Case Report: Clinical application of an in vitro prenylation assay in the diagnosis of an early-onset case of mevalonate kinase deficiency harbouring a novel MVK variant

Mevalonate kinase deficiency (MKD) is a systemic autoinflammatory disease caused by biallelic mutations in MVK. Individuals with MKD present with a recurrent fever syndrome, often including a skin rash, gastrointestinal symptoms and lymphadenopathy. The severity depends on the residual enzyme activity, which can be measured using an assay to confirm diagnoses in cases with non-confirmatory/novel MVK genotype. However, the assay is not widely available and utilises radioisotope, limiting its use in routine clinical care. More recently, the accumulation of unprenylated Rab GTPases in peripheral blood mononuclear cells, a downstream consequence of mevalonate kinase deficiency, has been described as an alternative diagnostic biomarker of MKD. We describe the utility of the Rab prenylation assay for the diagnostic workup of an infant with a novel MVK genotype presenting with fulminant autoinflammation. A seven-week-old girl, born to non-consanguineous White British parents, presented with features of haemophagocytic lymphohistiocytosis (HLH): fever, hepatosplenomegaly, bicytopenia, hyperferritinaemia, transaminitis, raised lactate dehydrogenase and C-reactive protein. She had a three-week history of fever and a generalised erythematous rash, with negative infectious work-up. Gene panel sequencing revealed biallelic trans MVK variants: MVK: c.151C > T (p.L51P), a previously described pathogenic variant; and MVK: c.1027C > T (p.L343P), a novel variant. A mevalonate kinase enzyme activity assay, requested via the reference laboratory in Amsterdam, confirmed 3% residual activity in the patient, consistent with MKD. However, this test result took 21 days to return, therefore a prenylation assay was performed in the meantime, revealing clear accumulation of Rab proteins in a blood sample from the patient, thus confirming pathogenicity of the variants and securing the diagnosis of MKD. The turnaround time of this assay was 2 days. We demonstrate the use of a protein prenylation assay in the diagnosis of a very early-onset case of MKD presenting with HLH, with a novel MVK genotype. This assay is quicker and simpler to set up in routine clinical care than measurement of mevalonate kinase activity, the current gold standard for MKD diagnosis. This case demonstrates the clinical utility of the prenylation assay for specific and timely MKD diagnosis and expands the genotypic spectrum of MKD.

Introduction

Systemic autoinflammatory diseases (SAIDs) are a family of disorders defined by uncontrolled activation of the innate immune system, leading to systemic inflammatory episodes. Mevalonate kinase deficiency (MKD) is a monogenic SAID caused by biallelic mutations in MVK, which encodes a crucial metabolic enzyme of the mevalonate pathway. MKD encompasses a clinical spectrum; the milder form (also previously known as Hyper-IgD and periodic fever syndrome, HIDS) is an early-onset recurrent fever syndrome, often accompanied by skin rash, gastrointestinal symptoms, hepatosplenomegaly and lymphadenopathy (1). A more severe form, mevalonic aciduria, additionally includes failure to thrive, developmental delay, facial dysmorphia and neurological symptoms. The severity of clinical features depends on the level of residual mevalonate kinase (MK) enzyme activity, which is determined by the particular combination of biallelic MVK variants. Mevalonic aciduria (MA) is associated with lowest residual activity (<0.5% of normal), while higher residual activity (0.5%–20% of normal) is associated with the periodic fever phenotype (2–4). Measurement of MK enzyme activity (5) is therefore particularly helpful to confirm MKD diagnoses in cases with a non-confirmatory/novel MVK genotype. However, this test is not widely available. Currently, the only reference laboratory offering an MK enzyme assay is in Amsterdam, Netherlands; the turnaround time is 3–6 weeks, and the assay requires shipment of a fresh blood sample. It is unlikely that this test will ever become available in UK routine clinical care since the assay utilises radioisotope, which is heavily restricted in clinical laboratory settings.

The build-up of unprenylated Rab GTPase proteins has been described as an alternative diagnostic biomarker of MKD (6, 7). MK is an enzyme in the mevalonate pathway, required for the synthesis of isoprenoid lipids that are essential for the post-translational prenylation and hence membrane localisation of numerous proteins, including Rab GTPases (8). In MKD, insufficient MK activity reduces the metabolic flux through this pathway, resulting in lack of isoprenoid lipid biosynthesis and thus defective prenylation of small GTPases including Rab, Rho and Rac proteins (9–11). Loss of membrane-bound prenylated proteins, or the accumulation of dysfunctional unprenylated proteins in the cytosol, appears to enhance activation of the pyrin and/or NLRP3 inflammasome and cause hypersecretion of IL-1β (12, 13). Defective protein prenylation is also recapitulated in genetic mouse models of MKD (14) and THP-1 cell line models (15). We have optimised a biochemical assay that allows detection of unprenylated Rab proteins in cells, by enabling in vitro prenylation of Rab proteins in cell lysates using recombinant prenyl transferase and a biotinylated isoprenoid lipid substrate (16). Normally, unprenylated Rab GTPases are barely detectable in cell lysates. Hence, the intracellular accumulation of unprenylated Rab GTPases is highly indicative of MKD, distinguishing it from other IL-1β-mediated diseases such as Cryopyrin-associated periodic syndromes (CAPS) and Familial Mediterranean fever (FMF) (6, 7) that are caused by variants in NLRP3 or MEFV, respectively. In theory, the prenylation assay could also help diagnose other genetic conditions in which disruption of the mevalonate pathway affects protein prenylation, for example in phosphomevalonate kinase deficiency caused by autosomal recessive mutations in the PMVK gene (17–19). Unlike the radioactive MK enzyme activity assay, the Rab prenylation assay does not require the use of radioactive reagents, and has a turnaround time of only two days, making it much more accessible in a routine clinical care setting.

Here, we describe the utility of the in vitro Rab prenylation assay in the diagnostic workup of an infant presenting with fulminant autoinflammation, with a non-confirmatory genotype suggesting possible MKD.

Methods

Ethical approval and consent

Full ethical approval for this study was given by the National Research Ethics Service, (ethics numbers 24/EM/0068 and 11/LO/0330)). Informed written consent/assent was obtained for the patient and her parents.

Genetic testing

Patient genetic screening was performed through the UK National Health Service (NHS) using a large, targeted panel for primary immune deficiency (https://panelapp.genomicsengland.co.uk/panels/398/). For Sanger sequencing confirmation, PBMCs were extracted from patient and parental blood by Lymphoprep separation, and then DNA isolated using Gentra Puregene Blood Kit (Qiagen). Variants were confirmed by PCR and Sanger sequencing using the following primers for MVK exon 3 forward: 5′-CACTGGCTGTATCCTTGAACT-3′ and reverse: 5′-TCCTTGCACTCACCCAGAAA-3′ and MVK exon 10 forward: 5′-ACCATCTGAATGCCCTCGG-3′ and reverse: 5′-GGAGCTGAGACCTGTTTCCA-3′.

MVK enzyme activity assay

Fresh patient blood was shipped to Amsterdam UMC laboratory, Amsterdam (https://www.amc.nl), where the standard MK enzyme activity assay was performed as previously described (5) in fresh patient lymphocyte lysates using the isotope R,S-[2-14C]-mevalonic acid and known Rf values of mevalonolactone, mevalonate 5-monophosphate and mevalonate 5-pyrophosphate.

In vitro Rab prenylation assay

PBMCs were isolated from patient, parent, and healthy control blood by Lymphoprep separation, and stored in liquid nitrogen. The prenylation assay was performed as previously described (10, 14, 16). Briefly, cell lysates were generated from cryopreserved PBMCs by sonication in buffer (50 mM HEPES, 50 mM NaCl, 2 mM MgCl₂, 100 μM GDP, protease inhibitor cocktail) and protein was quantified using a Pierce Rapid Gold BCA Protein Assay (Thermo Fisher Scientific). Unprenylated Rab proteins in cell lysates were prenylated in an in vitro reaction by incubating 10 mg protein from the lysates with 2 μM recombinant Rab geranylgeranyltransferase (GGTase II, Jena Bioscience), 2 μM Rab escort protein (Jena Bioscience), 0.5 μM biotin-tagged geranyl diphosphate (Jena Bioscience) and 2 mM DTT for 5 h at room temperature. Samples were then combined with Laemmli buffer supplemented with 10% beta-mercaptoethanol, boiled for 5 min at 95 °C, and separated by SDS-PAGE. Proteins were transferred to a PVDF membrane, washed with TBS-Tween (0.1%), blocked with 3% BSA, and incubated with streptavidin-HRP (Abcam) also in 3% BSA. Signal was detected using Amersham ECL Western Blotting Detection Reagent (Cytiva). Images were taken using a ChemiDoc Imager (Bio-Rad) and analysed using Image Lab software (Bio-Rad).

Case presentation

A seven-week-old girl, born at term via elective caesarean section to non-consanguineous white British parents from an uneventful IVF pregnancy, presented to the local hospital with features suggestive of haemophagocytic lymphohistiocytosis (HLH): fever, hepatosplenomegaly, bicytopenia (Haemoglobin (Hb):53 g/L (normal range:100–135 g/L), platelets:20 × 10*9/L (normal range:150–450 × 10*9/L), white cell count: 22.9 × 10*9 g/L (normal range: 6.00–18.00 × 10*9/L), lymphocytes:14.6 × 10*9 g/L (normal range:3.00–13.50 × 10*9 g/L), neutrophils:5.1 × 10*9 g/L (normal range:1.00–8.50 × 10*9 g/L), hyperferritinaemia (6,000 ug/L, normal range:9.5–75.7 ug/L), transaminitis (ALT: 690 U/L, normal range:12–47 U/L), raised lactate dehydrogenase (LDH) (911 U/L, normal range: 163–452 U/L) and a raised C-reactive protein (CRP) (60 mg/L, normal range: 0–5 mg/L). Parents reported illness onset at 4 weeks of life with fever and a generalised non-blanching erythematous rash (Figure 1A). During the first hospital admission with fever of unknown origin, she was extensively investigated for potential causes of sepsis and infectious screening was negative, including blood, urine, and cerebrospinal fluid (CSF) cultures and serum viral PCRs for Epstein–Barr virus (EBV), cytomegalovirus (CMV), adenovirus, enterovirus, varicella zoster virus (VZV) and parvovirus. CMV and toxoplasma gondii IgM were negative, IgGs were positive reflecting placental transmission. CSF viral PCRs for herpes simplex virus (HSV) 1 and 2, adenovirus, parechovirus, VZV and enterovirus were negative. Viral respiratory nasopharyngeal swab was negative. Chest x-ray and echocardiogram were normal. Maternal TORCH screening was negative. There was no other family history of note. The patient received 10 days of intravenous antibiotics (ceftriaxone and acyclovir) with complete resolution of rash, and she was discharged on oral acyclovir. Acyclovir was stopped following negative serum HSV PCRs.

Figure 1

Figure 1. A very early onset case of mevalonate kinase deficiency (MKD) harbouring a novel MVK genotype. HC, healthy control; WT, wildtype. (A) Widespread erythematous, non-blanching rash at week 7 of age. (B) Sanger sequencing traces showing c.151C > T and c.1027C > T variants in the patient (II-1) and both parents (I-1 and I-2). (C) Pedigree and familial segregation of the MVK alleles with the affected individual marked in black and unaffected in white. (D) Following an in vitro prenylation reaction, cell lysate of PBMCs from the patient (II-1) shows clear accumulation of unprenylated Rab GTPases compared to two healthy controls and both heterozygous parents. The positive control (lane 1) shows THP-1 monocytic cells treated for 24 h with 0.5 mM simvastatin. (E) Densitometry analysis of the unprenylated Rab GTPase bands shows an increase in the presence of unprenylated Rab proteins in the patient PBMCs (II-1) compared with either healthy control (5.4-fold increase) or parent carrier (4.2-fold increase) PBMCs.

At her second hospital admission with fever, poor feeding and a new skin rash that started as small erythematous papules and rapidly progressed to target lesions that coalesced on extremities, trunk, and face, she was found to be anaemic with Hb of 53 g/L and platelets were low at 20 × 10*9/L. She also was noted to have new hepatosplenomegaly, oedema, deranged liver functions and clotting with low fibrinogen (<1,5 g/L (normal: 1,7−4,0 g/L) requiring repeated doses of Vitamin K. She was covered with empirical IV antibiotics (ceftriaxone and gentamicin), and repeated blood and CSF cultures were negative. Blood film showed leucoerythroblasts, polychromasia, left shift and blast cells. Magnetic resonance imaging of the brain was normal. Ultrasound abdomen showed hepatosplenomegaly. Both kidneys looked normal and there was no proteinuria. Extended respiratory viral nasopharyngeal aspirate was negative. She remained transfusion dependent for red blood cells and platelets with no clear underlying diagnosis and was thus transferred to Great Ormond Street Hospital (GOSH) under the paediatric haematology team for diagnostic work-up and further management. On her transfer to GOSH, differential diagnoses included infections, malignancies, primary HLH, primary immunodeficiencies, and genetic causes of bone marrow suppression. Immunology, infectious diseases, haematology, and oncology teams were involved in the multidisciplinary diagnostic work-up. Perforin expression and CD107a granule release assays were normal (data not shown). Immunoglobulins (Ig)G and A were mildly raised [IgG: 9.81 g/L (2.10-7.70 g/L), IgA: 1.66 g/L (0.05-0.40 g/L)], and IgM was normal. Lymphocyte subsets with memory panel showed normal lymphocyte distribution and T cell types were also normal. Soluble CD25 was elevated at 15,613 pg/mL (normal range: 0–2,500 pg/mL); however, lymphocyte subset activation assay demonstrated normal CD25 expression patterns, indicating increased immune activation rather than an intrinsic defect in CD25 activation on T cells (data not shown).

Ultrasound of the abdomen showed a bulky liver with increased periportal echogenicity. There was no intrahepatic lesion with normal directional flow observed in the portal vein and hepatic veins. The gallbladder wall appeared thickened and oedematous with increased vascularity. Spleen was enlarged at 7.5 cm with the upper limits for age of 6 cm. There was moderate volume of anechoic free fluid, bilateral external iliac lymph nodes, largest in the left measuring 8 mm in short axis. A repeat chest x-ray was normal. Repeat echocardiogram confirmed normal intracardiac anatomy.

Bone marrow aspiration showed no phenotypic evidence of a haematological malignancy and no histopathological evidence of haemophagocytosis. Peripheral blood flow cytometry was normal. Metabolic investigations were normal apart from the urine organic acid test which showed presence of mevalonolactone. Blood electron microscopy demonstrated no ultrastructural indication of a storage disease. Extensive infectious work-up at GOSH showed a positive CMV IgM, positive CMV IgG and a positive serum CMV PCR with a viral load of 146,177 copies/mL whole blood. Other serum viral PCRs were all negative, including parvovirus, HIV, HHV-6, EBV, HSV 1 and 2, parechovirus, VZV, enterovirus and hepatitis A and C. Leishmania serology and culture on the bone marrow aspirate were negative. Although positive CMV PCR and CMV IgM were explained with postnatally acquired infection, the decision was to start treatment with an antiviral medication (valganciclovir) since CMV can be a potential trigger of an inflammatory process and HLH. Ophthalmology assessment for CMV retinitis and audiology test were both normal. There were no clinical signs of congenital CMV infection. After initiating antiviral treatment, CMV viral load started to reduce with improvement in blood counts (Hb and platelets) allowing her to be transfusion-independent and HLH parameters including ferritin, liver enzymes, fibrinogen and LDH normalised. Patient was discharged with pending genetic testing and oral valganciclovir treatment was stopped after a two-week course. Despite resolution of clinical symptoms and stability of blood counts, CRP remained mildly elevated around 25 mg/L (normal:0–20 mg/L).

Due to very early presentation with HLH features, the NHS England genetic panel for “Primary immunodeficiency disorders” was requested. This revealed biallelic MVK variants: MVK: NM_000431: c.151C > T (p.L51P), a rare variant previously observed in MKD (20, 21); and MVK: NM_000431: 1027C > T (p.L343P), a novel variant. The variants were inherited biparentally (in trans), as confirmed by parental Sanger sequencing (Figures 1B,C). At this stage, the patient was initiated on treatment with anti-interleukin 1 (IL1), anakinra administered as daily subcutaneous injections at a dose of 3 mg/kg/day. This led to the complete resolution of symptoms, normalisation of blood counts, including an increase in Hb from 86 g/L to 101 g/L (normal:100–135 g/L), a rapid reduction in CRP levels from 40 mg/L to 5 mg/L over 14 days, and normalisation of ferritin from 1,190 ug/L to 46.7 ug/L (reference range 9.5–75.7 ug/L). At the age of 9 months, she was converted from anakinra to canakinumab (4 mg/kg monthly) and remains well in both clinical and serological remission on this treatment at the time of writing, now 17 months of age.

To investigate the pathogenicity of the patient's variants, an MK enzyme activity assay (5) was requested via a reference laboratory in Amsterdam, since the test is not currently available in the UK. To the best of our knowledge, this is the only laboratory offering this test in Europe. After 21 days the results were returned, confirming 3% residual enzyme activity [level: 6 pmol/(min.mg protein), reference range: 125–395] in the patient, consistent with MKD.

The enzyme activity test result took 21 days to return, therefore an in vitro Rab prenylation assay (6) was performed in the meantime. This revealed a clear accumulation of unprenylated Rab GTPases in the patient PBMCs, compared with both healthy controls (5.4-fold increase) and carrier parents (4.2-fold increase) (Figures 1D,E). This result was consistent with previous observations for MKD patients (6, 7), confirming the pathogenicity of both variants MVK:c.151C > T (p.L51P) and MVK:1027C > T (p.L343P), and securing the diagnosis of MKD. The turnaround time of this assay was two days.

Discussion and conclusions

We describe a case of early-onset MKD harbouring compound heterozygous MVK variants, one of which was previously described in a single symptomatic case (p.L51P), and the other novel (p.L343P). Our patient first presented at 4 weeks of life with fever and skin rash and then developed features of HLH at week 7, remained transfusion-dependent which led to her transfer to a quaternary centre and extensive investigations including genetic testing. To verify the pathogenicity of the two MVK variants, two functional assays were performed on PBMC samples—analysis of residual MK enzyme activity and accumulation of unprenylated Rab GTPase proteins. The results of both tests were consistent with MKD, confirming the pathogenicity of both variants; however, the Rab prenylation assay had a turnaround time of 2 days when performed in-house, compared with 21 days for the outsourced MK enzyme activity assay.

The development of clinically relevant functional tests to confirm genetic diagnoses has been relatively slow in comparison to advances in molecular genetics. This is particularly pertinent in the field of SAIDs, where many new diseases continue to be described, outpacing routine clinical laboratory functional assay development. Although MKD is well described and common pathogenic MVK mutations such as p.V377I are well characterised and highly prevalent in MKD patients [128/144 (86.0%) harbouring at least one copy in the largest cohort described (1)], some patients still present with compelling clinical phenotypes but non-confirmatory genotypes, mandating confirmation by measurement of residual MK enzyme activity. Despite this, availability of the MK enzyme activity assay is extremely limited, costly (when shipping costs of fresh samples are included), and time-consuming (3–6 weeks turnaround time). Simpler, cheaper and more rapid assays to confirm MKD, such as the in vitro Rab prenylation assay (6, 7, 10), are therefore needed.

Our case illustrates this point well, since she harboured one copy of an entirely novel MVK variant (p.L343P) combined with another known pathogenic variant (p.L51P), inherited biparentally. Our patient experienced severe gastrointestinal involvement with slow weight gain and vomiting since the start of her symptoms, and continuous rather than “periodic” autoinflammation, possibly explained by the relatively severe combination of MVK alleles. Audiological and ophthalmological evaluations of the patient were within normal limits, and neurodevelopmental milestones were appropriate for her age. There were no dysmorphic features suggestive of mevalonic aciduria, hence the patient was discharged from the care of the metabolic team. Ter Haar et al. found that absence of the p.V377I genotype had significant correlation with continuous course of disease, severe gastrointestinal involvement, and musculoskeletal involvement. Our patient's autoinflammatory phenotype suggested that neither the p.L51P or p.L343P variants have a severe enough impact on the protein structure to cause mevalonic aciduria (MA), consistent with both variants being outside “hotspot” regions of the enzyme associated with MA (residues 8–35 and 234–338) (22). However, the patient did show features consistent with HLH, which is a severe and unusual feature in MKD (23–25). Our patient did not have any primary HLH or primary immunodeficiency-related gene mutations, and we conclude that HLH in this instance was driven by MKD which can be associated with HLH with or without infectious triggers.

As we demonstrate in this case report, the MK enzyme activity assay and the Rab prenylation assay are both helpful in the diagnosis of MKD. Whilst reduced MK enzyme activity is diagnostic of MKD, the prenylation assay may theoretically detect insufficiency in other stages of the same metabolic pathway, for example in cases of PMVK deficiency (17–19). Whilst the gold standard MK enzyme activity has many strengths, there are also many practical limitations (Table 1). Most importantly, the MK enzyme activity assay is rather impractical for routine care, since it requires shipping of fresh sample to Amsterdam with a turnaround time of several weeks. It is unlikely that this assay will ever become routinely available in the UK since the use of radioisotopes is heavily restricted in routine clinical laboratory settings. In severe and life-threatening presentations with HLH features and persistent autoinflammation, as observed in our case, starting treatment promptly before the MK enzyme activity result becomes available may be critical in controlling inflammation and preventing devastating outcomes, particularly when MKD is the highly suspected diagnosis.

Table 1

Table 1. Strengths and limitations of mevalonate kinase enzyme and Rab prenylation assays for routine diagnostic workup of suspected mevalonate kinase deficiency (MKD).

Another alternative assay used in the workup of MKD is the detection of mevalonic acid in urine samples. Although this may be of great importance to support a diagnosis, methodology influences the sensitivity and specificity of this test, and the timing of the assay in relation to disease activity is important. Urinary mevalonic acid levels are highly informative during symptomatic periods of MKD, with high sensitivity and specificity (26). This test, especially when using advanced techniques such as gas chromatography and mass spectrometry (which was the method used in this case) can provide a more specific indication of MKD. Overall, however, this test is a basic screening tool when genetic testing is unavailable, and often reported in a qualitative rather than a quantitative fashion, as in our case. Recent European guidelines emphasise that even when urinary mevalonic acid excretion is positive, analysis of the MVK gene remains essential to confirm the diagnosis of MKD (26).

Alternatively, the in vitro Rab prenylation assay does not rely on symptomatic periods to provide a positive result in MKD patients, and is simple to establish, requiring only western blotting equipment. The assay has a short turnaround time of two days and does not require the use of radioisotope. The assay can be performed using fresh or cryopreserved PBMCs, again making it much more accessible for use in routine clinical care. Although the extent of the protein prenylation defect appears to correlate with residual MK enzyme activity (7), analysis of a larger cohort is required to further define this relationship. The prenylation assay cannot, therefore, be used to predict the severity of disease. It can, however, be utilised in the diagnostic workup of cases when the MK enzyme activity assay is inaccessible or will delay the treatment of urgent cases with novel or non-confirmatory genotypes. The strengths and limitations of these assays for the diagnostic work-up are summarised in Table 1.

The rationale for the in vitro prenylation assay is that, in MKD, insufficient synthesis of isoprenoid lipids (due to the partial loss-of-function mutations in MVK), prevents the normal prenylation of a multitude of proteins, including numerous Rho/Rac/Rap1-family and Rab-family small GTPases. These unprenylated proteins can be detected by western blotting for unprenylated Rap1a, or by in vitro labelling using a prenylation assay with recombinant geranylgeranyltransferase I (GGTase I) or GGTase II (Rab GGTase (16). Although defective prenylation of specific GTPases such as Rac may play a role in triggering the inflammatory cascade in MKD [reviewed in (13)], we have previously shown that the detection of GTPases modified by GGTase I (such as Rac), or western blotting for unprenylated Rap1a, lacks sensitivity. In comparison, the in vitro prenylation assay with GGTase II to detect unprenylated Rab GTPases is at least 10-fold more sensitive, since PBMC have an abundance of different Rab GTPases that can be prenylated in vitro by GGTase II (16). Hence, unprenylated Rab proteins are the most sensitive surrogate marker of defective protein prenylation in general.

We demonstrate with this case the clinical utility of the in vitro Rab prenylation assay for specific and timely diagnosis of MKD and we expand the genotypic spectrum of MKD. We suggest that the prenylation assay has practical advantages over the traditional MK enzyme assay and will be increasingly relevant for the diagnostic workup of MKD and related genotypes as summarised in Table 1. Larger scale validation, with consideration of genotype-phenotype correlation matched with patient MK enzyme activity, will further expand our understanding of the strengths and limitations of this assay in routine practice.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Ethics statement

The studies involving humans were approved by HRA and Health Care Research Wales. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Author contributions

AB: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing, Methodology. OK: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Methodology. PT: Investigation, Writing – original draft, Writing – review & editing. MM: Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. CA-K: Investigation, Writing – original draft, Writing – review & editing. MR: Conceptualization, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. DE: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing. PB: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. AB was funded by Great Ormond Street Hospital Children’s Charity (GOSHCC) (W1181), Rosetrees Trust (OoR2022\100005), and the National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre (NIHR GOSH BRC) (17IR24). PB and DE acknowledge support from GOSHCC and NIHR GOSH BRC. All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is supported by the NIHR GOSH BRC. This work was funded in part by NHMRC project grant 2017/GNT1139644 to MJR and 2024/GNT2036891 to MAM. CA-K was supported through an Australian Government Research Training Program Scholarship.

Acknowledgments

We are grateful to the family who took part in this study.

Conflict of interest

PB has received institutional grants from: Novartis, SOBI, Roche, Chemocentryx and Novimmune; consultancy fees from Roche, Novartis and SOBI; and speaker fees from UCB. DE has received institutional grants from Lilly, Sobi, Roche and Pfizer.

The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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References

1. Ter Haar NM, Jeyaratnam J, Lachmann HJ, Simon A, Brogan PA, Doglio M, et al. The phenotype and genotype of mevalonate kinase deficiency: a series of 114 cases from the eurofever registry. Arthritis Rheumatol. (2016) 68(11):2795–805. doi: 10.1002/art.39763

PubMed Abstract | Crossref Full Text | Google Scholar

2. Haas D, Hoffmann GF. Mevalonate kinase deficiencies: from mevalonic aciduria to hyperimmunoglobulinemia D syndrome. Orphanet J Rare Dis. (2006) 1:13. doi: 10.1186/1750-1172-1-13

PubMed Abstract | Crossref Full Text | Google Scholar

3. Houten SM, Wanders RJA, Waterham HR. Biochemical and genetic aspects of mevalonate kinase and its deficiency. BBA Mol Cell Biol Lipids. (2000) 1529(1):19–32. doi: 10.1016/S1388-1981(00)00135-9

PubMed Abstract | Crossref Full Text | Google Scholar

4. Cuisset L, Drenth JP, Simon A, Vincent MF, van der Velde Visser S, van der Meer JW, et al. Molecular analysis of MVK mutations and enzymatic activity in hyper-IgD and periodic fever syndrome. Eur J Hum Genet. (2001) 9(4):260–6. doi: 10.1038/sj.ejhg.5200614

PubMed Abstract | Crossref Full Text | Google Scholar

5. Gibson KM, Lohr JL, Broock RL, Hoffmann G, Nyhan WL, Sweetman L, et al. Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria. Enzyme. (1989) 41(1):47–55. doi: 10.1159/000469050

PubMed Abstract | Crossref Full Text | Google Scholar

6. Munoz MA, Jurczyluk J, Mehr S, Chai RC, Arts RJW, Sheu A, et al. Defective protein prenylation is a diagnostic biomarker of mevalonate kinase deficiency. J Allergy Clin Immunol. (2017) 140(3):873–875.e6. doi: 10.1016/j.jaci.2017.02.033

PubMed Abstract | Crossref Full Text | Google Scholar

7. Munoz MA, Jurczyluk J, Simon A, Hissaria P, Arts RJW, Coman D, et al. Defective protein prenylation in a spectrum of patients with mevalonate kinase deficiency. Front Immunol. (2019) 10:1900. doi: 10.3389/fimmu.2019.01900

PubMed Abstract | Crossref Full Text | Google Scholar

8. Pereira-Leal JB, Hume AN, Seabra MC. Prenylation of rab GTPases: molecular mechanisms and involvement in genetic disease. FEBS Lett. (2001) 498(2–3):197–200. doi: 10.1016/S0014-5793(01)02483-8

PubMed Abstract | Crossref Full Text | Google Scholar

9. Henneman L, Schneiders MS, Turkenburg M, Waterham HR. Compromized geranylgeranylation of RhoA and Rac1 in mevalonate kinase deficiency. J Inherit Metab Dis. (2010) 33(5):625–32. doi: 10.1007/s10545-010-9173-8

PubMed Abstract | Crossref Full Text | Google Scholar

10. Jurczyluk J, Munoz MA, Skinner OP, Chai RC, Ali N, Palendira U, et al. Mevalonate kinase deficiency leads to decreased prenylation of Rab GTPases. Immunol Cell Biol. (2016) 94(10):994–9. doi: 10.1038/icb.2016.58

PubMed Abstract | Crossref Full Text | Google Scholar

11. Leung KF, Baron R, Seabra MC. Thematic review series: lipid posttranslational modifications. Geranylgeranylation of Rab GTPases. J Lipid Res. (2006) 47(3):467–75. doi: 10.1194/jlr.R500017-JLR200

PubMed Abstract | Crossref Full Text | Google Scholar

12. van der Burgh R, Pervolaraki K, Turkenburg M, Waterham HR, Frenkel J, Boes M. Unprenylated RhoA contributes to IL-1β hypersecretion in mevalonate kinase deficiency model through stimulation of Rac1 activity. J Biol Chem. (2014) 289(40):27757–65. doi: 10.1074/jbc.M114.571810

PubMed Abstract | Crossref Full Text | Google Scholar

13. Politiek FA, Waterham HR. Compromised protein prenylation as pathogenic mechanism in mevalonate kinase deficiency. Front Immunol. (2021) 12:724991. doi: 10.3389/fimmu.2021.724991

PubMed Abstract | Crossref Full Text | Google Scholar

14. Munoz MA, Skinner OP, Masle-Farquhar E, Jurczyluk J, Xiao Y, Fletcher EK, et al. Increased core body temperature exacerbates defective protein prenylation in mouse models of mevalonate kinase deficiency. J Clin Invest. (2022) 132(19):e160929. doi: 10.1172/JCI160929

PubMed Abstract | Crossref Full Text | Google Scholar

15. Politiek FA, Turkenburg M, Ofman R, Waterham HR. Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype. Front Immunol. (2024) 15:1379220. doi: 10.3389/fimmu.2024.1379220

PubMed Abstract | Crossref Full Text | Google Scholar

16. Ali N, Jurczyluk J, Shay G, Tnimov Z, Alexandrov K, Munoz MA, et al. A highly sensitive prenylation assay reveals in vivo effects of bisphosphonate drug on the Rab prenylome of macrophages outside the skeleton. Small GTPases. (2015) 6(4):202–11. doi: 10.1080/21541248.2015.1085485

PubMed Abstract | Crossref Full Text | Google Scholar

17. Berner J, van de Wetering C, Jimenez Heredia R, Rashkova C, Ferdinandusse S, Koster J, et al. Phosphomevalonate kinase deficiency expands the genetic spectrum of systemic autoinflammatory diseases. J Allergy Clin Immunol. (2023) 152(4):1025–1031.e2. doi: 10.1016/j.jaci.2023.06.013

PubMed Abstract | Crossref Full Text | Google Scholar

18. Jairaman A, Badiger VA, Raj S, Nair KV, Balan S, Narayanan DL. A novel homozygous variant in PMVK is associated with enhanced IL1β secretion and a hyper-IgD syndrome-like phenotype. Clin Genet. (2023) 105:302–7. doi: 10.1111/cge.14451

PubMed Abstract | Crossref Full Text | Google Scholar

19. Yıldız Ç, Gezgin Yıldırım D, Inci A, Tümer L, Cengiz Ergin FB, Sunar Yayla ENS, et al. A possibly new autoinflammatory disease due to compound heterozygous phosphomevalonate kinase gene mutation. Joint Bone Spine. (2023) 90(1):105490. doi: 10.1016/j.jbspin.2022.105490

PubMed Abstract | Crossref Full Text | Google Scholar

20. Infevers. INFEVERS Database Entry for MVK:c.151C>T:p.(Leu51Phe) (2011). Available online at: https://infevers.umai-montpellier.fr/web/detail_mutation.php

Google Scholar

21. Yamashita Y, Matsumoto S, Hiramoto R, Komori I, Tanaka T, Nishikomori R, et al. A 6-year-old girl diagnosed with mevalonate kinase deficiency who had hydrops fetalis and neonatal-onset cholestasis. Nihon Rinsho Meneki Gakkai Kaishi. (2017) 40(2):131–7. doi: 10.2177/jsci.40.131

PubMed Abstract | Crossref Full Text | Google Scholar

22. Browne C, Timson DJ. In silico prediction of the effects of mutations in the human mevalonate kinase gene: towards a predictive framework for mevalonate kinase deficiency. Ann Hum Genet. (2015) 79(6):451–9. doi: 10.1111/ahg.12126

PubMed Abstract | Crossref Full Text | Google Scholar

23. Rigante D, Capoluongo E, Bertoni B, Ansuini V, Chiaretti A, Piastra M, et al. First report of macrophage activation syndrome in hyperimmunoglobulinemia D with periodic fever syndrome. Arthritis Rheum. (2007) 56(2):658–61. doi: 10.1002/art.22409

PubMed Abstract | Crossref Full Text | Google Scholar

24. Schulert GS, Bove K, McMasters R, Campbell K, Leslie N, Grom AA. 11-Month-old infant with periodic fevers, recurrent liver dysfunction, and perforin gene polymorphism. Arthritis Care Res (Hoboken). (2015) 67(8):1173–9. doi: 10.1002/acr.22527. PMID: 25469482;25469482

PubMed Abstract | Crossref Full Text | Google Scholar

25. Jeyaratnam J, Faraci M, Gennery AR, Drabko K, Algeri M, Morimoto A, et al. The efficacy and safety of allogeneic stem cell transplantation in mevalonate kinase deficiency. Pediatr Rheumatol Online J. (2022) 20(1):56. doi: 10.1186/s12969-022-00716-4

PubMed Abstract | Crossref Full Text | Google Scholar

26. Lengvári L, Takács K, Lengyel A, Pálinkás A, Wouters CH, Koné-Paut I, et al. Mevalonate kinase deficiency: an updated clinical overview and revision of the SHARE recommendations. Front Immunol. (2024) 15:1466844. doi: 10.3389/fimmu.2024.1466844

PubMed Abstract | Crossref Full Text | Google Scholar