BACKGROUND AND PURPOSE: 

• Certain combinations of birth defects occur more often than expected by chance
• One such association is VACTERL  
  • Verterbral defects 
  • Anal atresia 
  • Cardiac defects 
  • TracheoEsophageal fistula 
  • Renal anomalies  
  • Limb abnormalities  
• Shi et al. (NEJM, 2017) performed genomic sequencing to identify the underlying genetics of birth defects that arise in combination and developed mice models to establish the function and role of these genetic changes 

METHODS: 

• Study participants:  
  • 4 patients with both congenital vertebral and heart malformations and their families  
  • Patients had other findings consistent with VACTERL, such as shortened limbs and renal anomalies (e.g., hypoplastic or solitary kidneys)
• 3 families underwent whole exome sequencing and 1 family underwent whole genome sequencing  
• In vitro enzyme activity and metabolite levels were measured to determine the function of the variant  
• Similar variants were then engineered in mouse models using CRISPR-Cas9 

RESULTS: 

Experiment 1: Genomic sequencing to find mutations  

• Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioygenase (HAAO) and kynureninase (KYNU) 
• Affected individuals were homozygous for the mutant allele (same deleterious mutation) or a compound heterozyote (two deleterious mutations)  
• Parents were heterozygous suggesting autosomal recessive pattern of inheritance
• These mutations were not found in other families who had birth defects, but not in this particular combination 

Experiment 2: In vitro enzyme activity measurements

• Activity of the truncated HAAO and KYNU enzymes were only 0-19% as active as the nonmutant enzymes 
• Levels of upstream metabolites of these enzymes were elevated and downstream metabolites were reduced in individuals with birth defects
  • Downstream metabolites are NAD+ (the oxidized form of NAD) and NAD(H) (the reduced form of NAD)

Experiment 3: CRISPR-Cas9 mouse models   

• Haao-null and Kynu-null (missing the functional gene) mouse models had similar congenital defects as patients  
  • Deficiency of downstream NAD and not excess of upstream metabolites were associated with birth defects

Experiment 4: Supplying additional niacin to the maternal mouse diet to prevent birth defects  

• There is an NAD ‘salvage’ pathway that can bypass KYNU and HAAO by converting dietary niacin (Vitamin B3) and other precursors into NAD 
• Niacin supplementation to maternal mice prevented birth defects in the ‘null’ offspring 

CONCLUSION: 

• NAD synthesis disruption caused deficiency of NAD and congenital malformations in both humans and mice 
• Niacin supplementation during gestation prevented malformations in mice 
• This study reinforces the importance of understanding birth defects from both a genetic and environmental perspective 
  • Similar tissues affected but variation in phenotype  
• The authors suggest that 10 times the US recommended dose of niacin may prevent further birth defects in these 4 families  

Learn More – Primary Sources:  

NAD Deficiency, Congenital Malformations, and Niacin Supplementation 

Related ObG Topics:

CRISPR and Human Genome Editing: Ready for Prime Time? 11 Professional Organizations Respond 

The Exome and Whole Exome Sequencing

The Genome and Whole Genome Sequencing