Clinical Description
The male neonate was born by cesarean section to a 19-year-old mother at 39+2 weeks of gestation. The baby weighed 4050 grams. There was neither consanguinity, nor a significant family history, and no abnormalities were detected during pregnancy. Both parents were healthy, with no recurrent infections. The birth was uneventful so he was discharged home soon after vaccination with Bacille Calmette-Guérin. He was breast fed, and his umbilical cord fell off within a week after birth. However, beginning at an age of 16 days, he suffered recurrent episodes (1–3 per day) of low to moderate grade fever (38.4℃–39.4℃), and a mild cough (four to five times every day). Physical examination on admission to the local hospital revealed no rash, jaundice, or lymphadenopathy. A complete blood count showed leukocytosis (30,390/µl; upper limit 20,000/µl) and elevated C-reactive protein levels (104.1 mg/L; normal range <10 mg/dl), and bilateral scattering opacities on chest X-ray. He was diagnosed with neonatal pneumonia and bacteremia, and prescribed cefotaxime and penicillin. However, his clinical condition worsened progressively, with persistent fever and respiratory distress; therefore, he was referred to our hospital and admitted to the neonatal intensive care unit.
Physical examination revealed tachypnea (70 breaths/min) and a fast heart rate (140 pulse-beats/min), as well as several small pustular rashes clustered on his face and forehead (Fig. 1a); these skin lesions enlarged when several scattered pustules emerged on the scalp, neck, and dorsum in the following week. Coarse breathing sounds were heard bilaterally, but the lymph nodes, spleen, and liver appeared normal. Blood analysis indicated leukocytosis and elevated C-reactive protein levels (Fig. 1b). Bone marrow aspiration revealed no findings suggestive of leukemic disorders (Fig. 1c). Levels of antibodies specific for toxoplasma gondii, rubella virus, cytomegalovirus, and herpes simplex virus (TORCH) were normal, although rough immunologic analyses indicated increased levels of immunoglobulins (Table 1). Bilateral diffuse scattering opacities were evident on chest X-ray (Fig. 1d). Further evaluations were performed to rule out sepsis; the results of which revealed sterile blood, cerebral spinal fluid, and sputum cultures, additionally, mNGS of peripheral blood was also carried out and showed negative pathogenic microorganism. Consultation with experts in neonatal infectious disease led to addition of empirical vancomycin and meropenem to broaden antibiotic coverage.
Because a low-grade fever persisted for 6 days after appropriate antibiotic treatment, we performed a computed tomography (CT) scan of the thorax, which revealed pulmonary nodules and masses scattered throughout the bilateral lung parenchyma (Fig. 1e); contrast CT revealed an inhomogeneous hyperintense soft tissue mass surrounding the aortocaval vasculature (Fig. 1f). The bilateral, diffuse, scattering nodules with halo signs, and consolidation and atelectasis in different lung areas, were suggestive of invasive pulmonary aspergillosis. Therefore, we obtained BALF samples and found an increased percentage of neutrophils in the fluid (88%; normal range, < 2%). In addition, a galactomannan (GM) assay was positive for both BALF and peripheral blood samples (Table 2). Ultimately, mNGS of the BALF identified Aspergillus fumigatus, Aspergillus terreus, and Streptococcus pneumoniae as the causative pathogens (Fig. 2). The patient was started on a course of voriconazole, which improved his conditions throughout the hospitalization period (about 2 months) (Fig. 1g). Following discharge, he received prophylactic trimethoprim-sulfamethoxazole and itraconazole.
Impaired Oxidative Burst in Phagocytes
The DHR flow cytometry assay is the standard diagnostic test for CGD as it correlates with NADPH oxidase activity, and has proved to be a rapid and sensitive screening test for CGD [28]. Because the neonate had invasive pulmonary aspergillosis and marked leukocytosis, suspicion of CGD was high. To assess the possibility of underlying CGD, we conducted an oxidative burst test, which revealed that the patient’s neutrophils showed negligible ROS generation when compared with cells from his parents (Fig. 3). Assembly of neutrophil NADPH oxidase at cell membranes is vitally important for release of ROS into the extracellular milieu, and for generation of intracellular ROS [29]. To further examine neutrophil function, we performed internal and external ROS assays. Simultaneous assay of respiratory burst activity by luminol-enhanced chemiluminescence showed a significant reduction in the neutrophil oxidase response to stimulation by a PMA, ionomycin, fMLF, or MKYMVM (Fig. 4). We also conducted quantitative determination of NETs formation, which usually relies on production of ROS from NADPH oxidase [30]; the results confirmed a marked reduction in NETs formation by the patient’s cells compared with cells from his parents (Fig. 5). Therefore, the neutrophils of this neonate showed considerable and serious defects in superoxide and NETs production, which are consistent with a diagnosis of CGD.
Gene Sequencing Identified a Novel Heterozygous CYBA Mutations
In an attempt to define the genetic cause of CGD, we performed WES, which detected a presumed homozygous variant, c.7G>T, in exon 1 of the CYBA gene, which results in a nonsense mutation (p.Q3X) [31-38]. This mutation was confirmed by Sanger sequencing; however, although such a heterozygous mutation was present in his father, it was not detected in his mother (Fig. 6a), which seemed implausible. CNV analysis detected a novel de novo heterozygous 200 kb microdeletion (chr16:88,621,282-88,839,560), which was not present in either parent (Fig. 6b). The microdeletion spanned at least eight encoding genes, including CYBA (Fig. 6c). Taken together, these findings indicate that the neonate harbored novel compound heterozygous CYBA mutations: a nonsense mutation (c.7G>T) and a novel de novo microdeletion of 200 kb at 16q24.2-q24.3. The mutations were ultimately responsible for the underlying pathogenic mechanism that inactivated NADPH oxidase.