Clinical manifestations
The first 4-month-old male proband (P1) with X-HIMG (Fig. 1A) was the first child in a non-consanguineous healthy family and delivered by cesarean section at full-term with a birth weight of 3800g. Apgar score for him was 10 scores at 1, 5 and 10 min after birth. This patient presented recurrent shortness of breath, hoarseness and cough for a month without apparent inducement. He was admitted to hospital on March 10, 2019, after ineffective treatment with Amoxicillin and Clavulanate Potassium for 4 days. Physical examinations: shortness of breath with 38 breaths/min, dyspnea with positive tri-concave sign, slightly perioral cyanosis, hoarseness, cough, body temperature 37.1 degrees, heart rate 152 beats/min, and blood pressure 84/55 mmHg. The oxygen saturation rate (SpO2) was about 78–85 percent without oxygen inhalation. During the emergency observation, the patient developed increased cyanosis and was given mask oxygen inhalation mixed with nebulized budesonide, intravenous infusion of erythromycin and methylprednisolone. The peripheral blood examination showed a high level of white blood cell (18.32*109/L, reference value: 5–12*109/L), and the chest CT (Computed Tomography, CT) indicated pneumonia. Subsequent laboratory examinations: the serum IgG < 1.360 g/L (Reference value: 1.39–9.34 g/L), IgA 0.04 g/L (Reference value: 0.03–0.78 g/L), IgE 16.70 IU/mL (Reference value: 0–60 IU/mL), IgM 0.23 g/L (Reference value: 0.04–1.2 g/L); the serum Cytomegalovirus DNA (HCMV-DNA) test 4.31E + 02 copies/mL, the bronchoalveolar lavage fluid of HCMV-DNA 2.25E + 05 copies/mL. Mycobacterium tuberculosis test was negative. Based on the clinical and laboratory findings, the preliminary diagnoses were severe pneumonia, acute respiratory failure and hypogammaglobulinemia. During hospitalization, the proband received Beta-lactam antibiotics including cephalosporin antibiotics, meropenem, and Piperacillin Sodium and Tazobactam Sodium against bacterial infections, fluconazole against fungal infections, erythromycin against atypical pathogens, and ganciclovir against HCMV infection. Other supportive therapies included mask oxygen inhalation, methylprednisolone for anti-inflammatory treatment and intravenous immunoglobulin (IVIg). The patient was discharged from the hospital when he got better.
The second male proband (P2) of HIES (Fig. 1B) was hospitalized two hours after delivered by cesarean section at 38 weeks with a birth weight of 3200g. Apgar score was 7 scores (Respiration, Appearance, Activity, -1) at 1 minute without intrauterine fetal anoxia and premature rupture of membrane, 8 and 9 scores at 5 and 10 min with positive-pressure ventilation. The patient presented perioral cyanosis, shortness of breath with 55 breaths/min, groaning, and not crying. Physical examinations: body temperature 36.4 degrees, heart rate 110 beats/min, blood pressure 63/30 mmHg, and poor responses. Thick respiratory and wet rales were audible in both lungs and the fetal SpO2 was about 85 percent without oxygen inhalation. The cranial MR and chest CT examination separately indicated subarachnoid hemorrhage and pneumonia. In addition, the cornea of his both eyes were milky white and the pupils were invisible. The following ophthalmology ultrasonography described obvious corneal edema and thickening, narrow atrial angle in all directions and short axial diameter. The patient was diagnosed with congenital microphthalmia, congenital leucoma, and secondary glaucoma by ophthalmologist. He soon developed convulsion with regular shaking and increased muscle tension and was given phenobarbital for sedation and spasmolysis. His condition deteriorated rapidly with the treatment of oxygen inhalation, intravenous IVIg and cefotaxime anti-infections, manifesting as poor response, irregular breath and pale skin. Blood gas analysis indicated metabolic acidosis and hyperkalemia. Echocardiography showed a decreased left ventricular systolic function (the ejection fraction was 28%), which indicated a heart failure of the patient. His parents gave up continuing treatment and signed for discharge due to his severe symptoms and little hope of survival. The proband was the second child in this non-consanguineous healthy family, in which the first child was a female infant and died of asphyxia neonatorum at 7 days after delivery. However, WES analysis was not performed in the first child.
Genetic analysis
WES analysis revealed a novel hemizygous mutation in CD40L (c.257delA or deletion of adenine at nucleotide position 257) in P1. This frame shift mutation resulted in the substitution of glycine for glutamic acid at 86 codon of the protein causing the early termination of translation at downstream codon 9 (p.E86Gfs*9). Sanger sequencing was then utilized to verify the detected variant in CD40L by WES analysis (Fig. 2A) and showed that the hemizygous c.257delA mutation was inherited from his mother. Besides, this deletion mutation was not found in 100 irrelevant healthy people and was likely responsible for the X-HIGM phenotype.
We identified the compound heterozygous mutations in DOCK8, one in exon 14, c.1546C > G (substitution of cytosine for guanine at nucleotide 1546), resulting in amino acid change from proline to alanine at position 516 (p.P516A), another in exon 41, c.5355 + 6C > T(splicing) resulting in splicing change of amino acid. Both mutations were not described in HIES before. The c.1546C > G variant was predicted to be benign by PolyPhen-2 (score 0.082) but was predicted to be deleterious by PROVEAN (score − 5.668) and Mutation Taster (score 27). The c.5355 + 6C > T(splicing) variant was predicted by MaxEntScan, Spliceman and ASSP. MaxEntScan showed Maximum Entropy Model (MAXENT) 6.77, Maximum Dependence Decomposition Model (MDD) 10.48, First-order Markov Model (MM) 5.24, Weight Matrix Model (WMM) 6.50, comparatively, with scores of 7.58, 10.28, 5.31 and 5.27 in wild type. Spliceman suggested a ranking of 79% and ASSP suggested a score of 10.161 (Donor site cutoff: 4.5). The compound heterozygous mutations were independently inherited from his patents, both of which were not found in 100 irrelevant healthy people. His father was heterozygote of c.1546C > G mutation and his mother was heterozygote of c.5355 + 6C > T (splicing) mutation (Fig. 2B, C). Therefore, the co-segregation analysis in this family indicated that the compound heterozygous mutations were likely pathogenic in HIES phenotype.
Bioinformatic analysis of CD40L and DOCK8 mutations
The CD40L and protein sequences of different species, including Homo sapiens, Rattus norvegicus, Mus musculus, Canis lupus familiaris, Felis catus, Sus scrofa and Macaca mulatta, were obtained from the National Center for Biotechnology Information (NCBI) website. Multiple sequence alignment among these species were compared using DNAMAN software, demonstrating that the variant -p.E86- was located at a highly conserved sequence of CD40L (Fig. 3A). The DOCK8 and protein sequences were searched in Homo sapiens, Mus musculus species, Canis lupus familiaris, Macaca mulatta and Rattus norvegicus. The result indicated that the variant p.P516A was located at a highly conserved sequence of DOCK8 (Fig. 3B).