Association And Mutation Of FOXP2 Gene With Functional Articulation Disorders

IntroductionFunctional articulation disorder (FAD) is the most common speech disorder of children. It is also known as developmental articulation disorder or developmental phonological disorder. The estimated prevalence is 15.6% in children at age 3 and 3.8% at age 6. It is defined by developmentally inappropriate errors in speech production that reduce intelligibility. More than half of these children encounter later academic difficulties in language, reading, and spelling. The residual effects of preschool FAD may be life long.Although the etiology of FAD is unknown, there is a lot of literature suggesting that susceptibility to FAD is genetic, including familial aggregation studies and some twin studies.Forkhead-box P2 (FOXP2) gene (MIM 605317), located on 7q31, encodes a transcription factor containing a polyglutamine tract, a zinc finger, a leucine zipper motif, and a forkhead-box DNA binding domain. The G/A mutation in exon 14 of FOXP2 was found to cause a severe developmental disorder of verbal communication, involving profound articulation deficits, accompanied by linguistic and grammatical impairments in a large family known as the KE family. Lai CS et al have determined the detailed spatial and temporal expression pattern of FOXP2 mRNA in the developing brain of mouse and human. They found expression in several structures including the cortical plate, basal ganglia, thalamus, inferior olives and cerebellum. These data support a role for FOXP2 in the development of corticostriatal and olivocerebellar circuits involved in motor control. And it is likely to regulate gene expression in defined areas of developing lung, cardiovascular, intestinal, and neural tissue. The available expression data are compatible with a conserved role (or roles) for this gene in regulating development of neural circuitry underlying motor control and sensory-motor integration in mammals and birds. Structural and functional neuroimaging with gene expression studies supported a significant relationship between FOXP2 and the language neural system. All these studies suggest that the FOXP2 gene is involved in the development of the neural system that mediates the specific motor coordination necessary for speech. Moreover, Zeesman et al found a child with developmental apraxia of speech and mild cognitive delay who had a deletion of 7q31 that included FOXP2 gene. These findings indicate that FOXP2 is a candidate gene for speech and language disorder.In the present study, we attempted to investigate the association between the FOXP2 gene and FAD using the analyses of association and haplotype.Materials and methods1,SubjectsA total of 150 patients and 140 healthy unrelated controls of similar ethnic background were recruited from Shenyang, Shengjing Hospital, China Medical University. Of the 150 cases, were 109 male and 41 female. The mean age of the children at the time of testing was 6.45±1.98 years. All the patients fulfilled the criteria for functional articulation disorder. Physical examinations were performed and patients were excluded if they had any medical or genetic conditions which could be contributing.2,Methods(1)Genotyping of SNPsGenomic DNA was obtained from peripheral blood leukocytes using standard phenol-chloroform method. Five single nucleotide polymorphisms (SNPs) rs923875, rs2396722, rs1852469, rs17137124 and rs1456031 in the FOXP2 gene were selected from the dbSNP database. PCR primer pairs were based on references or designed by Primer Premier 5.0. PCR was performed and the products were digested with restriction enzymes Apall, VspⅠ, Trull, Afl II and RsaⅠrespectively. The samples were loaded on 2% or 2.5% agarose gels containing ethidium brodmide for electrophoresis at 100 V for 50 min. Gel were read blindly by two independent raters with discrepancies resolved by re-genotyping.6 random select samples for each SNP were tested again by direct DNA sequencing. PCR products were purified using a QIAQuick PCR purification kit (Qiagen, Germany). Direct sequencing of the samples was performed on an ABI 3730 DNA sequencer (Perkin Elmer, Foster city, California, USA). Sequencing results were compared with the reference human FOXP2 sequence and the results of RFLP.(2)Mutation analysisFor the moderate to severe patients, the G/A mutation of exon 14 was screened. PCR primer pairs were based on reference. PCR was performed and the products were digested with restriction enzyme Tail. The samples were loaded on 2% agarose gels containing ethidium brodmide for electrophoresis at 100 V for 50 min. Direct DNA sequencing was used in 10 samples to test the results of PCR-RFLP and other mutations.(3)Statistical analysisSignificance level was previously established at 0.05. The Hardy-Weinberg equilibrium for genotype frequencies was evaluated by the Chi-square test. The comparisons of allelic frequencies and genotype analyses between patients and controls were performed using SHEsis program online. The pairwise linkage disequilibrium (LD) and haplotype analysis were also estimated by the SHEsis program. The Bonferroni test was applied to correct for multiple comparisons.ResultsThe allele and genotype frequencies of all five SNPs of FOXP2 gene were analyzed. The genotype and allele frequencies are rs923875:AA 0.11, AC 0.51, CC 0.38, A 0.368, C 0.632; rs2396722:CC 0.29, CT 0.52, TT 0.19; C 0.551, T 0.449; rs1852469:AA 0.17, AT 0.58, TT 0.25, A 0.464, T 0.536; rs17137124:CC 0.40, CT 0.44, TT 0.16, C 0.621, T 0.379; rs1456031:CC 0.28, CT 0.53, TT 0.19, C 0.547, T 0.453, respectively. All of them were found to be in Hardy-Weinberg equilibrium in both patient and control samples. The significant differences of genotype and allele frequency distributions were detected between different populations.Statistical analyses of the SNPs showed that neither genotype nor allele frequency distributions were different between FAD patients and control subjects with the exception of the SNP rs1852469. In this case, there were significant differences in the genotype (P=0.001) and allele (P=0.002528) frequencies. These P values remained significant after Bonferroni correction (P=0.005; P=0.0126 respectively).To test for LD between the FOXP2 SNPs, D'values were calculated for all pairs of SNPs on patients and controls. We considered the SNPs with D'>0.5 to perform the haplotype analysis. Significant difference in the haplotype frequency was observed between patients and controls for the haplotype rs923875A/+rs2396722T/+ rs1852469T(P=0.0103).After the division in accordance with the degree of severity, neither genotype nor allele frequency distributions were different between mild FAD patients and control subjects; There were significant differences in the genotype (P=0.0021) and allele (P=0.0036) frequencies of SNP rs1852469 between moderate to severe FAD and controls. These P values remained significant after Bonferroni correction (P=0.0105; P=0.018 respectively).We considered 3 SNPs with D'>0.5 to perform the haplotype analysis. A risk haplotype was detected, rs923875A/+ rs2396722T/+rs1852469T (P=0.023) which was significantly associated to moderate to severe FAD.Alternatively, a protective haplotype rs923875C/+ rs2396722C/+rs1852469A (P=0.0145) was also identified. But the G/A change found in the KE family were not detected in any of the moderate to severe patients.Conclusion1. There are polymorphisms of SNPs rs923875, rs2396722,rs1852469,rs17137124 and rs1456031 among Liaoning population. They can be used as genetic markers for association or linkage analysis.2. The distributions of the five SNPs exhibit ethnic heterogeneity.3. There is an association between SNP rs1852469 and FAD. The individuals with haplotype rs923875A/+rs2396722T/+rsl852469T are more susceptible to FAD. The individuals with haplotype rs923875C/+rs2396722C/+rs1852469A are in relatively lower risk.4. The G/A mutation are not the direct factor of functional articulation disorder.