Neutropenia Through Enhanced Neutrophil Apoptosis and Secondary Necrosis in Wolfram Syndrome 1

Wolfram syndrome (WFS)(MIM #222300), a rare autosomal recessive disorder resulting from wolframin deficiency, is clinically characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) in its classical form (1). Wolframin, crucial for regulating endoplasmic reticulum (ER) stress and calcium homeostasis, is highly expressed in cells with mitochondria that have significant protein folding demands, such as pancreatic -cells and neurons, where its deficiency can induce ER stress-mediated apoptosis (2,3).By suppressing ATF6 in the unfolded protein response (UPR) (4,5), wolframin plays a vital role in cell viability.While its expression has been identified in various cells (6), other affected cell types whose roles in the disease are yet to be determined likely exist.Given neutrophils' reliance on proper protein folding for antimicrobial functions and their possession of both ER and mitochondria, they may be susceptible to ER stressinduced dysfunction.Indeed, there are certain diseases caused by ER stress-induced apoptosis in neutrophils, and furthermore, there are conditions where neutrophil apoptosis leads to neutropenia (7-9).This study investigated the impact of the WFS gene mutation on neutrophil survival and apoptosis in a patient with WFS1 who presented with unexplained chronic neutropenia.Complete blood counts (CBCs) were performed utilizing a Beckman Coulter DCH900 automated hematology analyzer to quantify blood cell populations and corroborate the observed neutropenia.Cell viability was assessed via Trypan Blue exclusion assay.Flow cytometric analysis was employed to quantify cellular apoptosis and necrosis, utilizing Annexin V/propidium iodide (PI) staining.Furthermore, the expression levels of apoptotic markers, specifically active caspase-3 and Bcl-2, were determined through flow cytometry.A 24-year-old Turkish male, born to healthy, unrelated parents, was diagnosed with isolated neutropenia at age nine.This diagnosis was made during an evaluation for the insertion of tympanostomy tubes due to serous otitis media that developed following a middle ear infection.During follow-up, the isolated neutropenia persisted (stable neutrophil count of 150-700/mm 3 ).Initial genetic and bone marrow evaluations were unremarkable: Karyotyping and the Chromosomal Breakage (DEB) assay were negative, as was Schwachman-Diamond syndrome (SDS) gene sequencing.Bone marrow examination showed no evidence of pathology in the myeloid compartment.During follow-up, he developed poor growth and diabetes mellitus (DM).Hyperglycemia was first observed in the patient at 16 years of age, progressing to insulin-dependent DM by age 24.Insulin therapy has been initiated recently.On admission, his anthropometric measurements were 70 kg for weight (3rd-10th percentile), 170 cm for height (10th percentile), and 55 cm for head circumference (25th percentile).Laboratory results showed persistent neutropenia (absolute neutrophil count [ANC] 300/mm 3 ) with a normal red blood cell picture and platelet count.Biochemical analysis revealed hyperglycemia (fasting glucose 293 mg/dL) and elevated HbA1c (10.4%); other biochemical tests were normal.Anti-neutrophil antibodies were negative.The patient declined a repeat bone marrow examination.Whole-exome sequencing subsequently identified a homozygous pathogenic variant in the WFS1 gene (c.1672C>T,p.Arg558Cys) according to the recommendations of ACGS and ClinGen guidelines (Figure 1).In silico analyses determined that the variant impairs the protein's structure and function.No other causative variants in any gene, including known cytopenia-associated genes, were detected.A family-based genetic analysis was not possible due to financial constraints.The patient's chronic neutropenia, which began in childhood, was not attributable to any underlying chronic disease, medication use, or infection.Bone marrow evaluation revealed no impairment in neutrophil production,