A 5-year-old Korean male patient who had recurrent fever (up to 39°C) with high EBV titer was transferred from an outside hospital for further evaluation of primary immunodeficiency (PID). At 3 years old, he had been hospitalized for a total of three times due to multiple episodes of mycoplasma pneumonia. At that time, his immunoglobulin G was nearly absent (10.4 mg/dl, reference range: 480–900 mg/dL for 3–5 years old). However, he had normal B cell counts (1,082 × 10⁹/L, reference range: 200–2,100 × 10⁹/L). His NK cell count (67 × 10⁹/L, reference range: 100–1,000 × 10⁹/L) was decreased, although his T cell count was normal. With a presumptive diagnosis of common variable immunodeficiency, Intravenous immunoglobulin (IVIG) treatment was started. At that time, he had visited the previous hospital due to recurrent fever and diagnosis of pneumonia and bronchiectasis. He was treated with cefotaxime for 3 days. His C-reactive protein (CRP) decreased from 5.31 to 2.2 mg/dl. He was found to have an EBV titer of 35,100 copies/ml. Regarding his family history, his uncle (older brother of his mother) died from meningitis at 3 years old (Figure 1). When admitted, his body temperature was 39.0°C, and his pulse rate was 124/min. However, his breathing sound was normal. Palpable lymph node enlargement and organomegaly (except swelling in both parotid gland sites) were not observed. He had mild leukocytosis (19,450 × 10⁹/L) with neutrophilia (13,830 × 10⁹/L) and monocytosis (1,670 × 10⁹/L). He showed a markedly increased CRP level (14.16 mg/dl). Due to continuous administration of IVIG before he was transferred, his immunoglobulin levels were within the normal range. Tests for EBV were performed, including serologic tests of EBV and quantitation of EBV. The serologic tests for anti-VCA, IgG, and anti-EBNA antibodies were positive. The EBV DNA titer measured with a Real-Q EBV Quantification Kit (BioSewoom, Seoul, Korea) was 530 IU/ml. Immunophenotyping of lymphocyte subsets and naïve/memory B and T cell subsets was performed. The results are shown in Table 1. His CD16+56+3- NK cell fraction was markedly decreased both in proportion and count (1.0%, 33 × 10⁹/L). Although the CD19 + B cell proportion and count were within the reference ranges as before, both switched/non-switched (CD10-CD27+IgD+/-) and differentiated (IgD-CD27-) memory B cells were rarely detected (2.3%, 30 × 10⁹/L) with increased IgD + CD27- naïve B cell fraction. T cells also showed a skewed distribution of CD45RA+CCR7+ naïve T cells (73.2%, 441 × 10⁹/L) with decreased CD45RO + memory T cells. Radiologic studies revealed low-density lesions in the thymus, spleen, liver, and T12 level of spine bone. Multiple cervical and mediastinal lymph node enlargement with swollen left parotid gland which needed to be distinguished between inflammatory sialadenitis and EBV-related lymphoma were observed (Table 1). The pathology of lymph nodes based on video-assisted thoracoscopic surgery revealed classic Hodgkin's lymphoma, nodular sclerosis type with positive EBV by in situ hybridization. With a diagnosis of Hodgkin's lymphoma, a combination chemotherapy of COPP-ABV including cyclophosphamide, vincristine sulfate (Oncovin), prednisone, procarbazine hydrochloride, doxorubicin hydrochloride (Adriamycin), bleomycin, and vinblastine sulfate was started at admission day 16. After five cycles of chemotherapy, the patient underwent thoracoscopic lobectomy to remove persistent pulmonary nodules suspected to be a fungal infection. The pathology findings were consistent with inflammatory nodules composed of histiocyte aggregates and some lymphocytes. He developed a headache and left side motor weakness. The brain MRI showed multifocal brain lesions suspicious of lymphoproliferative disorder or encephalitis associated with EBV infection. After four cycles of rituximab, the abnormal hypermetabolic lesions due to EBV-related LPD resolved, with EBV DNA undetectable in the cerebrospinal fluid. Currently, the patient is awaiting haploidentical peripheral blood stem cell transplantation from his father.

NGS for targeted panel of B cell and humoral immune deficiency, including 74 related genes including SH2D1A, was performed. Target enrichment with customized probes (IDT, Coralville, IA, USA) and subsequent massively parallel sequencing with NextSeq550 (Illumina, San Diego, CA, USA) were performed. The sequence reads were aligned to the reference genome, hg19, with decoy sequence using the BWA-mem algorithm implemented in BWA 0.7.17 (10). Duplicate reads were marked using Picard 2.19 (https://broadinstitute.github.io/picard/). Recalibration of base quality scores was performed using previously known sites (from dbSNP138, Mills and 1000G gold standard INDELs b37 sites, and 1000G phase1 INDELs b37) after removing duplicate reads using GenomeAnalysisTK-4.1.2.0 (GATK) (10). Variants were called using HaplotypeCaller in GATK, VarDict, and Strelka2 and annotated with ANNOVAR. All variants were interpreted based on the 2015 American College of Medical Genetics (ACMG) and Genomics and the Association for Molecular Pathology (AMP) guideline (11) and, additionally, the ClinGen Sequence Variant Interpretation Workgroup's recommendation (12).

Three missense variants in COPA, NLRC4, and DOCK8 genes, deletion/duplication, and missense variants in SH2D1A were identified. According to the 2015 ACMG-AMP guideline, the missense variants in COPA, NLRC4, and DOCK8 genes were classified as variants of uncertain significance. Variants of the COPA gene (NM_004371.3:c.3502A > G) had one moderate and support pathogenic evidences [low allele frequency (0.00119%) and a low rate of benign missense variant in this gene] but one support benign evidence [in silico analysis (REVEL 0.1)]. The variant of the NLRC4 gene showed no evidence. Only a heterozygous variant was detected in the DOCK8 gene with an autosomal recessive inheritance pattern and a low allele frequency (0.00080%). For SH2D1A, several variants including deletion/duplication and missense changes located at exon 2 were called. Called by only GATK with low depths (5-11X) but not with other callers (Vardict and Strelka2), it was assumed as a false positive calling at first. A manual review of BAM file using the Integrated Genome Browser was performed to exclude whether exon deletion was present. Visual inspection revealed soft-clipped reads which suggested a large deletion/insertion event at the 3′ of exon 2, including the flanking intron (Figure 2A). Targeted direct sequencing revealed that the variant had a deletion of 71 bp and an insertion of 16 bp across exon 2 with a flanking intron [NM_002351.4 (SH2D1A):c.162_201+31delinsTACAAGGACATATACA] (Figure 2B). Since deletion/insertion involved the splice donor site, RNA study using peripheral blood leukocytes was performed to investigate the effect of such deletion/insertion on splicing. Reverse transcription-PCR and complementary DNA sequencing were performed across exons 1–3 of SH2D1A using primers designed by the authors. Gel electrophoresis showed two aberrant mRNA transcripts. One transcript corresponded to exon 2 skipping (r.138_201del, p.Tyr47_Glu67del). The other lacked exon 2 and 55 bp of exon 3 by creating a new splice acceptor site (r.138_256del, p.Tyr47Ilefs^*17) (Figure 3). Finally, the variant could be classified as likely a pathogenic variant (PVS1, PM2). By targeted direct sequencing, it was confirmed that his mother was a heterozygous carrier, while his unaffected brother did not have this variant.