Study On The Mutation Spectrum And Pathogenic Mechanism Of ALPK3 Gene In Congenital Scoliosi

Background:Congenital scoliosis（CS）is a three-dimensional spinal malformation characterized by failure of vertebral formation or/and segmentation resulting from abnormal somitogenesis during embryonic development,which is caused by multiple reasons.The disease can be early-onset and progressive,leading to short stature,razor back and thoracic deformity.In severe cases,it may affect the patient’s cardiopulmonary function or daily physical activities and require surgical intervention.To date,the etiology of CS remains unknown,exploring the etiology of CS is of significant importance for its early diagnosis and precise interventions as well as predicting the progress of the disease in clinical practice,and it has been a research hot spot for physicians around the world in recent years.ALPK3 encodes alpha-kinase 3 from a family of atypical protein kinase.Alpk3（also known as Midori）is the murine ortholog of ALPK3,whose expression is restricted to the fetal and adult heart and adult skeletal muscle in mice.Patients with biallelic damaging homozygous or compound heterozygous variants may present with early-onset cardiomyopathy of dilated cardiomyopathy（DCM）and/or hypertrophic cardiomyopathy（HCM）in various severity and often accompanied with extracardiac manifestations like short stature,facial dysmorphisms,cleft palate,Webbed neck,joint contractures and progressive scoliosis/kyphoscoliosis.Since symptoms including craniofacial malformation,growth retardation,joint contracture and cardiovascular diseases are rather common comorbidities in CS patients,ALPK3 pathogenic variants may play an important role in CS’s development.There are no studies on the pathogenicity assessment of ALPK3 variants in CS cohort.Objects:We intend to screen out the pathogenic mutations of ALPK3 in CS patients through large scale Whole Exome Sequencing（WES）information of patients in CS cohort,and further systematically investigate its molecular biological function and possible mechanism in the pathogenesis of CS,so as to further expand the pathogenic gene spectrum of CS and provide new directions and insights for the early diagnosis and precise treatment of CS.Methods:Part 1:According to inclusion and exclusion criteria,we performed WES and data analysis based on 420 CS patients from DISCO（Deciphering Disorders Involving Scoliosis and Comorbidities）cohort after peripheral DNA extraction.Pathogenic/Likely pathogenic ALPK3 variants were identified through bioinformatics tools for deleteriousness prediction.Part 2:Alpk3 mutant mice were generated by CRISPR/Cas9 system and the pathological changes in mice’s spine were observed.We performed proteomic analysis of the paravertebral muscle from Alpk3-/-and wildtype mice.Part 3:We further investigated possible biological functions of differentially expressed proteins through functional enrichment analysis of GO（gene ontology）and KEGG（Kyoto Encyclopedia of genes and genes）.Results:Part 1:Among the 420 patients with CS,we screened out 2 patients with recessive compound heterozygous mutation of ALPK3（c.1148G>A/c.5338₅339delTG and c.4840C>T/c.3061C>T）through WES and bioinformatic analysis.The above mutations are considered to be likely pathogenic（LP）to CS and have not been reported elsewhere.Patients with ALPK3 deleterious mutations showed similar clinical phenotypes of cardiomyopathy complicated with craniofacial-skeletal malformations as cases described in previous published studies,such phenotypes include DCM or HCM to varying degrees,progressive cervical and thoracic/thoracolumbar spinal malformations,facial dysmorphic features like downslanting palpebral fissures,small oral orifices low-set ears,as well as Webbed neck,and joint contractures.Part 2:Alpk3 mutant mice were generated by CRISPR/Cas9 system.Micro-CT,skeleton stain revealed that a total of 45.5%（25/55）homozygous（Alpk3-/-）mice of different age showed abnormality in spinal structure including fail of formation or segmentation of vertebrae or progressive fusion in C1 and C2.The incidence of spinal deformity in Alpk3-/-group was much higher than that of in heterozygous（Alpk3+/-）and wild type group,with more severe malformation.In addition,the morbidity of Alpk3-/mice at different age varied.No obvious spinal abnormalities were found in neonatal group,while the incidence of spinal deformity increased during adolescence and sexually matured period（3-12 weeks postnatal）and remain steady after reaching adulthood.Part 3:Proteomic analysis of paravertebral muscles in Alpk3-/-mice and wild type mice were performed.A total of 79 differentially expressed proteins（16 upregulated and 63 down regulated）were identified.GO analysis showed that differentially expressed proteins were mainly involved in regulating biological processes like cell centrosome replication and aggregation,regulating molecular functions including transcription coactivator activity,transmembrane signal receptor activity and spermidine oxide reductase activity,and were mainly located at Golgi apparatus,excitatory cell synapse and other cellular components.KEGG enrichment analysis showed that pathways of splicing,glutathione metabolism and PPAR signaling are the most canonical pathways for differentially expressed proteins,STRING database showed that ALPK3 is connected to OBSL1,which was also a differential expressed protein between the two groups.Conclusion:ALPK3 may be an potential pathogenic candidate gene of CS.The mutation spectrum has been expanded by the recessive compound heterozygous mutation of ALPK3.Deleterious biallelic mutations of ALPK3 may lead to a series of CS characterized by cardiomyopathy combined,craniofacial-skeletal deformity,and progressive scoliosis/kyphoscoliosis.Alpk3 may play an important role in paravertebral muscle in murine,the loss of function of which my cause dysfunction in mechanical strength,differentiation,regeneration,lipid metabolism and other physiological processes of paravertebral muscle,contributing to the spinal deformities in Alpk3-/-mice postnatally.Alpk3 and obsll may have interaction or similar pathogenic mechanism in disease with skeletal deformities.Alpk3 mutant mouse model can well simulate the clinical manifestations of CS patients related to ALPK3 and serve as a powerful tool for further study of gene function and its pathogenesis.