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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1661
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 12 4889-4892
Copyright © 2007 by The Endocrine Society

BRIEF REPORT

Polymorphisms Identified in the Upstream Core Polyadenylation Signal of IGF1 Gene Exon 6 Do Not Cause Pre- and Postnatal Growth Impairment

Debora C. Coutinho, Rocio R. D. Coletta, Elaine M. F. Costa, Paulo R. Pachi, Margaret C. S. Boguszewski, Durval Damiani, Berenice B. Mendonca, Ivo J. P. Arnhold and Alexander A. L. Jorge

Unidade de Endocrinologia do Desenvolvimento (D.C.C., R.R.D.C., E.M.F.C., B.B.M., I.J.P.A., A.A.L.J.), Laboratorio de Hormonios e Genetica Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clinicas da Faculdade de Medicina, and Unidade de Endocrinologia Pediátrica (D.D.), Instituto da Criança, Hospital das Clínicas, Faculdade de Medicina, Universidade de Sao Paulo, 05403-900 Sao Paulo, Brazil; Ambulatorio de Seguimento de Prematuros da Santa Casa de Sao Paulo (P.R.P.), 01221-020 Sao Paulo, Brazil; and Unidade de Endocrinologia Pediatrica (M.C.S.B.), Departamento de Pediatria, Hospital de Clinicas da Universidade Federal do Parana, 01221-020 Curitiba, Brazil

Address all correspondence and requests for reprints to: Alexander A. L. Jorge, Hospital das Clinicas, Laboratorio de Hormonios, Av Dr Eneas de Carvalho Aguiar 155 PAMB, 2 andar Bloco 6, 05403-900 São Paulo, Brazil. E-mail: alexj{at}usp.br.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Few children born small for gestational age (SGA) with IGF1 mutations have been reported. One of these patients presented a mutation at 3' untranslated region (UTR) at exon 6, probably affecting the polyadenylation process.

Objective: The objective of the study was to sequence the IGF1 gene of children born SGA.

Patients and Methods: IGF1 (exons 1–6) was directly sequenced in 53 SGA children without catch-up growth. Allelic variant frequency of the identified IGF1 polymorphisms was assessed in a total of 145 SGA children and in 180 controls born with adequate weight and length and adult height SD score greater than –2.

Results: No mutations were identified in the IGF1 coding regions in SGA children. In contrast, six allelic variants were identified in the upstream core polyadenylation signal located in IGF1 3' UTR at exon 6. The frequency of the different allelic variants was similar in SGA children and controls. It is noteworthy that the same allelic variant, previously described as causing severe IGF1 deficiency, was also observed in homozygous (n = 4) and heterozygous state (n = 6) in normal height controls, corresponding to 4% of studied alleles. The three most frequently identified allelic variants of IGF1 3' UTR showed no effect on height SD score of adult controls as well as on birth characteristics in SGA children.

Conclusion: The polymorphisms identified in the upstream core polyadenylation signal at IGF1 exon 6 do not cause IGF1 deficiency as well as pre- and postnatal growth impairment, in contrast to previously reported data.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
IGF1, OMIM 147440, IS A CRITICAL factor for pre- and postnatal growth (1, 2). The major source of circulating IGF1 is the liver, but it is also ubiquitously produced in the body, with endocrine and paracrine action models (3). It is encoded by a single gene, the IGF1 (NM_000618.2), which comprises six exons, distributed along 84,647 bp in the long arm of chromosome 12 (12 q22-q23). There are two different classes of IGF1 mRNA, originated from different transcription start sites present in either exon 1 or 2, which also codify part of the signal peptide (4). Additionally, IGF1 presents alternative splice sites that generate three major mRNA sizes of 1.1, 1.3, and 7.6 kb (5). The 1.1- and 7.6-kb IGF1 mRNAs comprise exons 1 or 2 plus exons 3, 4, and 6, which share the same coding sequence and differ in terms of their 3' untranslated region (UTR) due to alternative polyadenylation signal sequences at exon 6 (6). These two mRNAs produce IGF1A peptide (153 amino acids), the most abundant IGF1 isoform (5). On the other hand, the 1.3-kb IGF1 mRNA comprises exons 1 or 2 plus exons 3, 4, and 5, which generate IGF1B peptide (195 amino acids), a less abundant IGF1 isoform.

Three IGF1 mutations in homozygous state have been described to date: Woods et al. (7) described the deletion of IGF1 exons 4–5, whereas Bonapace et al. (8) reported a mutation located at the polyadenylation site at the 3'UTR in exon 6 of the IGF1 gene in patients with IGF1 deficiency. Moreover, Walenkanp et al. (9) described an IGF1 missense mutation (V44M) that originates a biologically inactive IGF1 peptide. These patients share similar clinical features: intrauterine growth retardation followed by extremely short stature, mental retardation, microcephaly, and sensorineural deafness. These patients present high levels of basal and/or stimulated GH. Low or undetectable levels of IGF1 were observed in patients who carried disruptive IGF1 mutations, in contrast with high levels of IGF1 in those with the biologically inactive peptide.

In 1990 Lajara et al. (10) screened for alterations in the IGF1 gene; however, their negative results were not conclusive due to technique limitation. Two subsequent studies systematically analyzed the coding region of the IGF1 gene in children with short stature: one analyzed exons 3 and 4 of the IGF1 gene in small for gestational age (SGA) children (11), whereas the other analyzed exons 1–5 in short children, regardless of birth weight and length (12). Both studies used the single strand conformation polymorphism mutation screening method and identified no mutations (11, 12). Single strand conformation polymorphism can present false-negative results (13), which could have influenced the observed negative outcome. In the present study, we analyzed the IGF1 coding region by direct sequencing in children born SGA who did not present spontaneous catch-up growth. A highly polymorphic region in the exon 6 of IGF1 at 3'UTR was identified in patients and controls, including one allelic variant previously described in an IGF1-deficient child; the relevance of these allelic variants on prenatal growth was investigated.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
One hundred forty-five children born SGA that presented birth weight and/or length below –2 SD for gestational age (14) and uncomplicated neonatal period were selected. None of these children presented endocrine disorders, chronic diseases, skeletal alterations, or any syndrome except Silver-Russell syndrome (five cases). Fifty-three of the 145 SGA children that did not present catch-up growth, demonstrated by height SD score (SDS) less than –2 after 2 yr of age (15), were selected for the sequencing of IGF1 exon 1–6. The remaining 92 SGA children were included to assess the influence of allelic variants present in the exon 6 of IGF1 at 3'UTR on prenatal growth.

One hundred eighty healthy adults born at term with adequate birth weight and length and height SDS greater than –2 comprised the control group.

Molecular studies

Genomic DNA was isolated from peripheral blood leukocytes. Exons 1–6 of the IGF1 gene were amplified using intronic primers in the 53 SGA children (primer sequences and amplification protocols will be provided on request). In the remaining 92 SGA children and the adult controls, only exon 6 was amplified to assess the influence of allelic variants present in the 3'UTR of IGF1 on prenatal growth. PCR products were directly sequenced with the dideoxy chain-termination method using a dye terminator kit and analyzed in an ABI Prism 3100 automated sequencer (Applied Biosystems, Foster City, CA).

Statistical analysis

Weight, length, and head circumference at birth were expressed as SDS for gestational age and sex (14), whereas height was expressed as SDS for age and sex (15). Differences between groups were analyzed by t test or Mann-Whitney rank sum test for numerical variables and {chi}2 or Fisher exact test for nominal variables, as appropriate. Statistical significance was set at P < 0.05. Statistical analyses were performed using the SIGMAstat statistical software package (Windows version 2.03; SPSS Inc., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
No mutations in the coding region of IGF1 gene were identified in the 53 SGA children without spontaneous catch-up growth. It is notable that several allelic variants were identified in the exon 6 of IGF1 at 3'UTR in SGA children. These allelic variants are located 260 bp downstream of TAG termination codon of IGF1 gene, in a region that is involved in the polyadenylation process, which is called the upstream core polyadenylation signal (UCPAS) (Fig. 1Go).


Figure 1
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  		FIG. 1. Nucleotide sequence of the UCPAS located in exon 6 of the IGF1 at 3'UTR. Wild-type and six allelic variant sequences (numbered 1–6) are depicted. The natural and alternative polyadenylation signal motifs are underlined. Solid arrows indicate the nucleotide change(s) in each variant.

 
IGF1 exon 6 was sequenced in all remaining children born SGA as well as in controls. The six distinct allelic variants, numbered 1–6, in IGF1 UCPAS region were identified in comparison with the wild-type sequence (Fig. 1Go): variant 1, an A>T substitution in the first nucleotide comprising the AATATA polyadenylation signal motif; variant 2, a T>A substitution in the third nucleotide of AATATA motif; variant 3, a deletion of the second nucleotide in AATATA motif; variant 4, a substitution of A > T in the nucleotide 1 bp upstream of AATATA motif; variant 5, deletion of the third nucleotide in AATATA motif; and variant 6, a substitution of A > T in the nucleotide 2 bp upstream of AATATA motif together with allelic variant 1.

The wild-type sequence was the most frequently observed allele in children born SGA as well as in controls (Table 1Go): it was found in homozygous state in 60% of SGA children and 63% of the control group. The frequencies of identified allelic variants were similar in the controls and in children born SGA (Table 1Go).


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  		TABLE 1. Frequency of allelic variants observed in the UCPAS located in the exon 6 of IGF1 at 3'UTR

 
It is noteworthy that the allelic variant 2, previously described by Bonapace et al. (8) as the cause of IGF1 deficiency in one SGA child, was also observed in homozygous (n = 4) and heterozygous state (n = 6) in controls, corresponding to 3.9% of studied alleles. The heights SDS of the four adults homozygous for allelic variant 2 ranged from –0.1 to +2.4.

The influence of the most frequent allelic variants on birth characteristics in children born SGA and on adult height in controls was investigated. Children homozygous for the wild-type allele or those carrying one or two copies of allelic variant 1, 2, or 3 presented similar gestational age, birth weight, length, and head circumference SDS (Table 2Go). Similarly, no effect of these allelic variants on adult height was observed: height SDS of 0.2 ± 1.1 for wild type (n = 112), 0.2 ± 1.2 for allelic variant 1 carriers (n = 48), 0.3 ± 1.1 for allelic variant 2 carriers (n = 10), 0.2 ± 1.2 for allelic variant 3 carriers (n = 11); P = 0.9.


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  		TABLE 2. Clinical features at birth of children born SGA regarding the four most frequent genotypes in the UCPAS located in IGF1 exon 6

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The substantial genetic contribution to interindividual variation of circulating IGF1 levels is well recognized (16). In addition, low circulating IGF1 levels were observed in children born SGA (1). However, there have been few reports of mutations in IGF1 gene explaining pre- and postnatal growth retardation (7, 8, 9), and few studies have investigated the association between growth and a limited number of polymorphisms in IGF1 noncoding region (17). In the present study, the IGF1 coding region was analyzed in a group of children born SGA without catch-up growth. None of these children had mutations or polymorphisms in the IGF1 coding region. Conversely, a very polymorphic region in the 3' UTR sequence in IGF1 exon 6 was characterized. This region, located upstream of the polyadenylation addition site, comprehends an important region for the correct identification of the endonucleolytic cleavage of pre-mRNA and subsequent addition of poly(A) tail to the newly formed 3' end (18).

The poly(A) tail is critical for the regulation of mRNA stability and translation (19). One of the polyadenylation signals is the highly conserved AAUAAA hexamer or a close variant thereof (termed UCPAS) located 10–30 nucleotides upstream of the cleavage site (20). The {alpha}- and β-thalassemia are examples of human diseases caused by mutations in UCPAS regions (18).

In 2003 Bonapace et al. (8) analyzed the IGF1 gene in one child born SGA presenting clinical and laboratory findings compatible with isolated IGF1 deficiency. The molecular study of the IGF1 gene in this patient disclosed a homozygous nucleotide substitution (AATATA > AAAATA) in the UCPAS located in IGF1 exon 6. The fact that this was the only allelic variant found in IGF1, and its absence in 100 unrelated healthy controls, led the authors to conclude that this mutation was responsible for the patient’s phenotype (8, 18).

However, our molecular study of 145 SGA children and 180 adult controls identified several polymorphisms in IGF1 UCPAS, including the same allelic variant described by Bonapace et al. (8) identified in homozygous state in four controls from our cohort. It is noteworthy the AATATA > AAAATA change (allelic variant 2) yields an alternative AATATA motif 2 bp downstream from the physiological UCPAS hexamer (Fig. 1Go). Similar upstream or downstream dislocation of the UCPAS hexamer occurs in the other described allelic variants. The fact that children and adults harboring these allelic variants do not present a distinct phenotype in comparison with those homozygous for the wild-type allele (Table 2Go) proposes that IGF1 UCPAS variants do not have a major influence on birth characteristics in children born SGA or on adult height of healthy individuals born adequate for gestational age. This finding suggests that AATATA > AAAATA change is not responsible for isolated IGF1 deficiency observed in the patient described by Bonapace et al. It is likely that a nonscreened mutation in the intronic or in the promoter region of IGF1 gene could be responsible for the phenotype observed in their patient.

In conclusion, IGF1 is a well-conserved gene, and mutations in IGF1 are rare in children born SGA. However, a highly polymorphic region located in the upstream core polyadenylation signal at IGF1 exon 6 has been identified for the first time in the present study. Our clinical and laboratory data demonstrate that these polymorphisms do not cause IGF1 deficiency as well as pre- and postnatal growth impairment (8).


   Acknowledgments
 
We thank Ms. Sonia Strong for helping with the English language.


   Footnotes
 
This work was supported by grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (05/04726-0) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (142062/06-5, to D.C.C.; 301246/95-5, to B.B.M.; 300938/06-3, to I.J.P.A.; 306000/04-0, to M.C.S.B., and 307951/06-5, to A.A.L.J.).

Disclosure Statement: The authors declare that they have no competing financial interests.

First Published Online September 25, 2007

Abbreviations: SDS, SD score; SGA, small for gestational age; UCPAS, upstream core polyadenylation signal; UTR, untranslated region.

Received July 25, 2007.

Accepted September 19, 2007.


   References
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 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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