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Wu N., Ming X., Xiao J., et al. TBX6 null variants and a common hypomorphic allele in congenital scoliosis. N Engl J Med, 2015. 372: 341-50.

Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis. We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multicenter series of 42 persons with 16p11.2 deletions. We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10−6). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10−6). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis. Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed.

Wang K, Zhao S, Liu B, et al. Perturbations of BMP/TGF-beta and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM). Journal of medical genetics 2018;55:675-84.

Brain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10–18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM. We performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model. We identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, ENG, and three damaging variants in novel candidate genes: PITPNM3, SARS and LEMD3, which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (TIMP3, SCUBE2, MAP4K4, CDH2, IL17RD, PREX2, ZFYVE16 and EGFR) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance (MAP4K4 with ENG, RASA1 with TIMP3 and SCUBE2 with ENG) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-β) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM. Our study highlights the specific role of BMP/TGF-β and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.

Liu J, Zhou Y, Liu S, et al. The coexistence of copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) at a locus can result in distorted calculations of the significance in associating SNPs to disease. Human genetics 2018;137:553-67.

With the recent advance in genome-wide association studies (GWAS), disease-associated single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) have been extensively reported. Accordingly, the issue of incorrect identification of recombination events that can induce the distortion of multi-allelic or hemizygous variants has received more attention. However, the potential distorted calculation bias or significance of a detected association in a GWAS due to the coexistence of CNVs and SNPs in the same genomic region may remain under-recognized. Here we performed the association study within a congenital scoliosis (CS) cohort whose genetic etiology was recently elucidated as a compound inheritance model, including mostly one rare variant deletion CNV null allele and one common variant non-coding hypomorphic haplotype of the TBX6 gene. We demonstrated that the existence of a deletion in TBX6 led to an overestimation of the contribution of the SNPs on the hypomorphic allele. Furthermore, we generalized a model to explain the calculation bias, or distorted significance calculation for an association study, that can be ‘induced’ by CNVs at a locus. Meanwhile, overlapping between the disease-associated SNPs from published GWAS and common CNVs (overlap 10%) and pathogenic/likely pathogenic CNVs (overlap 99.69%) was significantly higher than the random distribution (p<1x10-6 and p=0.034, respectively), indicating that such co-existence of CNV and SNV alleles might generally influence data interpretation and potential outcomes of a GWAS. We also verified and assessed the influence of colocalizing CNVs to the detection sensitivity of disease-associated SNP variant alleles in another adolescent idiopathic scoliosis (AIS) genome-wide association study. We proposed that detecting co-existent CNVs when evaluating the association signals between SNPs and disease traits could improve genetic model analyses and better integrate GWAS with robust Mendelian principles

Yang N, Wu N, Zhang L, et al. TBX6 compound inheritance leads to congenital vertebral malformations in humans and mice. Human molecular genetics 2018.

Congenital vertebral malformations (CVM) are associated with human TBX6 compound inheritance which combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. < 50%) as a potential mechanism of 6 TBX6-associated CVM. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found ten (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVM, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.

Zhu Q.#, Wu N.#, Liu G., et al. Comparative analysis of serum proteome in congenital scoliosis patients with TBX6 haploinsufficiency - a first report pointing to lipid metabolism. J Cell Mol Med, 2018. 22(1): p.533-545.

Congenital scoliosis (CS) is a three-dimensional deformity of the spine affecting quality of life. We have demonstrated TBX6 haploinsufficiency is the most important contributor to CS. However, the pathophysiology at the protein level remains unclear. Therefore, this study was to explore the differential proteome in serum of CS patients with TBX6 haploinsufficiency. Sera from nine CS patients with TBX6 haploinsufficiency and nine age- and gender-matched healthy controls were collected and analysed by isobaric tagged relative and absolute quantification (iTRAQ) labelling coupled with mass spectrometry (MS). In total, 277 proteins were detected and 20 proteins were designated as differentially expressed proteins, which were submitted to subsequent bioinformatics analysis. Gene Ontology classification analysis showed the biological process was primarily related to ‘cellular process’, molecular function ‘structural molecule activity’ and cellular component ‘extracellular region’. IPA analysis revealed 'LXR/RXRactivation’ was the top pathway, which is a crucial pathway in lipid metabolism. Hierarchical clustering analysis generated two clusters. In summary, this study is the first proteomic research to delineate the total and differential serum proteins in TBX6 haploinsufficiency caused CS. The proteins discovered in this experiment may serve as potential biomarkers for CS, and lipid metabolism might play important roles in the pathogenesis of CS.

Liu S.#, Wu N.#, Zuo Y., et al. Genetic Polymorphism of LBX1 Is Associated With Adolescent Idiopathic Scoliosis in Northern Chinese Han Population. Spine (Phila Pa 1976), 2017. 42(15): p.1125-1129.

Congenital scoliosis (CS) is a three-dimensional deformity of the spine affecting quality of life. We have demonstrated TBX6 haploinsufficiency is the most important contributor to CS. However, the pathophysiology at the protein level remains unclear. Therefore, this study was to explore the differential proteome in serum of CS patients with TBX6 haploinsufficiency. Sera from nine CS patients with TBX6 haploinsufficiency and nine age- and gender-matched healthy controls were collected and analysed by isobaric tagged relative and absolute quantification (iTRAQ) labelling coupled with mass spectrometry (MS). In total, 277 proteins were detected and 20 proteins were designated as differentially expressed proteins, which were submitted to subsequent bioinformatics analysis. Gene Ontology classification analysis showed the biological process was primarily related to ‘cellular process’, molecular function ‘structural molecule activity’ and cellular component ‘extracellular region’. IPA analysis revealed 'LXR/RXRactivation’ was the top pathway, which is a crucial pathway in lipid metabolism. Hierarchical clustering analysis generated two clusters. In summary, this study is the first proteomic research to delineate the total and differential serum proteins in TBX6 haploinsufficiency caused CS. The proteins discovered in this experiment may serve as potential biomarkers for CS, and lipid metabolism might play important roles in the pathogenesis of CS.

Liu G, Liu S, Lin M, et al. Genetic polymorphisms of GPR126 are functionally associated with PUMC classifications of adolescent idiopathic scoliosis in a Northern Han population. Journal of cellular and molecular medicine 2018;22:1964-71.

Wang K, Zhao S, Zhang Q, et al. Whole-exome sequencing reveals known and novel variants in a cohort of intracranial vertebral-basilar artery dissection (IVAD). Journal of human genetics 2018;63:1119-28.

Chen J, Zhu Q, Liu G, et al. Fat Mass and Obesity-Associated (FTO) Gene Polymorphisms Are Associated with Risk of Intervertebral Disc Degeneration in Chinese Han Population: A Case Control Study. Medical science monitor : international medical journal of experimental and clinical research 2018;24:5598-609.

Xu Q.#, Wu N.#, Cui L., Wu Z., and Qiu G., Filamin B: The next hotspot in skeletal research? J Genet Genomics, 2017. 44(7): p.335-342.

Lin J., Zhou Y., Liu J., Chen J., Chen W., Zhao S., Wu Z., and Wu N.*, Progress and Application of CRISPR/Cas Technology in Biological and Biomedical Investigation. J Cell Biochem, 2017. 118(10): p.3061-3071.

Chen J., Liu J., Zhou Y., Liu S., Liu G., Zuo Y., Wu Z., Wu N.*, and Qiu G.*, Molecular therapeutic strategies for FGFR3 gene-related skeletal dysplasia. J Mol Med (Berl), 2017. 95(12): p.1303-1313.

Liu J., Zhou Y., Qi X., Chen J., Chen W., Qiu G., Wu Z.*, and Wu N.*, CRISPR/Cas9 in zebrafish: an efficient combination for human genetic diseases modeling. Hum Genet, 2017. 136(1): p.1-12.

Chen Y., Liu Z., Chen J., Zuo Y., Liu S., Chen W., Liu G., Qiu G., Giampietro P.F., Wu N.*, and Wu Z.*, The genetic landscape and clinical implications of vertebral anomalies in VACTERL association. J Med Genet, 2016. 53(7): p.431-7.

Chen W., Liu J., Yuan D., Zuo Y., Liu Z., Liu S., Zhu Q., Qiu G., Huang S., Giampietro P.F., Zhang F., Wu N.*, and Wu Z.*, Progress and perspective of TBX6 gene in congenital vertebral malformations. Oncotarget, 2016. 7(35): p.57430-57441.

Liu S., Wu N., Liu J., Ming X., Chen J., Pavelec D., Su X., Qiu G., Tian Y., Giampietro P., and Wu Z., Novel NTRK1 Frameshift Mutation in Congenital Insensitivity to Pain With Anhidrosis. J Child Neurol, 2015. 30(10): p.1357-61.

Wu N., Yuan S., Liu J., Chen J., Fei Q., Liu S., Su X., Wang S., Zhang J., Li S., Wang Y., Qiu G., and Wu Z., Association of LMX1A genetic polymorphisms with susceptibility to congenital scoliosis in Chinese Han population. Spine (Phila Pa 1976), 2014. 39(21): p.1785-91.

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Please find at: ((((Nan Wu[Author]) OR Zhihong Wu[Author]) OR Guixing Qiu[Author])) AND peking union medical college hospital

Department of orthopedic surgery, Peking Union Medical College Hospital (PUMCH), Peking Union Medical College (PUMC) & Chinese Academy of Medical Sciences (CAMS)

Beijing Key Laboratory for Genetic Research of Skeletal Deformity

Medical Research Center of Orthopedics, Chinese Academy of Medical Sciences

Tel: +86-10-69152809

E-mail: dr.wunan@pumch.cn

Address: Department of orthopedic surgery, Peking Union Medical College Hospital, No.1 Shuaifuyuan, Dongcheng District, Beijing 100730, China

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