| Background and Objects of the StudyIdiopathic scoliosis (IS) is a three-dimensional spinal deformity, which manifests as the coronary scoliosis accompanied with vertebral rotation The etiology remains unclear. Previous studies focused on histological factors such as cartilage, skeleton, joint, nerve, and muscle, but remain inconclusive In recent years, considerable studies have indicated that genetic factors played an important role in the pathogenesis of IS. These studies also showed a high genetic heterogeneity of either autosomal dominant inheritance with a major gene or multifactorial modes of inheritance. Autosomal dominant inheritance X-linked dominant inheritance, and multifactorial inheritance are commonly reported. In this study, we investigated214IS families recuited in Southwest Hospital (Chongqing, China) by using the genetic epidemiological method and analyzed effects of genetic factors and the mode of inheritance through patients'age, incidence, familial aggregation, and heritability, as well as traits of hereditary diseases.IS related susceptible genes currently being researched includ melatonin receptor1A gene, melatonin receptor1B gene, Matrilin-1(MATN1) gene, y-1-syntrophin (SNTG1) gene, COL1A1, COL1A2, COL2A1, elastic fiber, and aggrecan gene. Studies showed that these genes are related with IS but can not explain all the IS patients. Chromosomal regions involved in susceptibility to IS includ6p,10q,18q,6p,6q,17p11.2,8q,9q,16q,19p13,19p13.3,15q25-26,9q31.2,9q34.2,17q25.3-qtel,12p, and Xq23-26. There are considerable genes in these foci. However, the specific susceptible genes for IS still need to be identified by deepened investigation. Two scholars have reported pathogenic foci on chromosome19p13. Furthermore, Chan et al defined the critical region of IS in the vicinity of D19S216, flanked by D19S894and D19S1034. By April2009,213genes had been identified in19p13.3in HUGO (Human Genome Organization). We screened SH3GL1, GADD45B, and FGF22in the vicinity of D19S216on chromosome19p13.3as IS candidate genes, and conducted a study using the case-sibling and case-relatives control designs on the basis of families. In this study, primer pairs were designed according to SH3GL1, GADD45B, and FGF22exons for PCR amplification, cloning, and sequencing. Sequence alignment among siblings and relatives was conducted and the protein structure was predicted.Research Method1.Genetic epidemiologic survey:Families of probands were examined by trained investigators using an independently designed IS genetic epidemiologic questionnaire, and the family tree was mapped. Investigation methods included the face-to-face inquiry in clinic and wards and/or telephone inquiry. Before entering in this study, subjects were informed about the study's objective and provided with the informed consents. Investigated subjects were IS probands and parents. The detailed subjects included the first-degree relatives (probands, their parents, children and siblings), the second-degree relatives (grandparents, uncles, and aunts), and the third-degree relatives (cousins). The patients'general situation (including the health history) and scoliosis history were reviewed. Suspected diseased individuals and susceptible individuals were confirmed by inspection, Adam's Bend Forward Test, and radiographs. IS diagnostic criteria:Diseased individuals were identified based on a Cobb angle of more than10°in the standing anteroposterior radiographs and susceptible individuals were identified in case of a Cobb angle of less than10. Children less than14years old with a normal phenotype were regarded as non-diseased relatives. Statistical analysis:Familial aggregation was analyzed using the sample and population rate-comparing U test; AIS heritability was estimated with the Falconer regression method;2. Analytic method of sequence alignment of exons of candidate genes SH3GL1, GADD45B, and FGF22:Six pairs of primers were designed using sequences of10exons of SH3GL1as the template. Two pairs of primers were designed and synthesized using sequences of four exons of GADD45B as the template. One pairs of primers were designed and synthesized using sequences of three exons of FGF22as the template. Each pair of primers was independently amplified with PTC-100PCR. PCR products were extracted and purified. PCR products were extracted and purified. Sequence alignment of siblings and relatives were analyzed using Chromas software and DNA sequence analysis software Vector NTI Advance10.3. Research Results1. Epidemiological Investigation ResultsInvestigated IS patients (including probands) had an onset age of3to20years,78.91%between10and14years old with a mean age of11.8years. The ratio of males to females was1:2.59. The overall incidence of IS in the investigated families was11.82%. IS incidence was10.01%for first-degree relatives,2.55%for second-degree relatives,1.76%for third-degree relatives, and the overall incidence of IS in the relatives was109/2518×100%=4.33%. The heritability was77.68±10.39%for the first-degree relatives (h12),69.89±3.14%for the second-degree relatives (h22), and62.14±11.92%for the third-degree relatives (h32). The weighted mean h12h22h32of the first-, second-, and third-degree relatives h2was49.17±2.92%2. Sequence AlignmentCloning sequence alignment analysis of10exons of SH3GL1of56IS patients showed gene mutation sites in all patients and a total of12mutant alleles in the2nd,4th,5th,6th,8th, and10th exons, of which10mutations were located in the coding region and two (both in the10th ex on) in the noncoding region Cloning sequence alignment analysis of the four exons of GADD45B of56IS patients indicated gene mutation sites in all the56patients and a total of three mutant alleles in the1st,3rd, and4th exons. Cloning sequence alignment analysis of the three exons of FGF22of56IS patients showed gene mutation sites in all the56patients and a total of two mutant alleles in the1st and3rd exons3. Prediction analysis of protein sequenceThe515th alleles of SH3GL1was CC in93relatives, TT in five of the56probands (8.93%)(proband and the mother with IS, proband and the father with IS, and another male proband)(Figure2) and CC in the remaining51probands (91.07%). If the proband's gene base is T, the stop codon (TAG) is formed and the protein reading frame also changes and encodes truncated proteinsConclusions:1. AIS was a genetic disease of multifactorial inheritance:Investigated AIS patients had an onset age of3to20years,78.91%between10and14years old with a mean age of11.8years., suggesting that females are more susceptible to this disease than males. A significant difference in incidence was noted between the total investigated families and the control population, promoting the familial aggregation of AIS. The incidence of AIS in the first-and second-degree relatives and all relatives was significantly higher than that of the control population (1.04%). U test results showed that the incidence of AIS in the first-and second-degree relatives and overall AIS incidence were significantly higher than that of the general population, illustrating the familial aggregation of AIS. In our study, heritability IS occurrences in the first-, second-, and third-degree relatives was77.68±10.39%,69.89±3.14%, and62.14±11.92%, respectively. Furthermore, all incidences of IS above60%suggest that genetic factors playe an important role in IS pathogenesis.2. Gene SH3GL1was one of the virulence genes of AIS.11mutations in SH3GL1,3mutations in GADD45B, and2mutations in FGF22did not induce ORF translocation or amino acid changes and therefore are nonsense mutations. The515th alleles of SH3GL1was CC in93relatives, TT in five of the56probands (8.93%)(proband and the mother with IS, proband and the father with IS, and another male probands)(Figure3), and CC in the remaining51probands (91.07%). If the proband's gene base is T, the TAG is formed and the protein reading frame also changes and encodes truncated proteins affecting the structure of proteins. This protein may be the diseased protein of IS, demonstrating that SH3GL1is possibly one of the main gene candidates..
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