Background Congenital vertebral malformations (CVMs) manifest with abnormal vertebral morphology. Genetic factors have been implicated in CVM pathogenesis, but the underlying pathogenic mechanisms remain unclear in most subjects. We previously reported that the human 16p11.2 BP4-BP5 deletion and its associated TBX6 dosage reduction caused CVMs. We aim to investigate the reciprocal 16p11.2 BP4-BP5 duplication and its potential genetic contributions to CVMs.
Methods and results Patients who were found to carry the 16p11.2 BP4-BP5 duplication by chromosomal microarray analysis were retrospectively analysed for their vertebral phenotypes. The spinal assessments in seven duplication carriers showed that four (57%) presented characteristics of CVMs, supporting the contention that increased TBX6 dosage could induce CVMs. For further in vivo functional investigation in a model organism, we conducted genome editing of the upstream regulatory region of mouse Tbx6 using CRISPR-Cas9 and obtained three mouse mutant alleles (Tbx6up1 to Tbx6up3 ) with elevated expression levels of Tbx6. Luciferase reporter assays showed that the Tbx6up3 allele presented with the 160% expression level of that observed in the reference (+) allele. Therefore, the homozygous Tbx6up3/up3 mice could functionally mimic the TBX6 dosage of heterozygous carriers of 16p11.2 BP4-BP5 duplication (approximately 150%, ie, 3/2 gene dosage of the normal level). Remarkably, 60% of the Tbx6up3/up3 mice manifested with CVMs. Consistent with our observations in humans, the CVMs induced by increased Tbx6 dosage in mice mainly affected the cervical vertebrae.
Conclusion Our findings in humans and mice consistently support that an increased TBX6 dosage contributes to the risk of developing cervical CVMs.
- congenital vertebral malformation
- chromosomal microarray analysis
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XR, NY and NW contributed equally.
JS, FZ and PL contributed equally.
Contributors XR, NY, NW and FZ designed the study. XR, NY, NW, XX, WC, LZ, YL, R-QD, SD, SZ, SC, L-PJ and LW conducted experiments. XR, NY, NW, XX, WC, LZ, YL, R-QD, SD, SZ, SC, L-PJ, LW, JZ, ZW, LJ, GQ, JRL, JS, FZ and PL analysed data. XR, NY, NW, JRL, JS, FZ and PL wrote the manuscript. JS, FZ and PL supervised the study.
Funding This work was supported by National Natural Science Foundation of China (31571297, 31625015, 31771396, 31521003, 81822030, 81472046, 81772301, 81672123 and 81772299), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), Shanghai Medical Center of Key Programs for Female Reproductive Diseases (2017ZZ01016), Beijing Natural Science Foundation (7172175), CAMS Initiative Fund for Medical Sciences (2016-I2M-3-003), the US National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS R35 NS105078) and the US National Human Genome Research Institute/National Heart Lung and Blood Institute (NHGRI/NHLBI) supported Baylor Hopkins Center for Mendelian Genomics (BHCMG), grant UM1 HG006542.
Competing interests JRL has stock ownership in 23andMe, is a paid consultant for Regeneron Pharmaceuticals and Novartis and is a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases and bacterial genomic fingerprinting. PL is an employee of Baylor College of Medicine and derives support through a professional services agreement with Baylor Genetics. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis and clinical exome sequencing offered at Baylor Genetics.
Patient consent for publication Not required.
Ethics approval Declaration of Helsinki and approved by the Ethical Committees of the centers participating in this study.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.
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