Fatal Cytomegalovirus Infection in an Adult with Inherited NOS2 Deficiency

Abstract

Background: Cytomegalovirus (CMV) can cause severe disease in children and adults with a variety of inherited or acquired T-cell immunodeficiencies, who are prone to multiple infections. It can also rarely cause disease in otherwise healthy persons. The pathogenesis of idiopathic CMV disease is unknown. Inbred mice that lack the gene encoding nitric oxide synthase 2 (Nos2) are susceptible to the related murine CMV infection.

Methods: We studied a previously healthy 51-year-old man from Iran who after acute CMV infection had an onset of progressive CMV disease that led to his death 29 months later. We hypothesized that the patient may have had a novel type of inborn error of immunity. Thus, we performed whole-exome sequencing and tested candidate mutant alleles experimentally.

Results: We found a homozygous frameshift mutation in NOS2 encoding a truncated NOS2 protein that did not produce nitric oxide, which determined that the patient had autosomal recessive NOS2 deficiency. Moreover, all NOS2 variants that we found in homozygosity in public databases encoded functional proteins, as did all other variants with an allele frequency greater than 0.001.

Conclusions: These findings suggest that inherited NOS2 deficiency was clinically silent in this patient until lethal infection with CMV. Moreover, NOS2 appeared to be redundant for control of other pathogens in this patient. (Funded by the National Center for Advancing Translational Sciences and others.).

Figure 1.. Homozygous Frameshift Mutation in NOS2 in a Patient with Fatal Cytomegalovirus Infection.

Panel A shows the pedigree of a kindred and the genotype of the patient (P, indicated by a solid square) and sequenced family members. Squares indicate male family members, and circles female members; WT denotes wild type. A slash indicates that the family member has died. Status was unknown for family member I.1. The abbreviation I391fs indicates an insertion in the codon encoding for isoleucine 391. Panel B shows a computed tomographic scan of the patient’s chest at the time of his initial presentation, revealing diffuse ground-glass opacities and septal thickening. Panel C shows the confirmation of genotypes by Sanger sequencing of genomic DNA samples obtained from the patient and his family members, which indicated that the patient was carrying a homozygous single base-pair I391fs variant, which led to a frameshift mutation and formation of a premature stop after 26 additional codons. Panel D shows the functional domains of nitric oxide synthase 2 (NOS2) protein and the predicted truncated NOS2 protein that would result from the patient’s variant. BH4 denotes tetrahydrobiopterin, CaM calmodulin, FAD flavin adenine dinucleotide, and FMN flavin mononucleotide.

Figure 2.. Loss-of-Function NOS2 Mutation in the Patient.

Panel A shows a Western blot of lysates from HEK293T cells that overexpressed empty vector (EV) or vector containing wild-type (WT) NOS2, the patient’s mutation (I391fs), or catalytically inactive mutant (D280A) NOS2 open reading frames for 72 hours. Panel B shows a Griess assay of supernatant from the same transfection conditions as a measure of nitric oxide production by each NOS2 allele expressed. (Since measurement of nitric oxide is technically difficult because of its chemical reactions with many other molecules, its derivatives are often measured, as in the micromolar amounts of nitrite shown here.) T bars represent the standard deviation from the mean of three independently transfected wells.

Figure 3 (facing page).. Analysis of NOS2 Variants in the General Population.

Panel A shows a Griess assay of supernatants of HEK293T cells after overexpression with selected NOS2 alleles listed in Figure S2. The 33 variants were identified in two public databases of human genetic variation. All the alleles (with the exception of A544V, a very rare variant) showed production of nitric oxide at levels similar to that of wild-type NOS2. Panel B shows a Western blot of similarly transfected HEK293T cells to confirm the expression of each allele. For the I391fs mutant, also shown is a region of the blot around 52 kDa indicating the truncated protein. Panel C shows a comparison of the kinetics of nitric oxide production of HEK293T cells that overexpress an empty vector or vector containing A544V, wild-type, or I391Ifs NOS2 open reading frames. In Panels A and C, the T bars and I bars represent the standard deviation from the mean of three independently transfected wells.