PMC8044466_01
Morphological, cellular and molecular findings in the CHS patient. (A-C) Wright stains of peripheral smear showing a neutrophil, lymphocyte and eosinophil with giant intracytoplasmic granules. (D) Bone marrow aspirate. Intense vacuolization and purple inclusions are also observed in immature cells of the myeloid linage. PB: peripheral blood; BM: bone marrow. (E) Degranulation assay was decreased compared to the healthy control both with PHA and PHA + IL2. PBMCs were incubated with or without (resting cells) PHA either alone or with IL-2 for 4 hr at 37 C. Thereafter, cells were stained with fluorochrome-conjugated anti-CD56, and anti-CD107a mAbs. CD107a surface expression was gated on CD56 cells. (F) A flow cytometry-based NK cytotoxicity assay was performed by measuring cytotoxicity against K562 cell line in a 4-hour staining with propidium iodide. The shaded area indicates normal ranges of cytotoxicity tested in healthy individuals. The patient showed a cytotoxic function on the lower range of normality. (G) Family tree and sequence analysis of the patient and his parents. Sequence patterns showing the patient's mutation c.8380dupT in homozygosis, the normal sequence of the non-carrier father and the mother's sequence showing the mutation in heterozygosis.
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Intense vacuolization, sometimes with a purple inclusion, was also seen in all cell types ( Figures 1A-D ). NK cells showed defective granule exocytosis with low expression of CD107a on stimulated NK cells ( Figure 1E ) and a slightly reduced cytotoxic capacity ( Figure 1F ) evaluated by degranulation assay. Table 2, Supplementary Figure 1A ). PCR products were then sequenced by Sanger. The genotype analysis of the patient revealed a homozygous frameshift c.8380dupT (NM_000081.3) variant in the exon 32 ( Figure 1G ) that would lead to a premature stop codon (PTC) (NP_000072.2: p.Tyr2794Leufs*8). To determine the inheritance pattern, segregation analysis was performed revealing only the mother as the single carrier of this variant, whereas the father was wild-type ( Figure 1C ).
Schematic representation of the triggering molecular mechanism of CHS in our patient. From top to bottom: the CN-LOH detected by SNP-array (Cytoscan HD, Affymetrix) in chromosome 1 (1q41q44) is delineated as a purple rectangle, the dots depict the altered markers in the array and the green and grey bands represent the 93 OMIM genes affected, including the LYST gene. Below, the LYST gene (NG_007397.1) that encodes for the entire functional protein is depicted. The c.8380dupT (NM_000081.3) frameshift mutation is localized in the exon 32. A lollipop-like image reflects the resulting truncated protein owing to a PTC at 2,800 amino acid that would remove all conserved domains (PH, BEACH and WD40).
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The effect of the variant on the encoded protein was assessed by identifying and comparing conserved domains in both the reference and the resulting mutated LYST protein with the NCBI Conserved Domain Search Tool () ( Figure 2 ). This tool revealed that the variant identified might remove all conserved domains responsible for the protein function (PH, BEACH and WD40). Whilst no CNVs were observed in the parents' DNA, a CN-LOH on the telomeric region of the long arm of chromosome 1 encompassing 25Mb (1q41q44) was detected in the child, accounting for the candidate variant in homozygosis in the LYST gene ( Figure 2 ). A time-line summary of genetic investigations is represented in  Supplementary Figure 2 .