Clinical Features And Pathogenesis Of Congenital Cervical Fusion Deformities

Part 1 Clinical Manifestations and Genetic Analysis of Klippel-Feil Syndrome[Background]Klippel-Feil syndrome(KFS)represents a rare anomaly characterized by congenital synostosis of the cervical vertebrae because of a segmentation or formation defect.KFS patients are asymptomatic at the early stage and are easily missed diagnosed because of associated abnormalities or complications,which has brought great challenges to the early diagnosis and treatment of KFS.As the progression of KFS,congenital fusion deformity of the cervical vertebrae often alters the kinematics of the cervical spine in ways that may accelerate degenerative changes throughout the region.Therefore,patients with KFS usually complain of neck pain and may be at risk for cervical spondylosis due to a lack of early prediction methods.Furthermore,it is often associated with a variety of congenital diseases and other systemic malformations,resuling in serious physical and mental problems for patients and bringing a significant burden on families and society.At present,mutations in GDF6,MEOX1,GDF3,MYO18B and RIPPLY2 have been associated with KFS.Nevertheless,the basis of genetic predisposition to KFS is largely unknown due to the rarity of the disease;only a small number of KFS cases can be explained by the five specific pathogenic genes mentioned above.To further decipher the molecular basis of KFS at the exome level and provide the basis for early diagnosis and targeted therapy,we herein investigate the molecular findings of WES among a cohort of KFS patients/families,and we further analyze rare variants by using genetic burden analysis.[Objectives]The purpose of this study is to assess clinical features and radiologic parameters of KFS patients in Han Chinese ethnicity,and to present whole-exome sequencing(WES)findings,so as to provide valuable information on the diagnosis and prognosis of KFS.[Methods]We consecutively recruited 37 patients of Han Chinese ethnicity at Peking Union Medical College Hospital(PUMCH)who had been diagnosed with KFS.Demographic information,clinical symptoms on presentation,physical examination results and radiological assessments were obtained.Radiographic parameters of interest,such as the segmentation of congenitally fused vertebrae,the Samartzis classification of KFS(Type Ⅰ,single congenitally fused cervical segment;Type Ⅱ,multiple noncontiguous congenitally fused cervical segments;Type Ⅲ,multiple contiguous congenitally fused cervical segments)were also recorded.WES was performed after DNA extraction from peripheral blood for 37 KFS patients.WES data processing was performed with the in-house developed PUMP(Peking Union Medical college hospital Pipeline).(1)Additionally,all variants were compared to public databases.(2)Computational prediction tools were used to predict the conservation and pathogenicity of candidate variants.(3)A criterion was used to filter for rare genuine variants with a minor allele frequency(MAF)<0.001.(4)After filtering,we performed an initial analysis focusing on variants in reported pathogenic genes related to KFS.Additionally,96 genes related to vertebral segmentation defects were added to the candidate gene list.To extend the spectrum of known mutations contributing to KFS,we searched for exome-wide rare variants in 96 candidate genes related to vertebral segmentation defects using genetic burden analysis,compared with the in-house control database consisted of WES data from 534 unrelated Chinese individuals at PUMCH with no apparent skeletal anomalies.[Results]The study cohort consisted of 37 unrelated KFS patients.There were 20 males and 17 females,with a mean age at diagnosis of 12.6 ± 5.3 years.Among the 37 patients with KFS,there was a mean of 2.3 congenital fusion levels.The most commonly fused segments were C6-C7,C2-C3,and C3-C4.KFS has been characterized as a clinical triad of short neck,low posterior hairline,and limited cervical range of motion(ROM).In our cohort,limited cervical ROM was the most common clinical feature(17 patients,45.9%),followed by short neck(10 patients,27.0%)and low posterior hairline(six patients,16.2%).However,43.2%of patients did not show any typical manifestation of the clinical triad;furthermore,only 10.8%of patients showed all three signs in the clinical triad at the same time.According to the KFS classification criteria by Samartzis,the proportion of patients suffered from short neck and limited cervical range of motion in KFS type Ⅲwas significantly higher than that in KFS type Ⅱ and type Ⅰ.Moreover,the KFS patients presented with varied intraspinal and extraskeletal anomalies.There were 9 patients(24.3%)manifesting intraspinal deformities(syringomyelia,diastematomyelia,and tethered cord)and 12 patients(32.4%)presenting with extraskeletal manifestations.After WES data processing and variant interpretation,we primarily examined rare variants in five reported genes(GDF6,MEOX1,GDF3,MYO18B and RIPPLY2)associated with KFS and detected three variants of uncertain significance in MYO18B.Based on rare variant burden analysis of 96 candidate genes related to vertebral segmentation defects,we identified BAZ1B(P=0.00000002)as having the highest probability of association with KFS,followed by FREM2(P=0.0003),SUFU(P=0.0004),VANGL1(P=0.0072)and KMT2D(P=0.0214).In addition,seven patients were proposed to show potential oligogenic inheritance involving more than one candidate gene,the frequency of which was significantly higher than that in the in-house controls(P=0.00148).[Conclusion]Our study presents clinical features and WES findings from a large cohort of KFS patients to date.Studies have shown that patients with multiple contiguous cervical fusions are more likely to manifest short neck,limited cervical ROM,and clinical triad.Therefore,attention and closely follow-up should be paid for these patients.Beyond identifying known candidate genes,our analysis highlights five novel rare variants associated with cervical congenital fusion among KFS patients through genetic burden analysis,extending the spectrum of known mutations contributing to this syndrome.These results indicate a highly significant enrichment of predicted damaging genes and the potential oligogenic inheritance of KFS.Part 2 Proteomic Analysis of Klippel-Feil Syndrome[Background]Klippel-Feil syndrome(KFS)is a relatively rare and complicated condition that is characterized as congenital fusion of two or more cervical vertebrae.Although significant progress has been made in understanding the process of cervical vertebra fusion,there is a wide gap in understanding of the detailed mechanisms of KFS because of its rarity,heterogeneity,small pedigrees,and the broad spectrum of anomalies.In the first part of our study,we recruited 37 KFS cohort and identified novel genetic mutations at the exon level that might be associated with KFS.It is well acknowledged that the direct performers of biological functions are proteins.The results of the genome and transcriptome could influence a part of changes in genetic expression products;however,the study of the proteomics would truly reflect the pathophysiological state of the human body.Thus,it is essential to explore early specific biomarkers and design measures to prevent potentially fatal outcomes in KFS patients.Our previous comparative proteomics study by Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)explored the differential serum protein abundance levels of nine patients with TBX6 associated congenital scoliosis(CS)and nine healthy controls.It was conducted that lipid metabolism might play important roles in the pathogenesis of CS.Although both of CS and KFS present with congenital vertebral malformation,whether there are potential pathogenic mechanisms between CS and KFS is largely unknown.Therefore,proteomics by Data-independent acquisition(DIA)coupled with Q-Exactive mass spectrometry was used to compare the serum protein profiles of patients with KFS and healthy controls as well as patients with KFS and patients with CS(patients with known syndromes and cervical deformity were excluded),in order to explore the correlated pathogenesis between KFS and CS and provide possible reasons for the deformity located at cervical segments.[Objectives]The purpose of this study is to depict the differential protein expression profiles of KFS in serum and to identify potential biomarkers,so as to further investigate the pathogenesis of KFS.[Methods]We recruited eight patients of Chinese Han ethnicity with KFS,five patients with CS who had no known syndrome or cervical deformity,and seven healthy controls.All of five patients in the CS group manifested with congenital fusion of the thoracic or lumbar spine.Proteomic analysis by DIA was performed and the cutoff values were set as 1.42-fold for increased and 0.70-fold for decreased abundance and the differential changes were considered significant at P<0.05.The three main Gene Ontology(GO)categories were analyzed with Blast2GO and pathway analysis was performed using the Ingenuity Pathway Analysis(IPA)database.[Results]We performed proteomic profiling of eight patients with KFS and seven healthy controls.A total of 493 proteins were identified and 49 of them were differentially abundant in the patients with KFS compared with the controls.Among the differentially abundant proteins(DAPs),27 were significantly decreased and 22 were significantly increased.Furthermore,a total of 462 proteins were identified and 192 DAPs were differentially abundant in patients with KFS compared with patients with CS.Among them,118 decreased and 74 increased in abundance.Fifteen DAPs that were common in both comparisons were considered as candidate markers for KFS,including glyceraldehyde-3-phosphate dehydrogenase,galectin-3-binding protein,peroxiredoxin-1,CD 109 antigen,noelin,cadherin-5,membrane primary amine oxidase and eight immunoglobulins.A cluster analysis of the abundance levels of the 15 DAPs in patients with KFS,controls,and patients with CS showed that the abundance levels of eight immunoglobulins decreased significantly in patients with KFS or CS compared with the controls.Moreover,the decline in the abundance levels of immunoglobulins in patients with CS was more significantly than that in patients with KFS.We indicated that immunoglobulins may be valuable biomarkers for KFS,and the dysfunction of the immune system may be the underlying pathogenic mechanism of somitogenesis defects in the cervical spine.One of the 15 common DAPs,membrane primary amine oxidase,showed a more than 1.6-fold increase in patients with KFS compared with the controls.We proposed that membrane primary amine oxidase could be a candidate biomarker related to amino acid metabolism in the pathogenesis of KFS.Furthermore,the distribution of the DAPs in the canonical pathways was calculated using the IPA software.The IPA illustrated that the LXR/RXR and FXR/RXR activation pathways were common significant canonical pathways in the patients with KFS vs controls and patients with KFS vs patients with CS comparisons.All seven of the differentially abundant apolipoproteins(APOC3,APOA4,APOF,APOA2,APOE,APOH,and APOD)were involved these pathways.The differentially abundant apolipoproteins and LXR/RXR and FXR/RXR activation pathways related to lipid metabolism led us to strongly speculate that lipid metabolism was involved in the underlying pathogenesis of KFS.[Conclusion]This study provides the first proteomic profile for understanding the pathogenesis and identifying predictive biomarkers of KFS.We detected 15 DAPs that were common in both comparisons as candidate predictive biomarkers of KFS.The lipid metabolism-related canonical pathways of LXR/RXR and FXR/RXR activation together with seven differentially abundant apolipoproteins may play significant roles in the etiology of KFS and provide possible pathogenesis correlation between KFS and CS.We predict that the differentially abundant apolipoproteins detected in patients with KFS and patients with CS(APOA2,APOD,APOE,and APOH)could be significant biomarkers to investigate the possible mechanism associated with the vertebral segmental abnormalities located at cervical segments.