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. 2021 Oct 14;12(10):1615.
doi: 10.3390/genes12101615.

Disruptive NADSYN1 Variants Implicated in Congenital Vertebral Malformations

Jiachen Lin  1   2   3   4 , Lina Zhao  3   4   5 , Sen Zhao  1   2   3   4 , Shengjie Li  3   5 , Zhengye Zhao  1   2   3   4 , Zefu Chen  1   2   3   4 , Zhifa Zheng  2   3   4   5 , Jiashen Shao  1   2   3   4 , Yuchen Niu  3   5 , Xiaoxin Li  3   5 , Jianguo Terry Zhang  1   2   4   5 , Zhihong Wu  2   3   4   5 , Nan Wu  1   2   4   5
Affiliations

Disruptive NADSYN1 Variants Implicated in Congenital Vertebral Malformations

Jiachen Lin et al. Genes (Basel). .

Abstract

Genetic perturbations in nicotinamide adenine dinucleotide de novo (NAD) synthesis pathway predispose individuals to congenital birth defects. The NADSYN1 encodes the final enzyme in the de novo NAD synthesis pathway and, therefore, plays an important role in NAD metabolism and organ embryogenesis. Biallelic mutations in the NADSYN1 gene have been reported to be causative of congenital organ defects known as VCRL syndrome (Vertebral-Cardiac-Renal-Limb syndrome). Here, we analyzed the genetic variants in NADSYN1 in an exome-sequenced cohort consisting of patients with congenital vertebral malformations (CVMs). A total number of eight variants in NADSYN1, including two truncating variants and six missense variants, were identified in nine unrelated patients. All enrolled patients presented multiple organ defects, with the involvement of either the heart, kidney, limbs, or liver, as well as intraspinal deformities. An in vitro assay using COS-7 cells demonstrated either significantly reduced protein levels or disrupted enzymatic activity of the identified variants. Our findings demonstrated that functional variants in NADSYN1 were involved in the complex genetic etiology of CVMs and provided further evidence for the causative NADSYN1 variants in congenital NAD Deficiency Disorder.

Keywords: NADSYN1 gene; congenital NAD Deficiency Disorder; congenital vertebral malformation; exome sequencing.

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Conflict of interest statement

There is no competing interests to declare for all authors.

Figures

Figure 1
Figure 1
(A) Protein domain diagram of human NADSYN1 and the position of identified variants. The glutaminase domain is labeled in green, NAD synthetase domain in purple, P1 loop in yellow, P2 loop in pink, and ATP binding site in red, respectively. The RefSeq transcript sequence of NADSYN1 gene is NM_018161.5; PDB accession code: 6OFB. (B) Space distribution of variants in 3D model using Swiss-Model and PyMOL (https://pymol.org (accessed on 4 January 2021)). (C) Position of the variants (red) relative to an evolutionarily conserved region using ClustalW sequence alignment (https://www.genome.jp/tools-bin/clustalw (accessed on 8 April 2021)).
Figure 2
Figure 2
(A) Expression of NADSYN1 protein in cells transfected with wild type (WT) and mutant NADSYN1 plasmid. (B) The western blot shows the content of the NADSYN1 protein before and after protein purification. (C)Quantification of the NADSYN1 WT and mutant protein based on western blot results. Then band intensity was measured by ImageJ based on ImageJ software. (D) Nicotinamide adenine dinucleotide synthetase activity of purified mutant protein compared to wild-type (WT) NADSYN1 protein. Abbreviations: NC, negative control; ns, non-significant. The results are shown as the mean ± standard error of three independent experiments. p-Values < 0.05 (*), 0.0001 (****) were considered significant.
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