A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder

Abstract

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Keywords: Embryonic development; Genetic diseases; Mouse models; Reproductive biology; Therapeutics.

Figure 1. Pedigrees of families with individuals harboring biallelic NADSYN1 variants.

Squares indicate male individuals, circles female, triangles first-trimester deaths, solid symbols affected individuals, slashes deceased, and double horizontal lines consanguinity. NADSYN1 variant details are provided underneath individuals.

Figure 2. Positions of previously and newly identified NADSYN1 variants identified relative to functional protein domains.

Blue circles indicate variants identified in the current study and respective family (see Figure 1). Other colored circles with numerals indicate study origin of identified variants and the chronology of their identification, respectively. Previously published variants have been reported in refs. 7, 11–13. Solid and dashed lines distinguish missense from presumed loss-of-function variants due to altered reading frame and protein truncation, respectively.

Figure 3. Functional assessment of NADSYN1-variant proteins corresponding to gene variants identified in affected individuals.

(A) NADSYN1 activity of purified variant proteins compared with wild-type NADSYN1 protein in the presence of glutamine as substrate. One-way ANOVA with Dunnett’s post hoc test. (B) NADSYN1 activity of purified variant proteins compared with wild-type NADSYN1 protein in the presence of ammonia as substrate. One-way ANOVA with Dunnett’s post hoc test. (C) Average NADSYN1 activity of variant protein relative to wild-type protein activity with respect to positions of variant sites in functional protein domains. ***P < 0.001, ****P < 0.0001; n = 4 experiments. WT, wild-type NADSYN1 protein; Control, negative control (untransfected cell lysate).

Figure 4. Whole-blood and plasma NAD metabolomic profiles in individuals with biallelic NADSYN1 variants and their heterozygous parents.

(A) Simplified NAD biosynthesis pathway; genes in which biallelic pathogenic variants cause CNDD (cyan), and B₃ vitamers (green). (B and D) Partial least squares–discriminant analysis (PLS-DA) 2-dimensional score plots of proband and parental whole blood (B) and plasma (D). Affected individuals (red) and parents (cyan) are denoted by their pedigree IDs (see Figure 1). (C and E) Respective variable importance in projection (VIP) plots. The most discriminating metabolites are shown in descending order of their VIP scores. n = 3 probands and their 6 parents, respectively. AA, anthranilic acid; 3HK, 3-hydroxykynurenine; KYN, kynurenine; 1MNA, 1-methylnicotinamide; NA, nicotinic acid; NaAD, nicotinic acid adenine dinucleotide; NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide; NaMN, nicotinic acid mononucleotide; NAR, nicotinic acid riboside; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; 2PY, N¹-methyl-2-pyridone-5-carboxamide; 4PY, N¹-methyl-4-pyridone-3-carboxamide; XA, xanthurenic acid. See Supplemental Figure 1 for an overview of the NAD synthesis pathways and associated metabolites. Metabolite concentration values are provided in Table 3 and Supplemental Table 3.

Figure 5. Liver NAD and NAD-related metabolites in plasma of female mice of different Nadsyn1 genotypes under various dietary conditions.

All mice were pretreated with the Sufficient Diet for more than 21 days and then provided with the indicated diets for 17 days, after which they were dissected and liver tissue and plasma collected. (A) Liver total NAD. Statistical comparisons represent within-diet 2-tailed Student’s t test between Nadsyn1^+/– and Nadsyn1^–/– mice. *P < 0.05, **P < 0.01; n = 5–10 mice per condition. For numerical values, see Supplemental Table 9. (B and C) Partial least squares–discriminant analysis (PLS-DA) 2-dimensional score plots (B) and corresponding variable importance in projection (VIP) plots (C) comparing plasma metabolite levels between female Nadsyn1^+/– and Nadsyn1^–/– mice fed the Sufficient Diet; n = 6 mice per condition. (D) NAD-related metabolites with the highest VIP scores in mouse plasma. Nadsyn1 genotypes are shown below each graph and dietary conditions on top of graphs. Bars indicate mean ± standard deviation; n = 5–10 mice per condition. Values for the other measured NAD-related metabolites are summarized in Supplemental Figure 12. “< LOD” indicates below the limit of detection. See Supplemental Figure 1 for an overview of the NAD synthesis pathways and associated metabolites.