0.994 * * *
*
*
*
0.2 0.239 0.975 0.519 » 0.567 0.03 0.177 *
* *
*
*
1.5 14.5 1.8 1.5 15.0 5.0 12.0 6.3 2.1 4.4 1.9 9.6 3.1 6.5 0.8 3.8 5.0 23.0
0.177 6.7 1.173 0.356
3.5 7.8 0.155 0.6 = 1.414 1.3 1.262 =
=
1.8
6.4 =
0.566 0.97
1.116 0.155 1.030 1.8 0.2 0.567 2.7
= 3.7 4.0
1.9
0.994 5.2
0.239 0.975 0.519 0.62 = 0.177 0.88
Observed restric tion sites n
Poly mor phisms n
4 3 5 6 2 6 3 2 6 2 6 4 4 3 7 6 3 2
_ 1 -
1 -
known for all enzymes used, as the boundary sites fall outside the sequeneed region. The observed fragments and the number of exam ined cleavage sites are reported in the next columns. None of the 68 screened restriction sites of a minimum of 19 subjects were poly morphic. This number was chosen because it gives a 98% probability of detecting an autoso mal variant with an allele frequency of 10% or greater [6]. Considering both the previously known RFLPs [1] and the present results, a rough estimate of heterozygosity per nucleo tide was calculated according to Ewens et al. [7] as: ki “h k5 + kft
8 m 4+ 10m5+ 1 2 m 6
136
A set of 18 endonucleases was used, 4 rec ognizing 4-bp-long sequences, 2 recognizing 5-bp-long sequences and 12 recognizing 6-bplong sequences. 19 individuals or 38 homolo gous DNA segments were examined with each enzyme. 22 and 10 cleavage sites were detected by restriction endonucleases showing four and five base recognition sequences, respectively. All 38 homologous DNA segments were cleaved at the 22 and 10 sites; thus m4 = 22, K4 = 0 and m5 = 10, K5 = 0. With the restriction endonucleases recognizing 6 base sequences, 42 cleavage sites were found, 40 of which were cleaved in all 38 segments, thus m(, = 42, K*, = 2. By using the above reported equation, we estimated the fraction of sites in the DNA seg ment having 2 or more nucleotide types repre-
Lucarelli/Mantuano/Giorgi/Bartolotta/ Cardinale/Cappa/Palmarino
The Somatostatin Gene in IGHD 1
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* Expected fragment sizes not known (see text); = fragments not observed in either the patient or the con trol groups owing to the small molecular size.
M Be
Bx
M
P
T
E
1
1
1
1
1
1
a
e,
lH ll
|m
a p (h m |t m 1
Be Ev P / t M
1 ! iff
?
?
Et 1
1 E,
Bi W
Fig.1. Restriction endonuclease map of the 23-kb Bel I (Bc) - Eco RI (E,) region containing the SST gene. The line below the map corresponds to the DNA probe used in this work. The box delimits the structural portion of the gene. The polymorphic sites arc indicated below the map. M = MspI; E, = EcoRI; B, = BamHI; Bc = Bell; H = H indlll; P = PvuII; Ps = PstI; T = TaqI; Ev = EcoRV.
A map of the SST gene region was con structed using 9 different restriction enzymes for a total of 28 sites (fig. 1).
Sample Analysis The Restriction Data We studied 87 subjects with growth failure due to hGH deficiency (IGHD I). The entire sample was examined with 12 out of 18 en zymes for which the restriction pattern had been previously defined in normals (table 1). The used endonucleases were Avail, BamHI, BanI, EcoRI, EcoRV, H aelll, H indlll, MspI, PstI, PvuII, Rsal, TaqI. No unexpected frag ments or absence of fragments were observed in the patient group. The Polymorphisms Fig. 2 shows the autoradiograms of the EcoRI and BamHI polymorphic bands de tected by the pgHS7-2.7 probe. The alleles were designed Es (kb = 12), EF (kb = 6.4); Bs (kb = 14.5), BF (kb = 7.8,6.7). The control pop ulation composed of 611 subjects was ran domly collected from 11 different areas in Central and Southern Continental Italy and in the two main islands, Sicily and Sardinia. The
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sented to be 2/780 = 0.0026. These data imply that approximately 1 in 400 bases is polymor phic, on the average, over a SST gene region encompassing about 23 kb in total. The nucleotide diversity of the SST gene region in the Italian sample is close to those observed for the human gene clusters of (l-globin [8], apolipoproteins APO Al - APO C3 APO A4 [9] GH [10] and for the human a r anti-trypsin [II] and insulin [12] genes. Together, these data indicate that, on the average, 1 in 500 bp is different between any two randomly chosen chromosomes. In spite of the similarity in nucleotide variability, the extent of polymorphisms in these studies is different. The value of 2.7% resulting from our data, obtained comparing the number of RFLPs to the total number of screened sites, is similar to that reported for both the al-antitrypsin (1.1-2.6%) [11] and insulin (2.9%) [12] genes. Considerably higher values were re ported for the P-globin (9.3%) [11], apolipo proteins APO Al - APO C3 - APO A4 (9.4%) [9] and GH (4.3-4.5%) [10] gene clusters. It is noteworthy that for both the (i-globin and APO Al - APO C3 - APO A4 gene clusters, a relevant number of probes were used [9, 13] and that the GH cluster shows a high internal sequence homology [10].
b
a
Table 2. Restriction enzyme analysis for normals
and patients Genotypes
Normals
Patients
EcoRI“ 12/12 12/6.4 6.4/6.4
480 122b 9b
71 16b 0b
BamHI0 14.5/14.5 14.5/6.7-7.8 6.7-7.S/6.7-7.8
505 !02b 4b
76 lib 0b
" Fig. 2. RFLPs. a Genotypes of the EcoRI alleles. Lane 1: Es Ef; lane 2: Es Es; lane 3. Ef E f. b Genotypes of
the BaniHI alleles. Lane 1; B' B'; lane 2: B° B1; lane 3: B” Bs. The DNA fragment sizes are shown in kilobasc pairs on the right side of each autoradiogram.
Comparison between the genotype frequencies
X: = 0.42; d.f. = 1.
b Pooled classes in calculated the x~. c Comparison between the genotype frequencies X2 = 1.209; d.f. = I.
Table 3. RFLP haplotypes of the human SST locus from parental chromosomes in normals and patients
1 2 3 4
EcoRI
BaniHI
_
_
-
+
+ +
+
Haplotype frequency comparison:
-
normals observed
normals expected
patients observed
978 103 134 7
984.14 97.33 127.88 12.65
147 11 16 -
= 1.946, d.f. = 2.
distribution of the EcoRI and BamHI pheno types in the different samples was in HardyWeinberg equilibrium. A x2 test of heteroge neity indicated that the differences between the 11 groups were statistically not significant and consequently we pooled the data in order to obtain a control sample ethnically consis tent with the patients. The phenotype distri butions for both groups were in accordance with the expected values assuming Hardy-
138
Chromosomes frequencies
Weinberg equilibrium. Table 2 shows the ge notype frequencies: no significant differences were observed in the distribution of either RFL.Ps between patients and controls. The Haplotypes SST haplotypes were examined by deter mining the absence (-) or presence (+) of the polymorphic cleavage sites for both EcoRI and BamHI. They were either deduced di-
Lucarelli/Mantuano/Giorgi/Barlolotta/ Cardinale/Cappa/Palmarino
The Somatostatin Gene in IGHD I
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Haplotypes
rcctly from Southern blot analysis or, in the case of double heterozygotes, inferred from the pedigrees. Only 4 double heterozygote control subjects could not be haplotyped ow ing to the absence of pedigree information. The definition of each haplotype and their distributions among the patient and control groups are shown in table 3. The same table also shows the expected haplotypes for the control group, calculated as the product of the respective allele frequencies given the locus independence [4], A comparison of the two groups gave a x2 of 1.946 with 3 d.f., indicating that the observed differences in the frequen cies are again not significant.
Taken together, our results suggest, at least under the technical conditions used and al lowing for the studied sample size, that the SST gene does not appear to be involved in the genetic determination of 1GHD 1.
Acknowledgments Wc are grateful to Dr. G.I. Bell for generously sup plying us with the probe pgHS7-2.7, and to E. Schiattarella, A. Di Francesco and V. Salviati for excellent technical assistance. The work was supported by the Consiglio Nazionalc dclle Ricerche and by the ‘Fondazione Cenci Bolognctti’.
References 6 Skolnick MH. While R: Strategies for detecting and characterizing re striction fragment length polymor phisms (RFLPs). Cytogenet Cell Genet 1982:32:58-67.' 7 Ewens WJ, Spielman RS. Harris H: Estimation of genetic variation at the DNA level from restriction en donuclease data. Proc Natl Acad Sci USA 1981:78:3748-3750. 8 Chakravarti A. Buetow KH. Antonarakis SE. Waber PG, Boehm CD. Kazazian HH: Nonuniform recom bination within the human p-globin gene cluster. Am J Hum Genet 1984:36:1239-1258. 9 Amonarakis SE. Oeugèn P. Chakra varti A, Halloran SL, Hudson RR. Feisee L. Karathanasis SK: DNA polymorphism haplotypes of the hu man apolipoprotein APOAIAPOC3-APOA4 gene cluster. Hum Genet 1988;80:265-273. 10 Chakravarti A. Phillips JA, III, Mcllits KH. Buetow KH. Sceburg PH: Patterns of polymorphism and link age disequilibrium suggest inde pendent origins of the human growth hormone gene cluster. Proc Natl Acad Sci USA 1984:81:60856089.
11 Matteson KJ. Ostrer H.Chakravarti A. Buetow KH. O'Brien WE. Bcaudet AL, Phillips JA: A study of re striction fragment length polymor phisms at the human alpha-1-anti trypsin locus. Hum Genet 1985:69: 263-267. 12 Chakravarti A. Elbein SC. Permutt MA: Evidence for increased recom bination near the human insulin gene: Implication for disease associ ation studies. Proc Natl Acad Sci USA 1986;83:1045-1049. 13 Kazazian HH.Chakravarti A, Orkin SFL Amonarakis SE: DNA plymorphisms in the human (l-globin gene cluster: in Nei M. Koehn RK (cds): Evolution of Genes and Proteins. Sunderland. Sinauer, 1983, pp 137— 146.
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1 Naylor SL, Sakaguchi AY. Shen LP. Bell Gl. Rutter WJ,ShowsTB: Poly morphic human somatostatin gene is located on chromosome 3. Proc Natl Acad Sei USA 1983:80:26862689. 2 Zabel BU, Naylor SI- Sakaguchi AY, Bell Gl. Shows TB: High-resolution chromosomal localization of human genes for amylase, proopio melanocortin. somatostatin, and a DNA fragment (D3S1) by in situ hy bridization. Proc Natl Acad Sei USA 1983:80:6932-6936. 3 Shen LP. Rutter WJ: Sequence of the human somatostatin I gene. Sci ence 1984;224:168-171. 4 Lucarelli P. Mantuano F. Schiattarella E. Palmarino R: Evidence for linkage equilibrium between two RFLPs associated with the human SST locus. Hum Genet 1988:78:291292. 5 Kunkel LM. Smith KD, Boyer SH, Borgaonkar DS. Wachtel SS. Miller OJ, Breg WR, Jones HW. Rary JM: Analysis of human Y-chromosomespecific reiterated DNA in chromo some variants. Proc Natl Acad Sei USA 1977;74:1245-1249.
P. LucarcllP E. Mantuanoa PL. Giorgih E. BartoIottab G. Cardinaleh M. Cappac R. Palmarinoa
D N A Restriction Fragment Analysis of the Somatostatin Gene in Familial Isolated Growth Hormone Deficiency Type I
* Centro di Genética Evoluzionistica del CNR, Dipartimento di Genética e Biología Molecolare, Universitá ‘La Sapienza’, Roma; h Dipartimento di Pediatría, Universitá, Ancona; c Ospedale Pediátrico Bambino Gesü, Roma, Italia
Abstract The somatostatin (SST) gene was analyzed to detect possible molecular variations in subjects with familial isolated growth hormone deficiency type I (IGHD I). No gross alterations in restriction fragments were observed with 18 used enzymes. The association with two RFLPs closely linked to the SST gene was also negative, adding weight to the evidence that the SST gene is not involved in the etiology of IGHD I. The nucleotide var iability of a 23-kb DNA segment containing the SST gene and its flanking sequences was studied by restriction analysis of a sample of 19 Italians. The date suggest that ~1 in 400 bp is var iant in this region.
Introduction The somatostatin (SST) gene is relevant to human growth because it encodes a family of SST-related peptides with many important roles among which the well-recognized influ ence on the pituitary secretion of growth hor
mone (GH). It is now clear that GH secretion is the result of the interaction between the in hibitory effect of SST and the inducing effect of GH-releasing factor (GHRF). Therefore, variants of the SST gene may contribute to the etiology of some forms of dwarfism although, as yet, no association was reported between
Dr. Lucarel ¡ Paola Dipartimento di Genética e Biología Molecolare, Universitá ‘La Sapienza’ P. le Aldo Moro 5 1-00185 Rome (Italy)
© 1992 S. Karger AG, Basel 0001-5652/92/ 0422-0134 $ 2.75/0
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Key Words Somatostatin Isolated growth hormone deficiency type I Restriction analysis DNA polymorphisms
Materials and Methods Patients and Reference Population Eighty-seven patients were selected from the pop ulations attending the University Pediatric Clinics, An cona (57). and the Hospital ‘Bambino GesiV. Roma (30). A diagnosis of familial IGHD I was established us ing the following criteria: (1) stature below 3'd ccntilc for age and sex; (2) significantly delayed bone age: (3) peak GH levels below 10 ng/ml after stimulation by in sulin. clonidinc or /-dopa: (4) no demonstrable ana tomic cause for IGHD: (5) growth velocity below 4 cm/ year that responded to exogenous hGH; (6) all other pituitary functions normal; (7) normal thyroid func
tion; (8) hGH-N gene sequences grossly normal | man uscript in preparation]; (9) both parents within the normal height range. The control population included 611 healthy unre lated volunteers randomly collected from II different areas in 5 Italian provinces. Stature was not recorded. DNA Analysis Total genomic DNA was extracted according to Kunkel et al. [5] from fresh or frozen blood samples collected in EDTA, digested to completion with a va riety of suitable restriction enzymes chosen for gener ating resolvable fragments, fractionated by electropho resis on 0.6-2% agarose gels and blotted onto Hybond-N or Hybond-N+ membranes (Amersham). The probe, a 2,667-bp human DNA fragment subcloned in pBR322 and designed pgHS7-2.7 which includes the SST gene [1] was generously provided by G.I. Bell. Both the original recombinant or the genomic EcoRIH indlll fragment were p :P|-labelcd by nick translation to a specific activity greater than 2 • 10s cpm/pg. Rou tinely, 2.4 • 10'’cpm of the probe were used with 200 enr filters for 24 h. Filter hybridization was performed in 6x SSC, 0.5% SDS. 5x Dcnhardt’s solution al 68°C for 24 h and followed by high-stringency washes al 68 °C (twice in 0.2x SSC, 0.1% SDS for 1 h, twice in 0.1 x SSC. 0.1% SDS for 1 h). Bands were visualized by autoradiogra phy following 1- to 15-day exposure of an X-ray film (Kodak X-Omat super rapid) at -70°C .
Results and Discussion Restriction Site Polymorphisms and Nucleotide Variability Estimation o f the SST Gene Region Wc used the SST probe to search for fur ther polymorphic restriction site variations besides those previously reported by Naylor et al. [1]. Out of 20 tested restriction enzymes, 16 produced excellent digestion patterns and were subsequently applied to population screening. Table 1 shows the used enzymes and the expected fragment sizes calculated using the available sequence data of the SST gene and a computer word processor to search for DNA restriction sites. Obviously, the expected lengths of the external fragments were not
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SST deficiency or mutant SST molecule and human disease. Developments in molecular genetics have made it possible to detect variations between individuals at a single gene locus using cloned gene probes. The human SST gene has been cloned, sequenced and localized to the end of the long arm of human chromosome 3, region q28 [1-3]. Two polymorphic sites are tightly linked to the .SST gene [1] in spite of their proximity (only about 5.4 kb apart), they do not appear to be in linkage disequilibrium. The four resulting haplotypes are frequent enough to be suitable for clinical, anthropo logical and gene linkage studies (PIC = 0.28) [41. Our strategy was to look for relevant alter ations of the SST region by comparing the re striction map in and around the SST locus be tween individuals with familial isolated GH deficiency type I (IGHD I) and normal con trols. In this process, we have searched for fur ther restriction fragment length polymor phisms (RFLPs) near the SST gene and from these data the nucleotide variability was cal culated. We also investigated the frequency distributions of the alleles and haplotypes defined by EcoRI and BamHI restriction enzymes in patients and controls.
Table 1. Restriction enzymes used in the polymorphie survey of the SST locus Restric tion enzymes
No. Fragments, kb of expected bases
Avail BamHI Ban! Band Bell BgHI EcoRI EcoRV H aelll Hindlll MspI Narl Pst I Pvull Rsal Sau 961 TaqI Xhol
5 6 6 6 6 6 6 6 4 6 4 6 6 6 4 5 4 6
* 0.177 ♦ * 1.173 * 0.356 « ♦ 0.069 * * * * 0.131 * * 1.116 * 0.155 *
*
1.03
observed
* * 0.155 * 0.052 1.414 * 1.262 0.049 *
0.566 0.97
*
0.095 0.994 * * *
*
*
*
0.2 0.239 0.975 0.519 » 0.567 0.03 0.177 *
* *
*
*
1.5 14.5 1.8 1.5 15.0 5.0 12.0 6.3 2.1 4.4 1.9 9.6 3.1 6.5 0.8 3.8 5.0 23.0
0.177 6.7 1.173 0.356
3.5 7.8 0.155 0.6 = 1.414 1.3 1.262 =
=
1.8
6.4 =
0.566 0.97
1.116 0.155 1.030 1.8 0.2 0.567 2.7
= 3.7 4.0
1.9
0.994 5.2
0.239 0.975 0.519 0.62 = 0.177 0.88
Observed restric tion sites n
Poly mor phisms n
4 3 5 6 2 6 3 2 6 2 6 4 4 3 7 6 3 2
_ 1 -
1 -
known for all enzymes used, as the boundary sites fall outside the sequeneed region. The observed fragments and the number of exam ined cleavage sites are reported in the next columns. None of the 68 screened restriction sites of a minimum of 19 subjects were poly morphic. This number was chosen because it gives a 98% probability of detecting an autoso mal variant with an allele frequency of 10% or greater [6]. Considering both the previously known RFLPs [1] and the present results, a rough estimate of heterozygosity per nucleo tide was calculated according to Ewens et al. [7] as: ki “h k5 + kft
8 m 4+ 10m5+ 1 2 m 6
136
A set of 18 endonucleases was used, 4 rec ognizing 4-bp-long sequences, 2 recognizing 5-bp-long sequences and 12 recognizing 6-bplong sequences. 19 individuals or 38 homolo gous DNA segments were examined with each enzyme. 22 and 10 cleavage sites were detected by restriction endonucleases showing four and five base recognition sequences, respectively. All 38 homologous DNA segments were cleaved at the 22 and 10 sites; thus m4 = 22, K4 = 0 and m5 = 10, K5 = 0. With the restriction endonucleases recognizing 6 base sequences, 42 cleavage sites were found, 40 of which were cleaved in all 38 segments, thus m(, = 42, K*, = 2. By using the above reported equation, we estimated the fraction of sites in the DNA seg ment having 2 or more nucleotide types repre-
Lucarelli/Mantuano/Giorgi/Bartolotta/ Cardinale/Cappa/Palmarino
The Somatostatin Gene in IGHD 1
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* Expected fragment sizes not known (see text); = fragments not observed in either the patient or the con trol groups owing to the small molecular size.
M Be
Bx
M
P
T
E
1
1
1
1
1
1
a
e,
lH ll
|m
a p (h m |t m 1
Be Ev P / t M
1 ! iff
?
?
Et 1
1 E,
Bi W
Fig.1. Restriction endonuclease map of the 23-kb Bel I (Bc) - Eco RI (E,) region containing the SST gene. The line below the map corresponds to the DNA probe used in this work. The box delimits the structural portion of the gene. The polymorphic sites arc indicated below the map. M = MspI; E, = EcoRI; B, = BamHI; Bc = Bell; H = H indlll; P = PvuII; Ps = PstI; T = TaqI; Ev = EcoRV.
A map of the SST gene region was con structed using 9 different restriction enzymes for a total of 28 sites (fig. 1).
Sample Analysis The Restriction Data We studied 87 subjects with growth failure due to hGH deficiency (IGHD I). The entire sample was examined with 12 out of 18 en zymes for which the restriction pattern had been previously defined in normals (table 1). The used endonucleases were Avail, BamHI, BanI, EcoRI, EcoRV, H aelll, H indlll, MspI, PstI, PvuII, Rsal, TaqI. No unexpected frag ments or absence of fragments were observed in the patient group. The Polymorphisms Fig. 2 shows the autoradiograms of the EcoRI and BamHI polymorphic bands de tected by the pgHS7-2.7 probe. The alleles were designed Es (kb = 12), EF (kb = 6.4); Bs (kb = 14.5), BF (kb = 7.8,6.7). The control pop ulation composed of 611 subjects was ran domly collected from 11 different areas in Central and Southern Continental Italy and in the two main islands, Sicily and Sardinia. The
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sented to be 2/780 = 0.0026. These data imply that approximately 1 in 400 bases is polymor phic, on the average, over a SST gene region encompassing about 23 kb in total. The nucleotide diversity of the SST gene region in the Italian sample is close to those observed for the human gene clusters of (l-globin [8], apolipoproteins APO Al - APO C3 APO A4 [9] GH [10] and for the human a r anti-trypsin [II] and insulin [12] genes. Together, these data indicate that, on the average, 1 in 500 bp is different between any two randomly chosen chromosomes. In spite of the similarity in nucleotide variability, the extent of polymorphisms in these studies is different. The value of 2.7% resulting from our data, obtained comparing the number of RFLPs to the total number of screened sites, is similar to that reported for both the al-antitrypsin (1.1-2.6%) [11] and insulin (2.9%) [12] genes. Considerably higher values were re ported for the P-globin (9.3%) [11], apolipo proteins APO Al - APO C3 - APO A4 (9.4%) [9] and GH (4.3-4.5%) [10] gene clusters. It is noteworthy that for both the (i-globin and APO Al - APO C3 - APO A4 gene clusters, a relevant number of probes were used [9, 13] and that the GH cluster shows a high internal sequence homology [10].
b
a
Table 2. Restriction enzyme analysis for normals
and patients Genotypes
Normals
Patients
EcoRI“ 12/12 12/6.4 6.4/6.4
480 122b 9b
71 16b 0b
BamHI0 14.5/14.5 14.5/6.7-7.8 6.7-7.S/6.7-7.8
505 !02b 4b
76 lib 0b
" Fig. 2. RFLPs. a Genotypes of the EcoRI alleles. Lane 1: Es Ef; lane 2: Es Es; lane 3. Ef E f. b Genotypes of
the BaniHI alleles. Lane 1; B' B'; lane 2: B° B1; lane 3: B” Bs. The DNA fragment sizes are shown in kilobasc pairs on the right side of each autoradiogram.
Comparison between the genotype frequencies
X: = 0.42; d.f. = 1.
b Pooled classes in calculated the x~. c Comparison between the genotype frequencies X2 = 1.209; d.f. = I.
Table 3. RFLP haplotypes of the human SST locus from parental chromosomes in normals and patients
1 2 3 4
EcoRI
BaniHI
_
_
-
+
+ +
+
Haplotype frequency comparison:
-
normals observed
normals expected
patients observed
978 103 134 7
984.14 97.33 127.88 12.65
147 11 16 -
= 1.946, d.f. = 2.
distribution of the EcoRI and BamHI pheno types in the different samples was in HardyWeinberg equilibrium. A x2 test of heteroge neity indicated that the differences between the 11 groups were statistically not significant and consequently we pooled the data in order to obtain a control sample ethnically consis tent with the patients. The phenotype distri butions for both groups were in accordance with the expected values assuming Hardy-
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Chromosomes frequencies
Weinberg equilibrium. Table 2 shows the ge notype frequencies: no significant differences were observed in the distribution of either RFL.Ps between patients and controls. The Haplotypes SST haplotypes were examined by deter mining the absence (-) or presence (+) of the polymorphic cleavage sites for both EcoRI and BamHI. They were either deduced di-
Lucarelli/Mantuano/Giorgi/Barlolotta/ Cardinale/Cappa/Palmarino
The Somatostatin Gene in IGHD I
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Haplotypes
rcctly from Southern blot analysis or, in the case of double heterozygotes, inferred from the pedigrees. Only 4 double heterozygote control subjects could not be haplotyped ow ing to the absence of pedigree information. The definition of each haplotype and their distributions among the patient and control groups are shown in table 3. The same table also shows the expected haplotypes for the control group, calculated as the product of the respective allele frequencies given the locus independence [4], A comparison of the two groups gave a x2 of 1.946 with 3 d.f., indicating that the observed differences in the frequen cies are again not significant.
Taken together, our results suggest, at least under the technical conditions used and al lowing for the studied sample size, that the SST gene does not appear to be involved in the genetic determination of 1GHD 1.
Acknowledgments Wc are grateful to Dr. G.I. Bell for generously sup plying us with the probe pgHS7-2.7, and to E. Schiattarella, A. Di Francesco and V. Salviati for excellent technical assistance. The work was supported by the Consiglio Nazionalc dclle Ricerche and by the ‘Fondazione Cenci Bolognctti’.
References 6 Skolnick MH. While R: Strategies for detecting and characterizing re striction fragment length polymor phisms (RFLPs). Cytogenet Cell Genet 1982:32:58-67.' 7 Ewens WJ, Spielman RS. Harris H: Estimation of genetic variation at the DNA level from restriction en donuclease data. Proc Natl Acad Sci USA 1981:78:3748-3750. 8 Chakravarti A. Buetow KH. Antonarakis SE. Waber PG, Boehm CD. Kazazian HH: Nonuniform recom bination within the human p-globin gene cluster. Am J Hum Genet 1984:36:1239-1258. 9 Amonarakis SE. Oeugèn P. Chakra varti A, Halloran SL, Hudson RR. Feisee L. Karathanasis SK: DNA polymorphism haplotypes of the hu man apolipoprotein APOAIAPOC3-APOA4 gene cluster. Hum Genet 1988;80:265-273. 10 Chakravarti A. Phillips JA, III, Mcllits KH. Buetow KH. Sceburg PH: Patterns of polymorphism and link age disequilibrium suggest inde pendent origins of the human growth hormone gene cluster. Proc Natl Acad Sci USA 1984:81:60856089.
11 Matteson KJ. Ostrer H.Chakravarti A. Buetow KH. O'Brien WE. Bcaudet AL, Phillips JA: A study of re striction fragment length polymor phisms at the human alpha-1-anti trypsin locus. Hum Genet 1985:69: 263-267. 12 Chakravarti A. Elbein SC. Permutt MA: Evidence for increased recom bination near the human insulin gene: Implication for disease associ ation studies. Proc Natl Acad Sci USA 1986;83:1045-1049. 13 Kazazian HH.Chakravarti A, Orkin SFL Amonarakis SE: DNA plymorphisms in the human (l-globin gene cluster: in Nei M. Koehn RK (cds): Evolution of Genes and Proteins. Sunderland. Sinauer, 1983, pp 137— 146.
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1 Naylor SL, Sakaguchi AY. Shen LP. Bell Gl. Rutter WJ,ShowsTB: Poly morphic human somatostatin gene is located on chromosome 3. Proc Natl Acad Sei USA 1983:80:26862689. 2 Zabel BU, Naylor SI- Sakaguchi AY, Bell Gl. Shows TB: High-resolution chromosomal localization of human genes for amylase, proopio melanocortin. somatostatin, and a DNA fragment (D3S1) by in situ hy bridization. Proc Natl Acad Sei USA 1983:80:6932-6936. 3 Shen LP. Rutter WJ: Sequence of the human somatostatin I gene. Sci ence 1984;224:168-171. 4 Lucarelli P. Mantuano F. Schiattarella E. Palmarino R: Evidence for linkage equilibrium between two RFLPs associated with the human SST locus. Hum Genet 1988:78:291292. 5 Kunkel LM. Smith KD, Boyer SH, Borgaonkar DS. Wachtel SS. Miller OJ, Breg WR, Jones HW. Rary JM: Analysis of human Y-chromosomespecific reiterated DNA in chromo some variants. Proc Natl Acad Sei USA 1977;74:1245-1249.
