Clinical Features of Affected Families

Table 1. Table 1. Summary of Patient Clinical Features and the Identified DNA and Protein Variants.

The clinical features of the study participants are summarized in Table 1; details are provided in the Results section and Table S1 in the Supplementary Appendix. The four families include one consanguineous family from Iraq (Family A), two consanguineous families from Lebanon (Families B and C), and a family from the United States without a history of consanguinity (Family D) (Figure 1). There is no other history of congenital anomalies or intellectual disability in these families. The mother in Family B had insulin-requiring gestational diabetes. The mother in Family C had prepregnancy insulin-requiring diabetes, hypercholesterolemia, and a body-mass index (BMI, the weight in kilograms divided by the square of the height in meters) of 32.8. The mother in Family D had a BMI of 29.3. Patient A and Patient B were each one of dizygotic twins.

All the affected persons were born with vertebral defects predominantly affecting the thoracolumbar spine. Patients A and B had a spinal lipoma, which was associated with sacral agenesis in Patient A and with spinal dysraphism in Patient B. All the patients had cardiac defects: patent ductus arteriosus in Patient C, an atrial septal defect in Patient A, and hypoplastic left heart in Patients B and D. Patients A, B, and C had hypoplastic kidneys, and Patient D had a solitary left kidney with moderate chronic kidney disease. Patient C had rhizomelia, and Patient D had shortened long bones. Patients A and C had talipes. Patients A and B had sensorineural hearing loss. Patient A had a submucous cleft palate, a bifid uvula, and a laryngeal web with persistent laryngeal tracheomalacia. Patient B had palsy in the left vocal cord that was possibly iatrogenic. None of the patients had a tracheoesophageal defect. Patient C had an anterior anus. Patient C died at 4 months of age from restrictive respiratory disease due to spondylocostal defects and Patient B at 11 months of age from complications of hypoplastic left heart.

In addition to congenital malformations, postnatal growth and cognitive defects were evident. Patients A, B, and C had microcephaly. Patients A and D have extreme short stature. Patient A has moderate intellectual disability and behavioral issues at 12 years of age, and Patient D has speech delay at 3 years of age.

Pathogenic Variants in HAAO and KYNU

Figure 2. Figure 2. Synthesis of Nicotinamide Adenine Dinucleotide (NAD). NAD is synthesized de novo from the essential amino acid L-tryptophan and salvaged from nicotinamide. Tryptophan and niacin (vitamin B 3 ), which is supplied as nicotinic acid and nicotinamide, represent dietary inputs. Details on NAD synthesis are provided in Figure S1 in the Supplementary Appendix.

In the consanguineous families, variants were filtered according to a recessive inheritance model and a compound heterozygous inheritance model and on the assumption that a de novo mutation in the patient was a possibility. Additional filtering was used to select variants that were nonsynonymous, rare, and predicted to be damaging, and these variants were assessed for further evidence of disease causation (Tables S2 through S7 in the Supplementary Appendix). Of these, predicted loss-of-function variants in two genes (HAAO, encoding 3-hydroxyanthranilic acid 3,4-dioxygenase, and KYNU, encoding kynureninase) were identified in three consanguineous families and were prioritized for further analysis. HAAO and KYNU are enzymes of the kynurenine pathway and are involved in the synthesis of nicotinamide adenine dinucleotide (NAD) (Figure 2). Pathogenic variants in genes that are associated with NAD synthesis were not identified in the remaining 10 families. Neither HAAO nor KYNU has been associated with congenital malformation; however, a KYNU missense mutation (p.T198A) has been reported to be associated with hydroxykynureninuria (Online Mendelian Inheritance in Man [OMIM] number, 236800).14

Sanger sequencing confirmed variant segregation with disease (Figure 1, and Fig. S2 in the Supplementary Appendix). In Family A, the patient was homozygous for a c.483dupT variant in HAAO (ClinVar accession number, SCV000540919), leading to a stop codon (p.D162*). In Family B, the patient was homozygous for a c.558G→A variant in HAAO (ClinVar accession number, SCV000540920), leading to a stop codon (p.W186*). In Family C, the patient was homozygous for a c.170-1G→T splicing variant in KYNU (ClinVar accession number, SCV000540921), leading to a stop codon downstream (p.V57Efs*21). In all three families, the unaffected parents and siblings were either heterozygous for the mutation or homozygous for the reference allele, indicating a recessive inheritance pattern. In Family D, the patient was compound heterozygous for KYNU variants c.468T→A (ClinVar accession number, SCV000540922) and c.1045_1051delTTTAAGC (ClinVar accession number, SCV000540923), which were inherited from the father and mother, respectively; each variant results in a stop codon (p.Y156* and p.F349Kfs*4, respectively). The locations of the variants in HAAO and KYNU and their corresponding protein variants are shown in Figure S3 in the Supplementary Appendix.

In Vitro Activity of Mutant HAAO and KYNU

We tested the activity of the truncated enzymes. For HAAO, we quantified the conversion of 3-hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconate-6-semialdehyde (ACMS). For KYNU, we quantified the conversion of 3-hydroxykynurenine (3HK) to 3HAA. (The enzyme assays are described in the Supplementary Appendix.) We tested the identified HAAO and KYNU variant proteins as well as KYNU p.T198A, which is associated with hydroxykynureninuria but not congenital malformation.14 The specific activity of all the truncated HAAO and KYNU enzymes was 0 to 19% as high as the activity of nonmutant enzymes (Fig. S4 and Table S8 in the Supplementary Appendix). By contrast, the activity of KYNU p.T198A was 64% as high as the activity of the nonmutant enzyme.

Levels of Metabolites in the Kynurenine Pathway and NAD Levels

Table 2. Table 2. Alteration in Levels of Metabolites in the Kynurenine Pathway and in NAD Levels in Plasma from Patient B (Family Member II.4).

We predicted that loss of HAAO or KYNU activity would lead to increased plasma levels of metabolites upstream of these enzymes and reduced levels downstream. Patients A and B (homozygous HAAO stop codon) had upstream 3HAA levels that were 64 and 385 times the levels in unaffected heterozygous family members, respectively. Downstream of HAAO, levels of NAD+ (the oxidized form of NAD) and of NAD(H) (the sum of NAD+ and NADH, the reduced form of NAD) were one third to one quarter of the levels in unaffected heterozygous family members (Table 1 and Table 2, and Table S9 in the Supplementary Appendix). Plasma was not available from Family C. Patient D (compound heterozygous for KYNU truncating mutations) had an upstream 3HK level that was 161 times the level in unaffected family members and a downstream NAD(H) level that was one seventh of the level in unaffected family members.

Modeling of Disease in Mice Null for Haao or Kynu

We generated mice with a null allele for Haao or Kynu (Fig. S5 in the Supplementary Appendix). Enzyme assays confirmed that the edited alleles were null (Fig. S6 in the Supplementary Appendix). Intercrosses of mice that were heterozygous null for Haao or for Kynu produced embryos in the expected mendelian ratio of genotypes. Unexpectedly, all the embryos were normal (Tables S10 and S11 in the Supplementary Appendix). We next quantified metabolites upstream and downstream of Haao and of Kynu in adult mouse serum. Haao−/− mice had 3HAA levels that were more than 100 times the levels in Haao+/− mice and wild-type mice (Table S12 in the Supplementary Appendix). Similarly, Kynu−/− mice had 3HK levels that were more than 70 times the levels in Kynu+/− mice and wild-type mice, a metabolic finding consistent with our findings in humans (Table 1 and Table 2, and Table S9 in the Supplementary Appendix). By contrast, NAD(H) levels were similar in all the mice, regardless of genotype (Table S12 in the Supplementary Appendix). This suggested that elevated levels of metabolites upstream of HAAO or KYNU in humans did not cause congenital malformation and underscored a deficit in the NAD level as the cause.

NAD is produced by two pathways: one requires dietary tryptophan, and the other requires dietary niacin (Figure 2). The NAD de novo synthesis pathway catabolizes tryptophan through the kynurenine pathway, and the NAD salvage pathway converts niacin and other precursors into NAD (independent of KYNU and HAAO). The niacin status of mice as measured by the concentration of NAD in whole blood is at least four times that in humans, possibly because mice convert tryptophan to NAD more efficiently,15 and they consume more tryptophan or niacin per unit of body weight owing to a higher basal metabolic rate.16 In mice, reduced niacin status occurs only when both de novo synthesis of NAD is blocked and niacin is removed from the diet.17

During development, embryos receive niacin from the mother and generate their own. It is therefore possible that maternal niacin has a buffering effect and protects the null mouse embryos from the development of NAD deficiency. We therefore sought to reduce niacin levels in pregnant mice that were heterozygous for a null allele (Haao+/− or Kynu+/−). As a first attempt to mimic the reduced NAD levels in humans, these mice were fed a niacin-free diet during pregnancy. In litters from Haao+/− or Kynu+/− intercrosses, embryos had the expected mendelian ratio of genotypes and were phenotypically normal (Tables S11 and S13 in the Supplementary Appendix). This observation suggested that heterozygous null female mice (i.e., those with one normal and one null allele of Haao or of Kynu) that receive a niacin-free diet produce sufficient NAD from dietary tryptophan to sustain normal embryonic development.

Figure 3. Figure 3. Effect of Niacin Supplementation on Levels of NAD in Null Mouse Embryos. Female mice that were homozygous null for Haao or Kynu were mated with heterozygous male mice. Pregnant mice were fed a niacin-free diet supplemented with 5, 10, or 15 mg of nicotinic acid per liter of drinking water from embryonic day 7.5 to 9.5. Embryos were harvested at embryonic day 9.5, and levels of NAD(H) (the sum of NAD+ and NADH) were quantified. The difference between groups was tested with the use of a one-way analysis-of-variance test based on log 2 -transformed data, followed by Dunnett’s multiple-comparison test (all other groups vs. null mice receiving 5 mg of nicotinic acid per liter). The middle lines of the I bars indicate mean values, and the I bars ±1 SD.

To preclude maternal NAD production from the de novo synthesis pathway in addition to the salvage pathway, null female mice (Haao−/− or Kynu−/−) were mated with Haao+/− or Kynu+/− male mice and fed a niacin-free diet. All the embryos died, regardless of genotype (Tables S11 and S14 in the Supplementary Appendix). Death also occurred when the niacin-free diet was limited from embryonic day 0.5 to embryonic day 4.5, 5.5, or 6.5 (Tables S11 and S14 in the Supplementary Appendix). We determined that a niacin-free diet supplemented with 5 mg of nicotinic acid per liter of drinking water between embryonic day 7.5 and embryonic day 12.5 better sustained embryogenesis in null mothers. Despite a large number of resorptions, live null embryos were present (Tables S11 and S15 in the Supplementary Appendix). All the Haao+/− and Kynu+/− embryos were normal. By contrast, all the Haao−/− and Kynu−/− embryos had multiple defects, including defects in vertebral segmentation, heart defects, small kidney, cleft palate, talipes, syndactyly, and caudal agenesis (Fig. S7 and Tables S15 and S16 in the Supplementary Appendix). NAD levels in null mouse embryos were one half the levels in unaffected heterozygous littermates at embryonic day 9.5 (Figure 3). This observation indicates that in both mice and humans, loss of embryonic NAD leads to embryo defects and death. It is the embryonic NAD deficit that causes defects rather than the maternal deficit, because null mothers produce normal heterozygous embryos (Table S15 and Fig. S7 in the Supplementary Appendix).

To prove that NAD deficiency was disrupting embryogenesis, pregnant null mice were fed a niacin-free diet as before, and water was supplemented with 10 or 15 mg of nicotinic acid per liter. Because mice consume 1.3 liters of water per kilogram of food,18 we calculated that with this regimen mice would consume 14% of the niacin equivalent in complete mouse chow (90 mg per kilogram). Litters from these mice contained embryos with genotypes in the expected mendelian ratio. With 10 mg of nicotinic acid per liter, null embryos were normal except for kidneys that were 30% smaller than those in heterozygous controls; with 15 mg of nicotinic acid per liter, all the embryos were normal (Table S15 in the Supplementary Appendix). We also observed a dose-dependent increase in embryonic NAD(H) levels in response to maternal niacin supplementation (Figure 3), a finding consistent with the absence of a phenotype. These data show that embryo death and defects were specifically due to a deficit of NAD in embryos and that niacin supplementation prevented the disruption of embryogenesis.