EPHA1 gene SNPs analysis in population of Ukraine

Aim. Analysis the EPHA1 gene G1475A and G1891A alleles distribution in the population of Ukraine, and to study the protein secondary structure as the first step in the investigation of EPHA1 gene involvement in intellectual disability pathogenesis. Methods. Observation group consisted of 300 individuals, including 164 (54.6 %) male and 136 (45.3 %) female individuals. Polymorphic variants were detected using PCR followed by Kpn1 RFLP analysis for G1475A and ARMS PCR analysis for G1891A. Results. The data concerning EPHA1 genotypes and allelic variants distribution were obtained. The low frequency of 1475A allele (0,012) and the absence of 1891A allele (not previously described) were revealed in the population of Ukrainian. Conclusions. Assays for the detection of EPHA1 gene G1475A and G1891A SNPs based on ARMS and restriction analysis were developed and the preliminary data on distribution of G1475A and G1891A polymorphisms in the population of Ukraine were obtained.


Introduction.
In the frame of CHERISH project (no.223692) concerning the intellectual disability (ID) genetic basis, the next generation exome sequencing was conducted for a Ukrainian family (UKR 094) with two affected children (proband and his younger sibling) and their healthy non-consanguineous parents.The proband (UKR 263) is a 12 year old boy with ID (IQ = 43) and hyperactivity.His brother (UKR 264) is a 4 year old boy with febrile convulsions, ID and multiple congenital anomalies (Fig. 1).Both affected probands underwent physical and neurological examination, standard G-banding karyotype analysis and biochemical tests: aminoacids and acylcarnitines (TANDEM MS) as well as molecular genetic screening of common ID syndromes (FRAXA, FRAXE, FRAXF loci together with 15q11-q13 locus).Standard karyotype, biochemical and molecular genetic tests as well as array-CGH analysis (400 K resolution) were normal in both patients.
Exome analysis revealed several variants in either homozygous or compound heterozygous state in five genes.Among these genes, we decided to concentrate on the EPHA1 gene, where two non-synonymous substitutions were detected in both patients: G1475A (rs11768549) inherited from the mother and G1891A (has not been previously described) inherited from the father.The both non-synonymous SNPs have been identified in the coding region (exons 7 and 11) of the EPHA1 receptor gene.EPHA1 has been reported to be highly expressed in the neural tissues during embryogenesis and it seemed a good candidate for autosomal recessive inherited ID [1,2].
EPHA1 is the founding member of the erythropoietin-producing hepatocellular (Eph) receptors family, but little is known about its function.Eph kinases constitute the largest family of receptor tyrosine kinases, with 16 distinct members.The receptors are termed EPHA (EPHA1-8, only including those found in mammals) or EPHB (EPHB1-4, EPHB6) on the basis of se-quence homologies and depending on the subgroup of ligands that they bind [3].Ligand for the EPHA receptor is called ephrin-A.It has been recently demonstrated that EPHA/ephrin-A signalling has a proapoptotic effect on the embryonic cortex without affecting the proliferation or cell cycle progression [4].Ephrin and Eph receptor genes are implicated in the control of cell and axon guidance in neural systems of different species [1,2,5,6].
EPHA1 gene is located at chromosome 7q34 and contains 18 exons, two more than the related tyrosine kinase.The human EPHA1 gene is transcribed as a single 3.5 kb mRNA, and encodes a polypeptide of 984 amino acids with a molecular mass of 130 kDa [7].It is important to know that EPHA1 is characterized by high conservative level among many species, indicating that there may be an evolutionarily conserved function [7].It was shown that this gene is highly expressed in liver, lung, and kidney, but not in the adult brain.However, it is highly expressed in the spinal neural tube during neurulation [8].The role of EPHA1 during tissue morphogenesis is highlighted by the expression studies performed on mouse embryos, where the pattern of expression is dynamic throughout the immediate postimplantation, gastrulation, neurulation, and early somitogenesis periods [9].
In this study the analysis of single nucleotide polymorphisms G1475A and G1891A in EPHA1 gene was performed in population of Ukraine and it is the first step in the analysis of EPHA1 gene involvement in intellectual disability pathogenesis.To understand a biochemical effect of these substitutions we tried to predict possible changes in the EPHA1 protein structure.
Materials and methods.The DNA-samples were extracted from peripheral blood leucocytes of unrela-ted volunteer donors from different regions of Ukraine by standard methods [10].Informed consents were obtained from all individuals participating in our study.The observation group consisted of 300 individuals, including 164 (54.6 %) male and 136 (45.3 %) female individuals.This group may be considered representative for the estimation of DNA polymorphism frequency in autosomal genes [11,12].
An analysis of the G1891A polymorphism was performed by ARMS (amplification refractory mutation system) PCR.Specific oligonucleotide primers were complementary either to the wild type or to the sequence with substitution (Table 1).The PCR amplification was performed in a final volume of 15 ml containing 1Ṕ CR buffer, 1.5 mM MgCl 2 , 200 mM of each dNTP, 0.8 mM of each primer, 0.2 units of Taq-DNA polymerase and 200 ng of the DNA template.The cycling conditions for G1891A variant were as follows: initial denaturation at 94 o C for 5 min, 30 cycles consisting of denaturation at 94 o C for 30 s, annealing at 63 o C for 30 s, extension at 72 o C for 30 s, and a final elongation step at 72 o C for 5 min.
The presence of G1475A EPHA1 polymorphism was examined by PCR-RFLP (restriction fragment length polymorphism) analysis using the specific oligonucleotide primers described in the  Primers were designed using the web-based PRIMER 3.0 program (http://workbench.sdsc.edu).We used the «BLAST» program at http://www.ncbi.nlm.nih.gov/blast to check the primers specificity.Hypothetical RFLP results were tested using NEBcutter V2.0 (http://tools.neb.com/NEBcutter2).We also used the PSIPRED Protein Sequence Analysis Workbench (http://bioinf.cs.ucl.ac.uk/psipred) to predict the protein secondary structure.
Results and discussion.The Sanger sequencing of EPHA1 gene loci with substitutions was performed.Sequencing confirmed the results of the exome analysis of UKR 094 family members and revealed that the father is a heterozygous carrier for the C1891T polymorphism and the mother is a heterozygous carrier for the C1475T polymorphism (Fig. 2).The sample that had previously undergone Sanger sequencing was used as a positive control.RFLP analysis of the G1475A EPHA1 variants.The designed primers successfully amplified the exon 7 fragment (194 bp) of EPHA1 gene.The G to A transition in G1475A variant creates a restriction site for endonuclease KpnI.Thereby three different patterns could be observed for G1475A variant after the restriction digestion: a 194 bp band (for 1475A/A); a 194 bp, a 101 bp and a 93 bp bands (for 1475G/A); a 101 bp and a 93 bp bands (1475G/G) (Fig. 3).
ARMS for G1891A analysis is based on the observation that PCR amplification is inefficient or completely refractory if there is a mismatch between the 3' terminal nucleotide of a PCR primer and a corresponding template.This approach implies two PCR reactions.Amplification of the allele 1891G is accomplished using a complementary primer «wild type» (Table 1).Conversely, only the 1891A allele will be amplified if the 3' residue is complementary to the sequence with substitution (Table 1).Thus, as a result a normal (G1891G) individual generates PCR product only in the «normal» reaction; a heterozygote gives products in both reactions, and a homozygous A1891A individual gives amplification only in the «mutant» reaction.The results of analysis of individuals with different genotypes are presented in Fig. 4.
Based on the RFLP analysis of G1475A variant, individuals were classified into three groups: GG (1475G/ G), GA (1475G/A) and AA (1475A/A).Of the 300 analyzed samples from the present study, we have found 7 heterozygous carriers for the G1475A polymorphism only.Genotypes and allele frequencies of the G1475A  2. The observed genotype distributions showed no deviations from Hardy-Weinberg expectations.
The frequency of allelic variants of EPHA1 gene in our study is in agreement with the published data obtained within the International HapMap Project (http:// hapmap.ncbi.nlm.nih.gov/)(Table 2).
We did not identify G1891A substitution in any of 300 investigated volunteer donors.It was found only in the members of aforesaid Ukrainian family with ID in children.Previous reports have demonstrated a significant association of T3022C (rs11767557) polymorphism within this gene with Alzheimer's disease [14,15].EPHA1 has been reported to be highly expressed in the neural tissues during embryogenesis [3,4].
The first analyzed SNP -G1475A -exchanges don 492 from arginine (CGG) to a glutamine (CAG).The second one -G1891A -leads to an amino acid change at position 631 from glycine (GGA) to arginine (AGA).To understand a biochemical effect of these substitutions we tried to predict possible changes in the EPHA1 protein structure.A comparative analysis of the predicted secondary structure revealed that both amino acid substitutions lead to significant changes in the probability of the b-sheet forming.The change of Arg492 to Glu results in extending the 11 th and 12 th b-sheets in the fibronectin type III repeat of EPHA1 protein by 1 amino acid.The change of Gly631 to Arg results in extending of the 2 nd b-sheet in the tyrosine kinase domain by 1 amino acid.Thereby these substitutions may directly cause changes in protein solubility or binding with ligands or protein-partners.
In the frame of CHERISH FP7 project, the exome NGS was performed for two affected probands from Ukrainian family with moderate ID, which revealed two polymorphisms in the EPHA1 gene: G1475A and novel SNP G1891A.The assays for detection of these SNPs based on ARMS and restriction analysis were developed and the preliminary studies on the distribution of G1475A and G1891A polymorphisms in the population of Ukraine were conducted.Further casecontrol EPHA1 exons sequencing will be carried out to evaluate the involvement of the EPHA1 mutations in ID development.
tion was performed in a final volume of 25 ml containing 1 ´PCR buffer, 1.5 mM MgCl 2 , 200 mM of each dNTP, 1 mM of each primer, 0.2 units of Taq-DNA polymerase and 200 ng of the DNA template.The cycling conditions for G1475A variant were as follows: initial denaturation at 94 o C for 5 min, 30 cycles consisting of denaturation at 94 o C for 40 s, annealing at 55 o C for 40 s, extension at 72 o C for 40 s, and a final elongation step at 72 o C for 3 min.The amplified fragments were digested with KpnI.Digestion was performed in 15 ml reaction volume containing 1 X reaction buffer, 0.5 units of the restriction enzyme and 10 ml of purified PCR product, incubated at 37 o C overnight and analyzed by 6 % standard agarose gel electrophoresis.

Table 1
table.The PCR amplifica-Specific oligonucleotides, designed and synthesized in accordance to corresponding exon sequences of EPHA1 gene, were used as primers