Whole-Exome Sequencing Uncovers Surprising Genetic Causes of Childhood Hyperammonemia

When a child arrives in hospital with dangerously high levels of ammonia in the blood, doctors face one of the most urgent puzzles in metabolic medicine. Hyperammonemia can trigger severe neurotoxicity, cerebral edema and irreversible neurological injury, particularly in infants and young children, and its symptoms—poor feeding, vomiting, lethargy, seizures, developmental delay and even coma—overlap with common neonatal emergencies such as sepsis. The classical culprits are well known: urea cycle defects, organic acidemias and fatty acid oxidation disorders. But a new study from Iran shows how often the true cause lies far outside these familiar categories, and how whole-exome sequencing can find it.

Researchers at the Center for Comprehensive Genetic Services, a national referral facility affiliated with Shahid Beheshti University of Medical Sciences in Tehran, retrospectively analyzed five unrelated pediatric patients who presented with hyperammonemia as a prominent metabolic manifestation. All five were of Iranian origin, and their data were drawn from the laboratory database and medical records. The team applied strict inclusion criteria: whole-exome sequencing had to have identified variants establishing a molecular diagnosis consistent with each patient’s clinical presentation, while children whose elevated ammonia was attributable to clear secondary causes—acute hepatic failure, respiratory insufficiency, gastrointestinal hemorrhage, prolonged parenteral nutrition or drug effects—were excluded. The study protocol was approved by the local institutional review board and conducted in accordance with the Declaration of Helsinki, with written informed consent obtained from parents or legal guardians.

The technical pipeline was thorough. Genomic DNA was extracted from peripheral blood leukocytes using a salting-out procedure, and quality was checked on a NanoDrop spectrophotometer. Exonic regions and exon–intron boundaries were enriched with the SureSelectXT Human All Exon V6 kit, and sequencing was performed on an Illumina HiSeq4000 platform generating 101-base-pair paired-end reads. After trimming and quality control, reads were aligned to the human reference genome GRCh37/hg19 with the Burrows–Wheeler Aligner, processed with SAMtools and Picard, and variant calling followed Genome Analysis Toolkit best-practice workflows. Annotation used ANNOVAR, with filtering that removed synonymous changes and any variant with a minor allele frequency of 1 percent or higher in the 1000 Genomes, ExAC and gnomAD databases. Candidate variants were confirmed by PCR and Sanger sequencing, and interpretation followed the ACMG/AMP 2015 guidelines.

The results were striking in their diversity. Across the five patients, sequencing identified disease-causing variants in five different genes: FBXL4, GLDC, FUCA1, BSND and COL6A2. Three of the variants were novel—FBXL4 c.1702G>A; p.(Gly568Arg), BSND c.85G>T; p.(Asp29Tyr) and COL6A2 c.1999C>T; p.(His667Tyr). All variants were either biallelic or homozygous, consistent with autosomal recessive inheritance, and segregation analysis confirmed the expected inheritance patterns with no evidence of de novo occurrence. The affected genes spanned a remarkable range of biological functions: mitochondrial function, glycine cleavage, lysosomal degradation, renal tubular transport and extracellular matrix organization.

The clinical pictures were equally heterogeneous. The cohort comprised three males and two females, with a median age at diagnosis of 2.8 years and a range from 2 months to 9 years. Parental consanguinity was reported in two families. Common features included hypotonia in three patients, metabolic acidosis in three and elevated lactate levels in three, while two children had seizures and two had developmental delay. Additional manifestations included diarrhea, hypokalemia, muscle wasting and renal stone formation. The first patient, a 9-year-old boy, carried compound heterozygous FBXL4 variants—the novel p.(Gly568Arg) change alongside a previously reported p.(Arg435Gln) allele—and presented with hyperammonemia, elevated lactate, hypotonia and episodes of metabolic acidosis.

The remaining cases pushed the diagnostic boundaries even further. A 9-month-old boy harbored a homozygous, previously reported pathogenic GLDC variant, c.1940C>T; p.(Pro647Leu), and showed seizures, developmental delay, raised lactate and hyperammonemia—a profile consistent with non-ketotic hyperglycinemia, a disorder in which elevated ammonia is not considered typical and has been reported only in a limited number of cases. An 8-month-old boy from consanguineous parents was homozygous for an FUCA1 variant, c.923G>A; p.(Arg308His), a known cause of fucosidosis, and exhibited hyperammonemia alongside elevated urinary 3-hydroxyisovaleric acid. A 2-month-old girl from a consanguineous family carried the novel homozygous BSND variant and presented with hyperammonemia, hypotonia, diarrhea and hypokalemia. The fifth patient, a 5-year-old girl, carried the novel homozygous COL6A2 variant and showed hyperammonemia, increased lactate, muscle wasting and renal stones.

Several of these associations are genuinely unexpected. Fucosidosis, caused by FUCA1 deficiency, is classically characterized by neurological deterioration, and previous case series have rarely emphasized hyperammonemia—some case reports specifically documented normal ammonia measurements. The authors suggest one plausible explanation is intercurrent metabolic decompensation, in which catabolic stress transiently overwhelms the urea cycle. Similarly, no previous report has directly associated BSND variants, which cause Bartter syndrome type 4a and typically produce hypokalemia, metabolic alkalosis and polyuria, with hyperammonemia. In their patient, the team proposes that recurrent electrolyte imbalance, volume depletion, dehydration and catabolic crises may indirectly influence ammonia homeostasis by increasing proteolysis and nitrogen load, though they caution that a direct mechanistic relationship has not been established and causality remains uncertain.

The COL6A2 finding is the most speculative. Pathogenic variants in this gene are classically associated with Bethlem myopathy and Ullrich congenital muscular dystrophy, marked by progressive muscle weakness and contractures—features far removed from the metabolic crisis seen in this patient. The researchers hypothesize that increased muscle catabolism could elevate nitrogen turnover and ammonia production, overloading the urea cycle in genetically susceptible individuals. They explicitly frame this as a hypothesis-generating genotype–phenotype association rather than evidence of a direct causal relationship, and call for functional studies to determine whether chronic muscle catabolism contributes to hyperammonemia in collagen VI-related disorders. For FBXL4, by contrast, the mechanism is more grounded: mitochondrial DNA depletion syndrome 13 typically presents with encephalopathy, lactic acidosis and profound energy deficiency, and mitochondrial dysfunction is hypothesized to impair the ATP production needed for efficient urea cycle operation, allowing ammonia to accumulate during metabolic stress.

Beyond the individual diagnoses, the study illustrates why conventional biochemical testing can mislead. Metabolite screening is phenotype-driven and limited to predefined pathways, and elevated urinary 3-hydroxyisovaleric acid—nonspecific and potentially reflective of secondary mitochondrial energy disturbances or biotin-dependent leucine catabolism during severe metabolic stress—can prompt misclassification when the real diagnosis is a lysosomal storage disorder. The authors argue that lysosomal storage diseases belong in the differential diagnosis of unexplained hyperammonemia, and that whole-exome sequencing provides an unbiased platform to interrogate the entire coding genome and identify rare inborn errors of metabolism with non-classical presentations.

The team is candid about the limitations. The small cohort and retrospective design restrict generalizability, longitudinal follow-up was incomplete in some patients, and no functional assays were performed, precluding definitive proof of pathogenicity for the novel variants. Because functional validation was beyond the scope of the study, a direct mechanistic relationship between the identified variants and hyperammonemia cannot be established. In consanguineous families, the possibility of dual molecular diagnoses also cannot be excluded, meaning some phenotypic features may reflect more than one underlying genetic disorder. Nevertheless, the findings underscore the considerable genetic heterogeneity of pediatric hyperammonemia and support early genomic testing in children with unexplained or atypical biochemical profiles, with further functional studies in larger cohorts needed to confirm the novel variants and clarify their mechanisms.

Subject of Research: Whole-exome sequencing to identify molecular diagnoses in pediatric patients presenting with hyperammonemia

Article Title: Whole-exome sequencing reveals the molecular diagnoses of pediatric patients presenting with hyperammonemia

Article References: Hajati, R., Kamfar, S., Reshadmanesh, A., Saberi, M., Karimzadeh, P., Moosavian, T., Movafagh, A., Sayad, A., & Salehpour, S. (2026). Whole-exome sequencing reveals the molecular diagnoses of pediatric patients presenting with hyperammonemia. Discover Genetics and Evolution, 72(1), Article 5. https://doi.org/10.1007/s00294-026-01338-z

Image Credits: AI Generated

DOI: 10.1007/s00294-026-01338-z

Keywords: hyperammonemia, whole-exome sequencing, pediatric genetics, inborn errors of metabolism, FBXL4, GLDC, FUCA1, BSND, COL6A2, novel variants, autosomal recessive inheritance, molecular diagnosis