Clonal cytopenia of undetermined significance and atypical Behçet’s: the importance of zinc

Abstract

Atypical Behçet’s is recognised in myelodysplastic syndrome (MDS) cases and is associated with trisomy 8. Clonal cytopenia of undetermined significance (CCUS) is recognised as a precursor to MDS. Our case describes the presentation of atypical Behçet’s, in association with CCUS, post a Streptococcal infection. A mutation of a zinc finger RNA spliceosome, ZRSR2, is also described. Our patient initially presented with macrocytic anaemia, together with neutropenia and lymphocytopenia on routine monitoring. Later gastrointestinal symptoms together with oral and anal ulcerations developed. He was treated with oral zinc therapy and had resolution of recurrent oral ulcerations and significant reduction in severity of anal ulcerations. The functional impact of ZRSR2 mutation on spliceosome assembly is yet to be defined, but has been previously reported in CCUS with a clinical phenotype of macrocytic anaemia.

Background

There are several published cases of Behçet’s in myelodysplastic syndrome (MDS) patients occurring with trisomy 8.¹ Chromosome 8 includes genes for MYC (regulator and proto-oncogenes with effects on the immune system), defensins (intestinal antimicrobials), natural-killer cells transcription and zinc transporters.^2–4 Other specific mutations have not previously been described in MDS/clonal cytopenia of undetermined significance (CCUS) associated with atypical Behçet’s.

CCUS is a category of clonal haematopoiesis presenting as an unexplained, yet clinically meaningful cytopenia within haematopoietic stem cells carrying an acquired genetic mutation of at least a 2% variant allele frequency in a leukaemia-associated gene.⁵ As in MDS, resultant ineffective haematopoiesis is due to the proliferation of the mutant stem cell clone.⁵ Peripheral blood cytopenia/s manifest either due to increased apoptosis and inflammatory cytokines, or underlying autoimmune and inflammatory cell over-activity.⁶

Behçet’s is considered either an autoimmune vasculitis or autoinflammatory disorder whose cardinal features are recurrent mouth ulcerations (at least three times a year) together with at least two of the following clinical presentations: recurrent genital ulcerations; skin lesions; eye lesions; a positive pathergy test.⁷

Multisystem involvement including vascular, neurological, pulmonary, renal, cardiac and gastrointestinal disease has been described.⁸ While the aetiology remains unknown, a genetic predisposition, which includes an association with HLA-B51 is recognised.⁹ In this sub-population, subsequent exposure to a pathogen (such as Streptococcal antigens, Helicobacter pylori, herpes simplex virus or parvovirus B19), may trigger Behçet’s syndrome.¹⁰

In Behçet’s, the pathogenesis is linked to inflammatory signals including an increase in Th1 cytokines (interleukin (IL)-2, IL-12, IL-18 and interferon (IFN)-γ).¹⁰ Th17 cells (secreting IL-17) are another proinflammatory T-helper population that are implicated in the pathogenesis of Behçet’s.^{10 11} In early stage MDS, increase in T-helper subpopulations (including Th1, Th2 and Th17) are thought to mediate apoptosis of blood progenitor cells and contribute to peripheral blood cytopenia/s.¹² Therefore there may be shared pathognomic mechanisms contributing to the clinical manifestations in this case.

Case presentation

A retired man in his 60s with extensive travel history was investigated for significant neutropenia (0.2×10⁹/L–NR: 1.8–7.5×10⁹/L) after routine monitoring. He was referred to a haematologist and underwent bone marrow biopsy. This showed subtle granulocyte changes (Pelgeroid neutrophils) but was insufficient to meet the diagnostic criteria for MDS with normal cytogenetics. No treatment was indicated but the patient was monitored.

Two months later he developed severe diarrhoea and vomiting requiring hospitalisation. No infective cause was evident by stool examination (culture negative). He had raised inflammatory markers (C reactive protein (CRP) 140, erythrocyte sedimentation rate (ESR) 120) and his full blood picture showed macrocytic anaemia with monocytosis, lymphopenia but normal neutrophil count. Endoscopy and colonoscopy revealed severe pancolitis with deep serpiginous ulcers, mild ileitis, moderate proctitis and mild duodenitis (figure 1). Colon biopsies showed active ulceration and inflammation with neutrophils and eosinophils, some crypt abscesses, but no granulomas or vascultitis. These features were thought most in keeping with active inflammatory bowel disease (likely Crohn’s disease). During this period, he reported fevers (up to 38°), night sweats and large joint arthropathy. Treatment with prednisolone, Humira and sulfasalazine was commenced with initial symptomatic improvement.

Figure 1.

Colonscopic findings of serpiginous ulcerations (arrow) in transverse colon.

Three weeks later, he was readmitted to hospital due to recurrent fever, night sweats, weight loss, cough and sinus congestion. While no infective cause was found, differential diagnoses of pneumonia or sulphasalazine reaction were considered. He was treated with doxycycline and ceftriaxone. Repeat endoscopy/colonscopy was macroscopically normal and treatment with immunomodulatory agents was ceased.

Six weeks later, the patient suffered recurrent episodes of oral ulceration, fatigue, odonophagia and weight loss of 5 kg. A macrocytic anaemia was noted with raised ESR, but normal CRP and a polyclonal gammopathy. His anaemia was attributed to chronic inflammatory disorder. His fatigue, oral and peri-anal ulceration, and constipation, persisted for several years. Behçet’s was considered, but his pathergy test was normal. Treatment with prednisolone and colchicine was trialled, with no improvement. The patient remained a diagnostic dilemma following work-up for infections, vasculitis, hormonal/metabolic abnormalities and malignancies. The only positive finding was Streptococcal antibodies (Antistreptolysin O 1290 IU/mL–NR: less than 250 IU/mL). Antibiotics failed to improve his symptoms.

Approximately 1 year later, due to persistent macrocytic anaemia, moderate neutropenia and polyclonal gammopathy, a repeat bone marrow biopsy was performed. CCUS was diagnosed. Further investigations included telomere testing, Whipple’s serology, flow cytometry for paroxysmal nocturnal haemoglobulinuria and serum tryptase for mastocytosis.

Months later trialling zinc was considered due to recurrent ulcerations, resistant to steroid therapy and previous reports of successful trials in Behçet’s patients. Prior to commencement, zinc serum levels were at the lower end of normal range (9.9 µmol/L–NR: 9–19 µmol/L). Zinc therapy was prescribed at 30 mg/day, subsequently raising zinc serum levels to 13.5 µmol/L. The patient’s mouth ulcers improved significantly. He gained 4 kg due to resolved odynophagia.

Investigations

Immune biomarkers of MDS were examined for possible clinical trial involvement. As MDS was not confirmed on bone marrow assessment, the patient did not proceed with trial therapy. Results of T-helper subsets analysed with patient consent, revealed elevated levels of Th17 in response to immune stimulation (figure 2). Treatment with zinc therapy seemed to promote T-helper cell expansion, including Th17 cells, while in control MDS samples, Th17 cells are almost undetectable (figure 2A). The overall baseline inflammatory profile of this patient (figure 3) is in keeping with other MDS controls.

Figure 2.

T-helper cell populations in response to zinc therapy. Peripheral blood mononuclear cells (PBMCs) were cultured overnight and stimulated with staphylococcal endotoxin. PBMC subset were measured on on a FACS-BD flow cytometer using a customised T-cell detection panel. Transcription factors were used to enumerate various T-cell subsets (FOXP3, T-regulatory cells; T-bet, Th1 cells; GATA2, Th2 cells; RORgt, Th17 cells). (A) The ratio of T-helper subsets compared with T-regulatory cells was measured in our patient (noted by initials DA) at baseline and after exposure to zinc therapy. (B) T-regulatory cell populations (as % of total lymphocytes) did not change significant in response to zinc therapy in our patient (DA). (C) Th17 cell populations increased in response to zinc therapy (as % of total lymphocytes) in response to zinc therapy in our patient.

Figure 3.

Cytokine bead array performed on patient (noted by initials DA). Shown here are peripheral blood mononuclear cells stimulated in culture conditions. Results were measured from cultured supernatant daily on days 1–4. The result presented is a mean of the cytokine concentration across 4 days (with error bars for SD) to normalise the time to peak cytokine concentration (which is different depending on the cytokine measured). IFN, interferon; IL, interleukin; MDS, myelodysplastic syndrome.

Outcome and follow-up

Prednisolone has been successfully weaned without recurrence of aphthous ulcers, while zinc supplementation continues. Hydroxychloroquine was added at 200 mgper two times a day post commencement of zinc therapy. Recent full blood count reveals persisting macrocytic anaemia, neutropenia and mild raised ESR. He has developed a visual disturbance concerning for uveitis and ophthalmology referral has been arranged.

Discussion

This case represents a cluster of symptoms including: intestinal ulceration; recurrent oral aphthous ulcers; large joint arthropathy; polyclonal gammopathy; persistent cytopenia (with less than 10% bone marrow dysplasia); and clonal mutation of peripheral blood mononuclear cells. We surmise his condition as CCUS with atypical Behçet’s. Cytokine assays demonstrated significant elevation in Th17 cells. Zinc is known to partially modulate these pathways.

IL-17 is a proinflammatory cytokine, produced mainly by Th17 cells, and is very important for oral and intestinal mucosal defence. Cells producing IL-17 express transcription factor RORyt. Once induced by macrophages and dendritic cells, IL-17 produces growth factors (granulocyte colony stimulating factor (G-CSF) and granulocyte/macrophage colony stimulating factor (GM-CSF)) and chemokines CXCL1 and CXCL2, to cause neutrophilic migration to injury site or bacterial invasion.¹³ This, mimicks the pathergy response of Behçet’s. Th17 cells are found in active Crohn’s and Behçet’s intestinal lesions.¹⁴ IL-17 is integral in the innate response and aberrant IL-17 production is implicated in chronic inflammation and autoimmune disease. IL-17 production in the gut is essential for intestinal epithelial tight junction formation (preventing gut permeability), defensin secretion from Paneth cells and mucin production.¹⁵ Zinc, discussed later, is essential for Paneth cell function and antimicrobial peptide production. IL-17 antibodies (secukinumab, brodallumab) have been used to treat psoriasis and can exacerbate or initiate inflammatory bowel disease; probably due to their effects on permeability and defensin production; however, anti-IL-17 (seckinamab) is successful therapy for the oral lesions of Behçet’s.¹⁶

IL-17 levels may be increased in MDS patients, particularly in early stages.¹⁷ While the risk varies depending on the underlying mutational status, patients with CCUS are observed to have an increased rate of progression to MDS.¹⁸ In MDS, immune system dysregulation is thought to be a key driver of blood cell apoptosis, thereby causing peripheral blood cytopenia. Elevated ratios of Th17 cells to T-regulatory cells is a marker of immune dysfunction in early stage MDS.¹⁹ As Th17 cells mainly produce IL-17, this potentially explains our patient’s neutropenia and gastrointestinal inflammatory manifestations. Of note, TET2 deficiency also influences the differentiation of T-helper cells and T regulatory cells.²⁰ A TET2 mutation (frameshift deletion) was present in our patient; however, the implications of this are unclear.

Zinc is an essential trace element required for the function of over 300 enzymes and cellular transcription factors.²¹ Zinc deficiency states cause immune deficiency of both the adaptative and innate arms, reduced neutrophil function, hypogonadism, growth retardation and a distinctive dermatological condition—acrodermatitis enteropathica.²² Zinc effects on the immune system are multiple: reduced Th1 cell function (decrease in IFN-γ and IL-2); increase in Th2 response (increase in IL-4 and IL-10); reduction in T-reg numbers; and increase in Th17 response (increase in IL-17).²² These changes increase infective risk, autoimmune and allergic disease propensity.²³ A randomised double blind control trial of zinc supplementation significantly reduced the clinical manifestations of Behçet’s.²⁴ In a large cohort study, reduced zinc intake was inversely associated with Crohn’s disease risk and severity.²⁵ Zinc has a protective effect on the colon reducing diarrhoea and ulceration via zinc G coupled protein receptor.²⁶ It also stimulates colonocyte proliferation and growth.²⁶ It is important to note that zinc supplementation can have some serious consequences, such as myelodysplasia and myelopathy due to acquired copper deficiency.^27–29 Serum copper should be monitored if long term therapy is considered.³⁰

Spliceosome mutations are a common driver mutation in MDS.³¹ The ZRSR2 gene mutation on the X chromosome encodes a spliceosome whose function is to remove introns from mRNA.³² This only affects males. The mutation is a premature stop codon making the protein non-functional. A number of spliceosome proteins contain zinc finger motifs, interacting with ionic zinc. Chemicals that chelate zinc will inhibit spliceosome function.³³ Our patient’s ZRSR2 mutation encoded for a zinc finger protein (figure 4). While zinc supplementation remains unstudied in disease or animal model, it may have altered the immune response inducing oral ulceration and IL-17 production.

Figure 4.

Sanger sequencing of patient’s ZRSR2 variant. The black arrow highlights the point mutation (ATG)—creating a premature stop codon and affecting the encoding for a zinc finger protein.

Patient’s perspective.

Having initially been hospitalised on the basis of suspected MDS, the diagnosis of ‘severe’ Crohn’s disease instead emerged. I was started on Humira. At the time of my second hospitalisation, all signs of my intestinal ulcerations had disappeared, to the apparent surprise of my gastroenterologist, putting a question mark over the Crohn’s diagnosis.

However, some quite debilitating symptoms persisted. Principally these were ulcers of my mouth, throat and anal regions; varying frequently in intensity from time to time. My dentist also diagnosed necrotic gum disease, which was despatched successfully by laser therapy.

After extensive testing which yielded no definitive conclusions, Dr Prentice recommended I take extra zinc, despite my blood tests never indicating a deficiency. The effect was immediate. I have not experienced a sore throat or mouth ulcers since that time. Problems with my anal region remain substantially reduced. The results of my regular blood tests are essentially stable, showing only minor variations with no patterns of concern.

For me, it seems the extra zinc has been a game-changer.

Learning points.

• Autoimmune and autoinflammatory diseases occur with clonal haematopoietic syndromes and may precede the diagnosis.

• Behçet’s may be associated with myelodysplastic syndrome (MDS) and trisomy 8.

• Clonal cytopenia of undetermined significance may be a precursor to MDS and is common.

• Zinc is an important essential micronutrient with effects on the immune system and gastrointestinal tract.

• Spliceosome mutations occur commonly in MDS.

Ethics statements

Patient consent for publication

Consent obtained directly from patient(s)