A promoter variant in the OTC gene associated with late and variable age of onset hyperammonemia

Abstract

Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn error caused by loss of function variants in the OTC gene typically associated with severe neonatal hyperammonemia. Rare examples of late-onset OTCD have also been described. Here, we describe an OTC promoter variant, c.-106C>A, in a conserved HNF4a binding site, identified in two male siblings in Family 1 whose first and only recognized episodes of severe hyperammonemia occurred at ages 14 and 39 years, respectively. We identified the same OTC variant segregating in a large family with late-onset OTCD with variable expressivity (Family 2). We show that this OTC promoter variant reduces expression >5-fold in a dual-luciferase assay that tests promoter function. Addition of an upstream OTC enhancer increases expression of both the wild type and the c.-106C>A variant promoter constructs >5-fold with the mutant promoter still about fourfold lower than the wild type. Thus, in both contexts, the promoter variant results in substantially lower OTC expression. Under normal demand on urea cycle function, OTC expression in hemizygous males, although reduced, is sufficient to meet the demand for waste nitrogen excretion. However, in response to severe metabolic stress with attendant increased requirements on urea cycle function, the impaired promoter function results in inadequate OTC expression with resultant hyperammonemia. In the absence of precipitating events, hemizygotes with this allele are asymptomatic, explaining the late age of onset of hyperammonemia in affected individuals and the incomplete penetrance observed in some individuals in Family 2.

Keywords: OTC deficiency; late onset; noncoding DNA; promoter variant; regulatory DNA sequence.

Figure 1.. Results of biochemical testing

(A) Plasma ammonia levels for proband measured over time, hemodialysis was begun 14.5 hours after admission at constant 500 μmol/L. Peak ammonia in proband measured to be 438 μmol/L and dropped to normal range (1-32) after 72 hours. (B) Plasma amino acids, urinary orotic acid, and peak plasma ammonia of probands taken at admission are consistent with OTCD. (C) Orotic acid levels (mmol/mol creatinine) of proband, mother, and nephew (normal range 2.1 +/− 0.71) at baseline or following allopurinol challenge. Results demonstrate normal baselines for all three individuals, however allopurinol challenge demonstrated results consistent with OTCD and OTCD carrier for proband and mother, respectively, but not for the nephew.

Figure 2.. Co-segregation of c.−106 C>A with late onset OTCD phenotype

(A) PCR amplification and Sanger sequencing of OTC upstream sequence confirmed transmission of the c.−106 C>A variant from the mother to the proband and deceased brother. (B) Pedigree of Family 1. The proband (III-4) and his brother (III-3) carry the pathogenic allele. Individual IV-1 carries the alternate X inherited from II-2 and showed no signs of metabolic disease by age 8. (C) Pedigree of Family 2 living in Utah demonstrates transmission of OTCD allele from common ancestors diagnosed with late onset OTCD.

Figure 3.. The c.−106C>A variant is sufficient to impair promoter activity independent of haplotype background

(A) Sanger sequencing confirmed the presence of the c.−106 C>A variant upstream of the OTC transcription start site (TSS) and start methionine (ATG). Five additional variants were identified within 1kb upstream of OTC. All five variants are known polymorphisms and each has a population frequency of 0.28 reported in dbSNP. (B) The five SNPs identified on the pathogenic haplotype are found in high LD with one another, demonstrating that they are often inherited as a single unit. The pathogenic variant is a rare variant on this haplotype. (C) Dual luciferase assay demonstrated that the promoter allele cloned from the proband was significantly less active than the WT allele. Inclusion of the 500 bp enhancer (Enh) element increased function of both the WT and OTCD alleles, however the OTCD allele was not able to achieve activity significantly higher than the basal activity of the WT allele. (D) The c.−106C>A variant was introduced onto the WT haplotype (WT + SDM) while the c.−106C>A variant was reverted back to reference on the OTCD haplotype by site directed mutagenesis (OTCD + SDM). Introduction of the c.−106C>A variant lowered the function of the WT allele to levels comparable to the OTCD allele. Correction of the c.−106C>A variant restored function of the OTCD allele to WT levels. All values displayed as min – max and (median).