The Etiology Of RIPPLY1/2 Gene And Congenital Scoliosis

BackgroundCongenital scoliosis(CS)is a congenital spinal deformity caused by vertebral dysplasia or segmentation defect during the embryonic stage.Clinieally,a disease with a vertebral structural abnormality and farther causing a spinal Cobb angle greater than 10 degrees is defined as CS.Vertebral malformations in patients with CS often lead to rapid progression of scoliosis and can be combined with other problems such as impaired lung function due to thoracic developmental limitations.In addition to affecting physical health,the presence of deformity is also a major threat to the mental health of patients,.and subsequent examination and treatnent also increases the economic burden of the family and society.In 2015,Wu et al.found that the 16p11.2 copy number variation(CNV)combined with the sub-effect allele of the TBX6 gene caused a hemivertebra deformity,explaining nearly 11%cause of CS,which suggesting that there should be a better way to explore the genetics of CS from single nucleotide variants and copy number variations.There is a negative feedback regulation mechanism between RIPPLY1/2 gene and TBX6 gene in somite formation.Previous animal models and case reports have also suggested that RIPPLY1/2 gene mutations are associated with vertebral malformations.However,the etiology of RIPPLY1/2 and CS is still unclear.Objects and MethodsThe single nucleotide variants and copy number variations of RIPPLY1/2 gene in CS cohort were searched by whole genome sequencing technology to explore the relationship between RIPPLY1/2 gene and CS.First,337 CS patients of Peking Union Medical College Hospital who met the admission criteria were collected,and phenotypic collection and analysis were completed.Whole blood samples from all patients were collected.Then,DNA was extracted,and whole genome sequencing was performed.Rare variants of the RIPPLY1/2 gene were obtained after annotation,and pathogenicity was further analyzed.The CNV mutation of RIPPLY2 gene was searched,verified by IGV,qPCR,digital PCR technology,and pathogenicity was analyzed.Results1)A total of 3 3 7 patients with congenital scoliosis were enrolled in this study.81 patients with CS type I had an average age of onset of 3.33±0.47 years;47 patients with typeⅡ had an average age of onset of 6.60±0.74 years;type Ⅲ,209 cases,average age 4.96 ±0.34 years old.In terms of vertebral malformation,155 cases(46.0%)had hemivertebra deformity,64 cases(19.0%)showed butterfly vertebral deformity,72 cases(21.4%)had wedge-shaped vertebrae,and 256 cases(76.0%)had segmentation defect.2)In terms of SNV,three possible potential pathogenic mutation sites were found in the RIPPLY1/2 gene:RIPPLY1 c.156-1G>C,RIPPLY1 c.155+1G>T,RIPPLY2 c.325A>T(p.Ilel09Phe).3)In terms of CNV,we found 7 patients with CNV carrying 8 RIPPLY2 genes in the cohort.Among them,RIPPLY2 CNV carried by three patients was highly coincident with each other,chr6:84565801-84567900,chr6:84566001-84568200,and chr6;84566301-84568800.Conclusion1)In the CS cohort,intraspinai malformation is the most comnon systemic malformation,followed by cardiac malformation.The incidence of intraspinal malformation of CS type Ⅱ vas significantly higher than that of the rest,suggesting that segmental failure may be associated with intraspinal malformation.2)RIPPLY1 gene splicing site mutation c.156-1G>C and c.155+1G>T suggests that transcription is affected,and the missense mutation of RIPPLY2 gene c.325A>T(p.Ilel09Phe)is predicted to be highly pathogenic.3)Recurrent R1PPLY2 gene CNVs suggest that the CNV may be closely related to CS,but further research is needed.