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.)

CYTOMEGALOVIRUS (CMV) IS A HUMAN beta-herpesvirus transmitted by means of body fluids that is common throughout the world. The average age of primary infection and seroprevalence by age vary greatly according to geographic location. In some countries, primary infection usually occurs during childhood and seroprevalence is nearly 100% among teenagers, whereas in other countries primary infection can occur well into adulthood, even in the fifth or sixth decade of life, with seroprevalence reaching only 60 to 70% by the age of 60 years.¹

Primary infection may be asymptomatic but usually causes a short mononucleosis-like syndrome. Infection is lifelong, but healthy persons are generally able to maintain sufficient anti-CMV immunity to maintain continuous control of the latent virus.² In contrast, primary CMV infection in children or adults with primary T-cell immunodeficiencies can be severe and lead to manifestations such as pneumonitis, hepatitis, esophagitis, gastroenteritis, retinitis, and encephalitis.³ Similarly, in children and adults with acquired T-cell immunodeficiencies, such as human immunodeficiency virus (HIV) infection and immunosuppression related to bone marrow or solid-organ transplantation, severe CMV disease can develop,^4,5 and such persons are also prone to multiple infectious diseases. More rarely, life-threatening CMV infection occurs in previously healthy children or adults who are resistant to other infections and who have no overt immunologic anomaly.^6,7

Epidemiologic data on this phenomenon are limited mostly to case reports and small series,^6,7 with one single-center retrospective study showing a frequency of idiopathic CMV infection of approximately 1 in 50,000 hospitalized patients,⁸ which suggests that the prevalence of life-threatening CMV infection in the otherwise healthy general population is much lower, with an estimate of 1 case per million. We and others have previously found that various life-threatening viral infections in otherwise healthy patients can be caused by single-gene inborn errors of immunity.^9,10 We thus hypothesized that life-threatening CMV infection in otherwise healthy children and adults may be due to monogenic inborn errors of anti-CMV immunity that are redundant for the control of other pathogens. We tested this hypothesis by studying a 51-year-old patient with no notable medical or family history who died after 29 months of progressive CMV infection.

METHODS

STUDY OVERSIGHT

All the studies reported here were performed in accordance with institutional and municipal guidelines with oversight by institutional review boards. Approval for this study was obtained from the French Ethics Committee, the French National Agency for Medicine and Health Product Safety, INSERM in France, and the institutional review board at Rockefeller University.

CLINICAL PHENOTYPE OF THE PATIENT

A 51-year-old man who had been born to Iranian parents initially presented to a university-affiliated hospital in Tehran with a 3-month history of progressive dyspnea (Fig. 1A). (A detailed case report is provided in the Supplementary Appendix, available with the full text of this article at NEJM.org.) Computed tomography of the chest showed diffuse ground-glass opacities and septal thickening (Fig. 1B). Bronchoalveolar lavage was positive for CMV and Pneumocystis jirovecii. There were positive results on serum CMV polymerase-chain-reaction (PCR) assay (6000 copies per milliliter, with an assay sensitivity of 122.6 copies per milliliter), CMV IgM (4.2 AI [avidity index]; upper limit of normal range, 1.1), and CMV IgG (17 AI; upper limit of normal range, 1.1). Peripheral-blood flow cytometry revealed a decreased number of CD4+ T cells with a normal CD8+ T-cell count, a decreased number of B cells, and, in a later analysis, a decreased number of natural killer (NK) cells. Other blood counts were normal. These lymphopenias remained until his death.

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.

An immunodeficiency workup was otherwise negative, including negative results for antibodies against HIV and human T-lymphotropic virus types 1 and 2 and normal immunoglobulin levels. Additional viral serologic analyses showed no evidence of other acute infections, although the patient had evidence of previous infection with Epstein–Barr virus, varicella–zoster virus, and herpes simplex virus (Fig. S1A and S1B in the Supplementary Appendix). The presumptive diagnosis of primary CMV infection causing lymphopenia and secondary pneumocystis pneumonia was made. He was treated with trimethoprim–sulfamethoxazole and valganciclovir, with resolution of the pneumonia. By 10 weeks, the results were negative for CMV IgM and positive for CMV IgG, and the CMV count had decreased to 4000 copies per milliliter on PCR assay. The patient continued to receive valganciclovir (after sensitivity to ganciclovir was confirmed) but continued to have increased CMV counts on PCR assay.

Sixteen months after the initial presentation, CMV retinitis and stomatitis developed, with a serum CMV count of 8.1 million copies per milliliter on PCR; intravenous ganciclovir was initiated. Detailed immunophenotyping showed a normal level of CD8+ T cells, a decreased level of CD4+ T cells (all of which were naive [i.e., had not encountered their cognate antigens]), a decreased number of B cells (which were almost exclusively unswitched memory [IgD+IgM]), and a decreased number of NK cells (which were all immature [CD56+NKG2A+]) (Fig. S2). During the subsequent months, he had recurrent fevers and a second episode of retinitis.

Twenty-nine months after his first presentation, at the age of 54 years, he died of respiratory failure due to CMV pneumonitis, CMV encephalitis, and hemophagocytic lymphohistiocytosis. At that time, the serum CMV level was 30 million copies per milliliter on PCR assay. VirScan analysis¹¹ that was performed on plasma samples obtained at 16 months after the onset of disease and shortly before his death showed a dominant CMV humoral response and evidence of exposure to a variety of common human pathogens (Fig. S1B).

GENETIC AND IN VITRO STUDIES

We performed whole-exome sequencing on genomic DNA extracted from blood, using the Sure-SelectXT Human All Exon+UTR kit (Agilent), and pair-end sequencing on an Illumina HiSeq2500. We conducted in vitro assays by cloning open reading frames encoding NOS2 variants of interest, transfecting them into HEK293T cells, and performing Western blot analysis on cell lysates to assess protein expression and Griess assays on culture supernatants to assess nitric oxide production. Detailed methods of whole-exome sequencing, bioinformatics analysis, and in vitro assays are described in the Supplementary Appendix.

RESULTS

WHOLE-EXOME SEQUENCING

Whole-exome sequencing revealed an estimated homozygosity rate of 4.22%, which strongly suggested parental consanguinity. We thus hypothesized that a rare homozygous nonsynonymous variant could have caused the patient’s disease. Filtering of annotated variants identified five homozygous variants in five genes (Fig. S3A and S3B). Four of these variants were missense mutations in genes that are unrelated to human immunity (Fig. S3C). The fifth variant, with the highest Combined Annotation–Dependent Depletion (CADD) score and the only loss-of-function variant (i.e., frameshift, nonsense, or essential splice), was a homozygous frameshift mutation in nitric oxide synthase 2 (NOS2, c.1436_1437insT), which is predicted to lead to a premature stop codon in the oxygenase domain p.Ile391IlefsTer26 (herein called I391fs).

NOS2 encompasses 27 exons coding for a single NOS2 isoform consisting of 1153 amino acids, which catalyzes the formation of nitric oxide from arginine.¹² NOS2 messenger RNA and protein expression in humans is highest in intestinal tissue at the steady state¹³ and is expressed by human epithelial-cell lines treated with cytokines in vitro^14-16 (Fig. S4). In human disease, NOS2 expression has been found in pulmonary alveolar macrophages and granulomas obtained from patients with tuberculosis^17,18 and in macrophage-laden atherosclerotic plaques.¹⁹ In mice, Nos2 is strongly induced in macrophages under inflammatory states and has essential roles in protective immunity.¹² Although Nos2-deficient mice are best known for their vulnerability to intramacrophagic bacteria, fungi, or parasites,¹² they are also highly susceptible to CMV infection and have higher levels of CMV viremia and a higher risk of death than wild-type mice.²⁰

We confirmed that the patient did not have potential deleterious mutations in any known primary immunodeficiency genes²¹ nor in any human orthologues of murine genes associated with CMV susceptibility (Table S1), which further suggested that NOS2 was the best candidate. The NOS2 variant and familial segregation were confirmed by Sanger sequencing (Fig. 1A and 1C). None of the heterozygous family members (which included children between the ages of 22 and 38 years and a 60-year-old sister) reported any notable medical history. These findings suggested that the patient may have had autosomal recessive, complete NOS2 deficiency.

IMPAIRED FUNCTION ASSOCIATED WITH NOS2 I391FS ALLELE

The patient’s homozygous NOS2 I391fs variant is predicted to cause a frameshift mutation at isoleucine 391, leading to a synonymous change of that codon (ATC to ATT) and a premature stop after an additional 26 out-of-frame codons, truncating the protein to 417 amino acids (instead of 1153 in the wild-type protein) (Fig. 1D). To confirm the effect of this variant on NOS2 protein, NOS2 complementary DNA encoding wild-type, I391fs, or D280A (a catalytically inactive NOS2 allele²²) was overexpressed in HEK293T cells. Wild-type and D280A NOS2 produced protein at the expected molecular weight (131 kDa), whereas overexpression of NOS2 I391fs produced a protein with a decreased molecular weight, which was consistent with the predicted 47 kDa (Fig. 2A). This truncated NOS2 protein is predicted to lack the entire C-terminal reductase domain required for the formation of nitric oxide and thus be nonfunctional (Fig. 1D). Consistent with this prediction, the overexpression of wild-type NOS2 in HEK293T cells led to the production of micromolar amounts of nitric oxide, a finding similar to that in previous studies.²² No nitric oxide was detected after the overexpression of I391fs or D280A (Fig. 2B). These results showed that the patient carried a homozygous NOS2 variant encoding a truncated NOS2 protein that cannot produce nitric oxide. The patient was homozygous for this private loss-of-function NOS2 allele (i.e., not appearing in public databases) and thus had autosomal recessive, complete NOS2 deficiency.

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.

FUNCTIONAL BIALLELIC NOS2 VARIANTS IN HEALTHY CONTROLS

We first performed an analysis of the population genetics at the NOS2 locus, which showed that NOS2 is under moderately purifying selection and highly intolerant to homozygous loss-of-function variants. (Details are provided in the Results section in the Supplementary Appendix.) To prove that NOS2 deficiency is not common in the general population owing to missense mutations with an unknown effect on protein function, we characterized the expression and function of all NOS2 variants that are present or predicted to be present in homozygosity in more than 1 per million persons listed in public databases (Table S2). This group of variants included the 16 missense mutations found in homozygosity in 1 or more persons (Table S2A) and 17 other variants with a minor allele frequency of more than 0.001 that were identified in two public databases of human genetic variation: the Genome Aggregation Database (gnomAD)²³ and the Greater Middle East (GME) Variome²⁴ (Table S2B). This cutoff for minor allele frequency was used to test the heterozygous variants estimated to occur in the homozygous state in more than 1 per million persons (on the assumption of independent assortment).

When open reading frames encoding these 33 variants were overexpressed in HEK293T cells, all NOS2 variants led to detectable protein expression at levels similar to that of wild-type NOS2 (Fig. 3B). Moreover, all had intact catalytic activity and produced nitric oxide at a level similar to that of wild-type NOS2, with the exception of one mildly hypomorphic allele, A544V (Fig. 3A). This allele retained approximately 30 to 75% of wild-type function (Fig. 3C). The A544V variant is very rare (minor allele frequence, 3×10⁻⁵ in gnomAD) and was homozygous in only 1 person listed in gnomAD. The effect of this degree of residual NOS2 function on CMV susceptibility is unclear, and the clinical phenotype of the person listed in the database is unknown. These results show that autosomal recessive complete NOS2 deficiency is extremely rare and is expected to occur in the population in fewer than 1 per million persons. These experimental findings are consistent with the computational predictions of population genetics.

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.

DISCUSSION

In this study, we describe a patient with inherited complete NOS2 deficiency who died of complications from CMV infection 29 months after his initial presentation. Fatal CMV after primary infection in a previously healthy person is rare,^6-8 as is complete NOS2 deficiency in the general population. The patient’s NOS2 allele encodes a nonfunctional protein, and we identified no other candidate genetic lesions that could explain his disease. Furthermore, mice deficient in Nos2 are highly vulnerable to the related murine CMV.²⁰ Of note, neither the patient nor the Nos2-knockout mice manifested disease in the gastrointestinal tract, where Cybb (encoding gp91-phox, a phagocytic NADPH oxidase) has been shown to compensate for Nos2 loss.²⁵ Overall, our study fulfills the criteria proposed for attributing a clinical phenotype to a candidate genotype in a single patient.²⁶ We do not exclude the possibility of additional aggravating factors. There are other examples of causality between a human genotype and a specific viral infection occurring in otherwise healthy patients.^27-29

Although it is surprising that the patient was healthy until the age of 51 years, the timing of the onset of symptoms may be due to a late primary infection with CMV.¹ The patient reached the age of 54 years without any other severe infection, except a secondary pneumocystis pneumonia that was treated and did not recur. NOS2 plays a role in mouse and rat models of pneumocystis pneumonia, notably in animals depleted of CD4+ T cells,^30-33 a finding that offers a potential explanation for why this type of pneumonia was the only other infection in our patient despite confirmed exposure to many other pathogens. In contrast, mice lacking Nos2 are vulnerable to a number of viruses, bacteria, fungi, and parasites,¹² which highlights the complementary interest of studies of immunity in humans in the course of natural infections.³⁴ Nevertheless, it is possible that other NOS2-deficient patients may have other infections.

The mechanistic connection between NOS2 deficiency and the patient’s CMV phenotype will need further investigation. Whereas the function of Nos2 in murine host immunity is well characterized, that of human NOS2 has remained elusive and controversial.³⁵ In murine macrophages, the induction of Nos2 by interferon-γ leads to the production of reactive nitrogen species derived from nitric oxide, which is important for the destruction of intraphagosomal pathogens such as mycobacteria.¹² In addition, studies in mice have shown that Nos2-derived nitric oxide is a signaling molecule with important roles in regulating T-cell differentiation and effector function.³⁶ However, conditions for robust and reproducible induction of NOS2 have not been found in studies of in vitro–derived human macrophages despite clear detection of NOS2 in macrophages from patients with infectious or inflammatory disorders.^17-19 Similarly, the potential role of NOS2 in human T-cell signaling is poorly understood, as is its role in human hepatocyte, intestinal epithelial, and pulmonary epithelial cells.^14-16

We favor the hypothesis that NOS2 expression in epithelial cells that are infected by CMV is required for control of the infection.³⁷ Human NOS2 protein can be induced in epithelial cells in vitro by stimuli known to induce Nos2 expression in murine macrophages^14-16; it is likely that these stimuli are present during active CMV infection.³⁸ Moreover, exogenously added nitric oxide has antimicrobial activity against viruses including CMV,³⁹ and human NOS2 expression has shown antiviral activity against human parainfluenza virus 3.⁴⁰ It is also possible that NOS2 expression in myeloid cells is required in order to control CMV.⁴¹ An alternative hypothesis is that NOS2 mediates a T-cell signaling program that is essential for long-term T-cell homeostasis in humans, leading to the lymphopenia seen in our patient. However, it is also unknown how the patient’s CD4 lymphopenia fits in with the clinical presentation. This type of lymphopenia may be caused by NOS2 deficiency, CMV infection, or both; it is unlikely that it was caused by an independent, aggravating factor. Human NOS2 is important for protective immunity against CMV but is apparently otherwise redundant for defense against many common pathogens that infected our patient during his lifetime.