EP404: Evidence supporting the pathogenicity of the NADSYN1 c.1717G>A (p.Ala573Thr) variant in individuals referred for clinical testing

Abstract

Vertebral, cardiac, renal, and limb defects syndrome 3 (VCRL3; OMIM #618845) is an autosomal recessive multiple congenital anomaly syndrome caused by pathogenic variants in the NADSYN1 gene. NADSYN1 encodes a nicotinamide adenine dinucleotide (NAD) synthetase that is part of the de novo NAD synthesis pathway. Other genes involved in this pathway include HAAO and KYNU. Genetic variants in any of these genes result in reduced de novo NAD synthesis and cause congenital anomalies of the heart, kidney, and vertebrae. Of note, presence of these three features is also considered clinical evidence for vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities (VACTERL) association, which is often a diagnosis of exclusion (ie, other genetic conditions with overlapping features must be ruled out before assigning this diagnosis). The recommended approach for genetic testing in possible VACTERL patients is to either pursue a comprehensive testing option, such as exome or genome sequencing, or a panel approach with the most likely candidates. The NADSYN1 gene is included on a VACTERL Association and Related Disorders panel and several patients were noted to be homozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant, thus providing additional evidence for both the pathogenicity and high carrier frequency of this change. To date, we have identified five patients who are homozygous for the NADSYN1 (NM_018161.4) c.1717G>A (p.Ala573Thr) variant. One patient was identified through targeted testing, one through testing with the VACTERL Association and Related Disorders panel, and three through exome sequencing. The first four cases were from pregnancies evaluated for the differential diagnosis of VACTERL association via panel testing or exome sequencing. All four fetuses were reported to have cardiac malformation and segmental vertebral anomalies. Parental data was available for three of the four fetuses, and all parents tested were heterozygous for the NADSYN1 c.1717G>A (p.Ala573Thr) variant. All cases denied consanguinity. In contrast to the severe and early onset features of the other four patients identified, the targeted testing case was an adult patient who previously had the NADSYN1 c.1717G>A variant identified in a research study. This individual was noted to have cardiac defects, vertebral defects, and short stature. This variant is relatively common in non-Finnish Europeans with an allele frequency of 0.15% in the gnomAD v2 dataset. Five patients from four families with VCRL3 have been described in the literature. Of these patients, three had the c.1717G>A variant: two homozygous siblings and one compound heterozygote with a loss of function variant on the opposite allele. Parental sequencing data confirmed the biallelic variants in all reported patients. In addition, yeast model functional studies performed by Szot and colleagues demonstrated that the p.Ala573Thr change diminished NAD synthetase activity and reduced yeast viability, consistent with a loss of function mechanism. Taken together, although the NADSYN1 c.1717G>A (p.Ala573Thr) has a relatively high minor allele frequency, there is sufficient evidence for this variant to be classified as pathogenic using ACMG/AMP variant classifications and internal variant interpretation guidelines. The NADSYN1 c.1717G>A (p.Ala573Thr) variant should be interpreted as pathogenic given the 5 additional homozygous patients and parental segregation data presented here. Additionally, the diagnosis of VCRL3 in an adult patient suggests that although penetrance for NAD deficiency is complete, variable expressivity is observed and could be explained by other genetic or non-genetic factors modulating disease severity. Given the high population frequency of this variant, genetic testing for VACTERL association should always include evaluation of NADSYN1 to consider VCRL3 in the differential diagnosis.