ORIGINAL ARTICLES DERIVED FROM RESEARCH

Molecular analysis of Sonic hedgehog (Shh) in the etiology of nonsyndromic cleft lip and palate in Chilean case-parent trios

Ramiro J. Rincón R.¹; José Suazo²; Rafael Blanco C.²

¹ Professor, School of Dentistry, Universidad de Antioquia, Medellín, Colombia
² Professors, School of Medicine, Universidad de Chile, Santiago, Chile

SUBMITTED: FEBRUARY 28/2012-ACCEPTED: JUNE 12/2012

Rincón RJ, Suazo J, Blanco R. Molecular analysis of Sonic hedgehog (Shh) in the etiology of nonsyndromic cleft lip and palate in Chilean case-parent trios. Rev Fac Odontol Univ Antioq 2012; 24(1): 110-120.

ABSTRACT

INTRODUCTION: nonsyndromic cleft lip and palate (NSCLP) is one of the most common congenital malformations not only in Chile but also worldwide. It has a multifactorial inheritance pattern with interaction of several genes and the environment. Several experimental studies have proven the participation of Sonic hedgedhog (Shh) in the migration process of cells from the neural crest, in the epithelium-mesenchyme transformation, and in the formation of middle craniofacial structures during embryo development; an association between Shh variants and NSCLP is probable.
METHODS: the goal of this study was to evaluate both exonic and intronic regions adjacent to Shh, in a sample of 150 case-parent trios in order to find possible associations with NSCLP. The PCR-RFLP method was used to determine the presence of heteroduplex. Afterwards, the Conformation Sensitive Gel Electrophoresis (CSGE) technique was used to visualize DNA distortion at the heteroduplexes. As an alternative method, a single-nucleotide polymorphism (SNP) analysis was performed in order to determine NSCLP-Shh associations, by means of these SNPs: rs1233555 and rs1233556, located at the first Shh intron.
RESULTS: no heteroduplexes were found in any of the analyzed Shh segments in 150 trios; SNP analysis did not show associations between Shh and NSCLP either.
CONCLUSIONS: this lack of association may be due to the fact that SNP distribution frequency among Chilean population is different to that of reference populations, or because the number of SNPs analyzed was not sufficient, or even because this study did not include other Shh regions.

INTRODUCTION

Nonsyndromic cleft lip and palate is a congenital malformation produced by lack of fusion among the structures that generate the upper lip and secondary palate development.^1-3 In mammals, by the fourth week the face is formed by the frontal processes surrounded by first and second branchial arches. Later, the frontal process originates medial and lateral frontal-nasal processes, and the first branchial arch originates maxillary and mandibular processes. Nasomedial processes grow more than the lateral ones, fusing with maxillary processes to form the upper lip and the primary palate.⁴ In the internal section of the primitive mouth, the palatal shelves of the maxillary process rise and fuse at the middle line with the nasal septum to form the secondary palate.^{5, 6} Any disruptions during the fusion of these embryo structures produce fissures.^{7, 8} This pathology is classified as syndromic7 and nonsyndromic cleft lip and palate (NSCLP, OMIM 119530).

NSCLP shows the characteristics of a complex hereditary disease produced by genetic and environmental factors, which create phenotypic expression variability.⁹ NSCLP is the most frequent worldwide. Amerindian and Asian races are the ones with the most rates of NSCLP.^{10, 11} In Chile, NSCLP presents an average rate of 1.5 per 1.000 alive newborns, and it is most frequently found among males.¹² The Chilean population of lower socioeconomic status presents a greater composition of Amerindian ethnic groups and higher NSCLP rates.^{13, 14} This anomaly has become a public health problem in Chile due to its social and economic impacts, as it increases its attention and rehabilitation costs.

The following processes are implicated in the development of upper lip and palate: cell migration from the Cranial Neural Crest (CNC),^{15, 16} epithelial-mesenchymal transformation,17 and formation of middle craniofacial structures.18-20 These processes are regulated by the expression of transcription factors and secretionor cell-surface molecules.^{2, 9, 17, 21-25} Sonic hedgedhog (Shh) is a form of signaling probably associated to the formation of upper lip and palate.^{9, 21, 23, 24, 26, 27}

Studies on fish^{19, 27} and mice²⁸ have shown that alterations or deficiencies in Shh block migration of CNC, producing phenotypes similar to NSCLP in humans. It has been observed that Shh in mice is associated to the epithelial-mesenchymal transformation, creating palatal fissures when Shh is mutated.²³ In chicken^{5, 29} and fish,³⁰ Shh alterations produce deficient middle craniofacial structures –phenotypes that are compatible to the holoprosencephaly syndrome,^{31, 32} along with labial-palatal fissures.

In humans, the Shh gene is located on the 7q-36,3 chromosome region, it is constituted by three exons and two introns, and has a length of 29,4 kb³. Molecularly, the sonic hedgehog (Shh)³³ protein behaves like an intercellular signaling molecule, which is synthetized by a precursor that suffers self-catalytic reaction,³⁴ and both cholesterol and palmitic acid covalently adhere to Shh.^34-38 It is believed that addition of lipids, cholesterol, and palmitic acid constrains Shh mobility in extracellular environments.^{39, 40} Sonic hedgehog is essential to the normal development of many organs, and it is a factor for holoprosencephaly.^{34, 41} Shh is expressed in the epithelium of the branchial arches.^{42, 43} Shh alterations in mice lead to phenotypes similar to that of NSCLP.^{8, 23}

These antecedents lead to the hypothesis that Shh would be associated to the NSCLP phenotype in humans. To verify this hypothesis, a sample of caseparent trios was used in order to identify alleles, genotypes, and haplotypes transmitted from parents to their descendants and therefore to establish ShhNSCLP association.

MATERIAL AND METHODS

Patients sample. Once all the participants signed a consent form, previously approved by the Ethics Committee of Universidad de Chile's School of Medicine. DNA was extracted from peripheral blood samples of 150 male patients suffering NSCLP and from their parents by using the modified protocol described by Maniatis.⁴⁴ NSCLP was diagnosed by a geneticist.

Primers were designed to amplify eight segments between 200 and 400 pb (table 1) of the exonic and intronic regions adjacent to Shh exons. These segments were amplified by means of PCR. The Primer 3 software was used to design primers.

The amplified samples were denaturalized at 94 °C and annealed at 64 °C. Possible mutations during the annealing step form mismatch creating deformations in the conformation of amplified segments which are detected by gels sensitive to these deformations, MDE-CSG gels.

In case of finding heteroduplexes, sequencing of the respective amplified segment was performed.

SNP analysis. SNP analysis was an alternative to heteroduplex analysis. Two SNPs were analyzed and their selection was performed by using the NCBI database and the International Project HapMap, having into account their frequency among Europeans and Asians, given the ethnic composition of Chilean population. The software DNA for Windows was used to find the restriction enzymes of these two SNPs. Also, the four primers for amplification of these two SNPs were designed. In order to determine presence of these two SNPs in our case-parent trios samples, we used NlaIII restriction endonucleases for rs1233 555 and Sau3A1 for rs 1233556 (table 2) .

Statistical analysis. Analysis of alleles, genotypes, and haplotypes was performed in order to determine Shh-NSCLP associations, by means of TransmissionDisequilibrium Test (TDT), odd ratio, and likelihood function. The UNPHASED software was used for these analyses.

The alleles of SNPs rs1233555 and rs1233556 were C > T and C > T, respectively. figures 3A and 3B show the results of NlaIII and Sau3A1 restriction endonuclease enzyme digests on nine amplified segments of SNP rs1233555 and eight amplified segments of SNP rs1233556 respectively.

NlaIII digest of the amplified segments of SNP rs1233556 may be observed in the agarose gel of figure 3A; the obtained genotypes were CT, TT and CC. Sau3A1 digest of the amplified segments of SNP rs1233555 may be observed in the agarose gel of figure 3B; the obtained genotypes were CT and CC. Combination of genotypes of both SNPs resulted in haplotypes CC, CT, TC and TT. These haplotypes did not show significant results in terms of association to NSCLP (table 3).

The statistical analysis of alleles, genotypes, and haplotypes did not show their preferential transmission from parents to the affected progeny. These results suggest that no linkage disequilibrium was detected between the two SNPs analyzed in this study. Therefore there was not detected an association between NSCLP and the selected markers (table 3).

DISCUSSION

The purpose of this study was to demonstrate that Shh is associated to NSCLP.

Initially, we used MDE-SGS gels but did not observe bands delayed to ADN migration of eight segments of Shh of 150 trios. This led to the analysis of two SNPs on the first intron of this gene, which presented experimental evidences in the development of upper lip and palate. Analyses of the SNPs did not show Shh-NSCLP associations.

This lack of association between gene Shh and NSCLP among alleles, genotype and haplotype may be due to the fact that the analyzed regions are not involved in such association, but maybe the nonanalyzed regulatory regions up and downstream have a direct or indirect influence on Shh-NSCLP associations. This influence has been suggested by experimental studies which demonstrated that Shh is implicated in cell migrations from Cranial Neural Crest Cells (CNCC), in the formation of middle cranial structures, and in the epithelial-mesenchymal transformation. These events intervene in the differentiation and formation of embryo structures that originate the upper lip and palate.

Mutations and haploinsuficiencies of this gene in fish, chicken, and rats alter cell migration from the cranial neural crest or the positional induction of cranial ecto-mesenchyme cells, affecting the mechanisms of induction, initiation, and execution of specific programs of differentiation among CNCCs and generating phenotypes similar to that of NSCLP in humans.^{16, 23, 27} But mutations of Shh are not the only ones that influence these conditions, there are also the ones modulated by transcriptional regulators which activate several genes that interact with Shh.

For instance, at a later stage of migration and induction of CNCCs, interaction of Shh with fibroblast growth factor 10 (FGF10) affects epithelial-mesenchymal transformation, a key process in the production of labial-palatal fissures.⁷ Another example is the case of bone morphogenetic protein 4 (BMP4), that induces Shh expression in the epithelium of the medial edge epithelia of the palatal shelves enabling their growth and fusion.²¹ Therefore, mutations or functional deficiencies of these Shh-interacting genes may alter such relations and produce the NSCLP phenotype, even if the Shh is not affected. It is also probable that slight Shh modifications severely influence regulation of other genes that participate in the etiology of NSCLP. These regulations of Shh by other genes or vice versa indicate an epistatic relation among these genes in the etiology of NSCLP.^{45, 46} Another important aspect to consider is the presence of this gene in the holoprosencephaly syndrome in humans, suggesting that Shh would be critical for craniofacial development, depending on the embryo's stage and environment. Shh severe mutations or during early embryo stages could lead towards syndromes accompanied by labial-palatal fissures, while mutations or weak interactions with other genes in advanced embryo stages would result in NSCLP only. We could therefore speculate that mutations or haploinsuficiencies of Shh are not the only requisites for producing NSCLP. Nevertheless, in disagreement with the present study, Orioli et al found out Shh mutations and polymorphisms associated to NSCLP.⁴⁷ It is possible, however, that sample size and a rigorous control of some factors inherent to the Chilean population were the factors that produced different results in terms of ShhNSCLP associations among the study populations in both studies.

Similarly, maybe the number of SNPs used was insufficient, or it was necessary to include other SNPs located at Shh-regulating regions in order to determine associations of this gene with NSCLP, or distribution of these SNPs in Chilean population present a frequency that differs from that of the reference populations, so it impeded clearly detecting a possible role of Shh in NSCLP.

Nevertheless, it is necessary to analyze cisregulatory regions and Shh-promoting regions which could also contain variants that influence the NSCLP phenotype.

We could then conclude that this lack of association may be due to the fact that SNP distribution frequency among the Chilean population differs from that of reference populations, or that the number of SNP used was not sufficient, or that other nonanalyzed Shh regions needed to be included.

CORRESPONDING AUTHOR

REFERENCES