Heterozygous OTULIN-variant related fasciitis and skin necrosis treated by TNF inhibition.

doi:10.21203/rs.3.rs-3575283/v1

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Heterozygous OTULIN-variant related fasciitis and skin necrosis treated by TNF inhibition.

https://doi.org/10.21203/rs.3.rs-3575283/v1

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published 22 Dec, 2023

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Here we describe an adult female with severe fasciitis and skin necrosis who carried a private, predicted deleterious missense mutation in OTULIN in heterozygozity. OTULIN is a cellular regulator of deubiquitination that has been shown to play a key role in intrinsic immunity against staphylococcal α-toxin. The patient was treated with broad spectrum antibiotics and multiple surgical explorations were conducted without clinical response. Since autoinflammation was the predominant clinical feature, TNF inhibition was started with a good clinical response. We show for the first time that excessive inflammation in autosomal dominant OTULIN deficiency can be effectively treated by TNF inhibition.

Otulin

Deubiquitinase

Auto-inflammation

Innate immunity

OTULIN is a deubiquitinating enzyme that plays an essential role in the regulation of inflammation by posttranslational protein modification [1-4]. Ubiquitination is the process whereby polyubiquitin chains are conjugated to intracellular proteins [5]. Depending on the type of polyubiquitin chain, ubiquitination regulates diverse cellular processes. Linear polyubiquitin chains are non-degradative and serve as a regulator of immune-related signaling. Polyubiquitination can be reversed by deubiquitinating enzymes, such as OTULIN [5]. OTULIN has specific activity towards linear ubiquitin, and is a negative regulator of NF-kB signaling [6].

In mice, a deleterious homozygous OTULIN variant leads to premature abortion. An inducible pathogenic variant shows a profound pro-inflammatory response, resulting in multiple organ failure and death within a few days [3]. In humans, few patients have been identified with bi-allelic deleterious variants in OTULIN, which causes the autosomal recessive, autoinflammatory syndrome called OTULIN-related autoinflammatory syndrome (ORAS). ORAS is characterized by fever, panniculitis, colitis, arthritis and marked neutrophilia and increased concentrations of inflammatory parameters. These symptoms occur directly after birth and are severe and potentially lethal. Although treatment with IL-1 inhibition or corticosteroids is not successful, inhibition of TNF is very effective [3, 7, 8].

Heterozygous deleterious variants, causing autosomal dominant OTULIN deficiency by means of haploinsufficiency, have recently been shown to underlie severe adult-onset skin or pulmonary necrosis, typically triggered by infections with Staphylococcus aureus [9]. Patients carrying these variants do not suffer from early-onset severe autoinflammation, have normal leukocyte counts and an intact regulation of NF-kB signaling. Instead, these patients suffer from a cell-intrinsic defect of their immunity to the staphylococcal virulence factor a-toxin in nonhematopoietic cells [9].

Here, we present a female adult patient with a new heterozygous OTULIN variant with severe sterile fasciitis and skin necrosis that was treated effectively by TNF inhibition.

This concerns a 31-year-old female with IgG2 deficiency during childhood. From the age of seven months till the age of four years she was admitted four times with pneumonia and lung abscesses. Cultures turned out negative. She received intravenous immunoglobulin supplementation until the age of nine years. After discontinuation, the complicated infections and IgG2 deficiency did not reoccur.

At the age of 31 a small garden fence fell on her right calf. The skin remained intact, but a hematoma occurred. After 17 days suddenly an increase of swelling, redness, and pain occurred (Fig 1A). The general practitioner started with oral flucloxacillin under the suspicion of cellulitis. Three days later the complaints remained, so incision and drainage were performed (Fig 1B) and the next day she was admitted to a regional hospital because of fever and deterioration of symptoms. Because of a suspicion of necrotizing fasciitis, surgical exploration was performed. During surgery, diffuse inflammation and necrosis were encountered and she was transferred to the intensive care unit (ICU) for hemodynamic and respiratory support. Cultures, taken while treated with antibiotics, remained negative. Pathology reported infiltrates with neutrophils, but no pathogens were seen. She was treated with broad-spectrum antibiotics and received intravenous immunoglobulins. In total four more surgical explorations were performed in the next week due to clinical deterioration. The cultures from these explorations also remained negative. CRP values increased above 200 mg/l and leukocytes rose to 60x10^9/l (almost exclusively neutrophils). Interestingly, also other minor trauma, such as intravenous catheter insertions, became inflamed as well; a positive pathergy phenomenon (Fig 1C). She was admitted to the ICU for a total 9 days. Under the suspicion of an autoinflammatory reaction the patient was started on corticosteroids and was transferred to our academic center. As treatment with corticosteroids was not effective, they were ceased and treatment with IL-1 receptor antagonist (Anakinra 300mg intravenously, once daily) was started. However, within 48 hours her clinical situation deteriorated again requiring ICU admission for hemodynamic support and a new surgical exploration. At that point we restarted prednisone (60mg) and added infliximab (300mg intravenously, a TNF inhibitor) instead of anakinra, after which the fever disappeared within two days, and CRP value (197 mg/L) and leukocyte counts (53.7x10^9/L) normalized within a week. She could be transferred to the ward again after two days. Vacuum-assisted closure of the wound was performed after which a splint skin graft could be placed, and the patient was discharged two weeks later after a second dose of infliximab (Fig 1D). Steroids were gradually phased out over four months, and she received two more infliximab administrations with a 4-week interval in our out-patient clinic. No new episodes of fever occurred after cessation of treatment and the skin graft and wound healed in several weeks.

2.2 Genetic analysis

Given the phenotype and severity of the symptoms, we considered an inborn error of immunity (IEI) and performed whole-exome sequencing (WES) by analyzing a panel of genes associated with IEI (474 genes) [10, 11]. This analysis revealed a private, heterozygous missense variant in OTULIN (Chr5(GRCh37):g.14690272C>T NM_138348.6:c.719C>T p.(Ala240Val)). The variant affected a highly conserved nucleotide position (PhyloP = 7.66), was predicted deleterious (REVEL = 0.27, CADD = 24.9) [12, 13], and was not reported in population databases such as gnomAD [14]. An ultrarare variant resulting in a different amino acid change at the same position (c.719C>G, p.Ala240Thr, minor allele frequency 4.0*10^-6 with a single allele count in gnomAD) is severely hypomorphic in a functional assay of NF-kB inhibition (Fig. 1E) [9]. Moreover, the patient’s variant was located in the functional ovarian tumor deubiquitinase (OTU) domain, in close proximity to inframe deleterious variants described in ORAS (p.Tyr244Cys) [8] and OTULIN haploinsufficiency (p.Asp246Val) [9] (Fig. 1E). We classified our patient’s variant (by the 5-tier classification) as likely pathogenic (class 4) according to guidelines of the Association for Clinical Genetics Science and the American College of Medical Genetics and Genomics (ACMG) [15, 16]. Segregation analysis showed that the p.(Ala240Val) variant was inherited from the mother, who was unaffected. Her mother had high titers of neutralizing antibodies toward α-toxin [9].

We describe a case of an adult female patient with a heterozygous OTULIN variant who suffered from a prolonged period of skin and fascia inflammation after a blunt trauma, and who showed a favorable clinical response to TNF inhibition.

Reflecting on this case, several points can be addressed. Genetic analysis showed a novel heterozygous OTULIN variant that we classified as a class 4 variant, being likely pathogenic, given the strong in-silico predictions and the clinical overlap with the previously described severe, necrotizing phenotype. Heterozygous deleterious OTULIN variants are typically associated with severe S. aureus-infections due to a-toxin mediated cell death, although S. aureus could not be cultured in all so far described OTULIN haploinsufficient patients [9]. Indeed, also in our patient, no pathogen was encountered despite extensive cultures, which could be due to pretreatment with flucloxacillin by the general practitioner. Nonetheless, long-term treatment with broad-spectrum antibiotics effective against S. aureus could not cease the inflammatory response in our patient either. Remarkable were also the positive pathergy reactions to the venous catheters and other minor ‘trauma’ on the other extremities. A deleterious variant in OTULIN on the other allele was considered, but specific genetic review did not show a second variant in trans. Although the patient displayed severe autoinflammation and extreme neutrophilia, this was not consistent with classical ORAS, which manifests with severe symptoms directly after birth. She experienced lung abscesses at a young age, which may have been due to autoinflammation or may have been triggered by microbes. The episodes in childhood were all treated with antibiotics and not with anti-inflammatory agents. The OTULIN variant was inherited from the unaffected mother. The absence of severe symptoms in the mother is probably due to incomplete penetrance, because the clinical penetrance of autosomal dominant OTULIN deficiency is estimated at around 30%. One driver of incomplete penetrance in OTULIN haploinsufficiency is the presence of protective, pre-existing α-toxin neutralizing antibodies, which indeed were detected in the mother [9].

In OTULIN haploinsufficient patients, blood leukocyte subsets are developmentally and functionally unaffected, and show a normal pattern of cytokine production in vitro [9]. An accumulation of linear ubiquitin is present in the patients’ primary dermal fibroblasts, but TNF-receptor dependent NF-kB signaling in these cells is intact. Their tissue inflammation and necrosis are triggered by the staphylococcal virulence factor a-toxin [9]. In this presented patient case, no S. aureus was cultured but there was a prolonged and increased inflammatory state up to several weeks after the initial trauma. This could be due to several factors. Firstly, S. aureus may have been involved in the initial inflammation, while additional factors at later time might have caused the persistent hyperinflammatory state. Especially if these factors are endogenously generated by cell necrosis, such as alarmins, an inflammatory loop could occur resulting in severe inflammation. However, no such connection between OTULIN and alarmins has been described in literature so far. Secondly, although in OTULIN haploinsufficiency no defect in TNF-receptor dependent NF-kB signaling was shown in fibroblasts under experimental inflammatory conditions [9], it could be that in a state of extreme inflammation, such as in this described patient, the gene dosage-dependent accumulation of linear ubiquitin does result in a self-amplifying inflammatory loop.

In the presented case there was a clear state of hyperinflammation with negative bacterial cultures. Initial attempts to inhibit the inflammatory response with corticosteroids and IL-1 receptor antagonist were ineffective, like in ORAS patients [4]. TNF inhibition did however induce a quick and sustained response, highlighting the apparent pivotal role of TNF in mediating the hyperinflammatory state in this patient with a heterozygous OTULIN-variant. Concluding, in patients with severe inflammation, neutrophilia, and tissue necrosis a defect in OTULIN could be the underlying cause. We now show that also in patients with a heterozygous variant, TNF inhibition should be considered to counteract the detrimental consequences of the persistent inflammatory response.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the study conception and design. All authors took part in the clinical care and diagnosis and treatment of the patient, except for Tristan van de Linden and András Spaan who did the functional analysis and measurement of a-toxin antibodies. The first draft of the manuscript was written by Rob Arts and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

Additional data generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This is an observational study, so no ethical approval is required.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that the patient provided informed consent for publication of the images in Figure 1.

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Journal Publication

published 22 Dec, 2023

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Editorial decision: Major Revisions Needed
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You are reading this latest preprint version

Heterozygous OTULIN-variant related fasciitis and skin necrosis treated by TNF inhibition.

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1.1 Introduction

2.1 Case report

3.1 Discussion

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