Molecular Studies on Preproghrelin Gene : Signal Peptide , Ghrelin Coding Region and Variants – A Brief Report Focusing on rs 34911341 Polymorphism

Ghrelin has been massively described as a central and peripheral factor which induces effects on appetite, carbohydrate metabolism, gastrointestinal motility and pancreatic secretion (1). Furthermore, ghrelin has been found to significantly decrease thyroid hormone concentrations (2). Thus polymorphisms found in the ghrelin coding region of the human preproghrelin gene (GHRL) have been associated with a different number of clinical effects. It has been reported that GHRL is composed of 4 exons on the short arm of chromosome 3 (3). However, Seim et al (4) suggest a new molecular structure for this gene. Thus, this study aimed to clarify GHRL and its products by a simple representation, highlighting the main polymorphisms described for GHRL up to this moment, specifically for the coding region related to the signal and ghrelin peptides of the isoform 1 (major isoform).


Molecular studies on GHRL
The PubMed (http://www.ncbi.nlm.nih.gov/pubmed),SciELO (http://www.scielo.org/php/index.php)and Science Direct (http://www.sciencedirect.com)databases were used to investigate published studies about genetic variations found in dbSNP.All the reference SNPs (rs) found in dbSNP were searched in all these databases.

Results and Discussion
Molecular Structure of GHRL The GHRL gene (ghrelin/obestatin prepropeptide [Homo sapiens (human)] NCBI: Gene ID: 51738, OMIM: 605353, NCBI Reference Sequence: NG_011560.1) is located at 3p25.3 region and is composed of 7198 base pairs (Figure 1A).According to the sequence found in NCBI, GHRL presents 4 exons.However, Seim et al (4) proposed, after experiments, a new molecular structure for this gene.Compared with the sequence found in NCBI, Seim et al ( 4) proposed an exon called -1, which has the same sequence of the exon 1 (NCBI); a new exon 0, which has 736 bp and is found in intron 1 (NCBI); and an extended exon 1 (E1), with 188 bp, also located in intron 1 (4).

GHRL Products
The product of the GHRL is a ghrelin precursor called preproghrelin, a peptide which is composed of 117 amino acid residues (isoform 1).Post-translational processing might lead to different products such as a 23-amino acid signal peptide and a 94-amino acid segment, called proghrelin (1).Proghrelin is metabolized in ghrelin (desacyl ghrelin, represented by 24-51 amino acids residues) and C-ghrelin, also called obestatin, (76-98 amino acid residues) (1) (Figure 1B).Furthermore, in order for ghrelin to come into its full activity, it is essential that this peptide should undergo modifications (5).This process consists of an acylation, a chemical process in which ghrelin receives an octanoyl group at the third serine amino acid residue (Ser3) (1).This acylation reaction is catalyzed by ghrelin O-acyl transferase (GOAT) and its product is the acyl ghrelin (Figure 1C).

GHRL Polymorphisms -Signal Peptide and Ghrelin Coding Regions Until now, 11 single nucleotide polymorphisms were
This scientific article was made in order to make the molecular structure of GHRL less difficult for readers, as well as to show the described variants in specific gene regions.found, described in the region of the GHRL which encodes the signal peptide and 23 variants in the ghrelin coding region (Table 1).

Report Highlights
After linkage disequilibrium analysis, only 4 markers would be enough to map the entire GHRL gene.The rs73125655, rs26802, and rs42451 variants are found in introns and rs696217 in exon 2.
Only one (rs34911341) of the described variants in Table 1 was found in PubMed, SciELO and Science Direct databases.We considered that a brief report of this variant is relevant since it has been associated with severe diseases such as diabetes mellitus, obesity, and hypertension.

The Polymorphism rs34911341
The Arg amino acid residue at position 51 of the preproghrelin peptide was considered important for endoprotease recognition and action (6).The endoprotease catalyzes the proteolytic cleavage within the ghrelin formation process (3,7).However, it is not clear whether this genetic variation really changes the activity or biological properties of ghrelin (5).
A negative association was found between the rs34911341 polymorphism and type 2 diabetes mellitus (T2DM) and obesity in Danish and Malaysian populations (8,9); moreover, similar results were observed regarding this polymorphism in German (10) and Italian (11) studies investigating obesity.
Conflicting results were observed in other studies.The Arg51Gln variant was identified in 6 (all heterozygotes) Swedish obese subjects, with a prevalence of 6.3%, whereas it was not observed among the control subjects (7).Positive association signals were also observed among the rs34911341 variant and other metabolic disorders, such as T2DM in a Finland population (P = 0.009) (6); gestational diabetes in a European-Brazilian population Molecular studies on GHRL (P = 0.0001) (12); hypertension in the Finnish population (P = 0.003) (6) and Caucasians (P = 0.0084) (13).

Conclusion
In this scientific article, we focused on the comparison of the described molecular structure of GHRL (NCBI and Seim et al), making it somewhat more informative for readers.Furthermore, in this first part, we also highlighted the main, or perhaps all reported GHRL genetic variations associated with signal and ghrelin peptides that might be useful to the ones interested in this gene and encourage other researchers in this field.Of the described variants, only rs34911341 has been associated with metabolic disorders, such as diabetes, hypertension and obesity.

Figure 1 .
Figure 1.Representations of the Molecular Structures of GHRL and its Products.A: Simplified comparison and representation of the GHRL from NCBI and Seim et al.The gray horizontal bars represent the whole gene and introns.The numbered boxes (black), the exons.E1: extended exon 1. B: GHRL products found after transcription and translation processes, as well as posttranslational modifications.C: Products formed after GOAT catalysis at Ser3 residue.

Table 1 .
Variants Found in Signal Peptide and Ghrelin Coding Regions and Their Characteristics *Unlike the others polymorphisms described in table 1, only for this variant were found studies in the literature.Obtained data from dbSNP (http://www.ncbi.nlm.nih.gov/snp).