Investigation of different genomic variants in familial

Mediterranean fever cases with monoallelic MEFV mutation

M. Kocabey1, T. Cankaya1, M.T. Bayram2, A. Ulgenalp1, A.O. Caglayan1,
O. Giray Bozkaya3
1
Department of Medical Genetics, 2Department of Paediatric Nephrology, 3Department of Paediatric
Genetics, Dokuz Eylul University Faculty of Medicine, Balcova, Izmir, Turkey.

Abstract
Objective
Familial Mediterranean Fever (FMF) is an inflammatory disease characterised by periodic fever and concurrent
episodes of serous membrane inflammation. FMF is considered to be inherited in autosomal recessive manner and
biallelic mutations in the MEFV gene are associated with the disease. However, approximately 20-25% of patients
only have a single mutation in MEFV gene, which creates confusion in differential diagnosis of many patients.
This study aimed to reveal rare variants that may act in conjunction with the single pathogenic MEFV variant in
the pathogenesis of FMF.

Methods
We performed whole exome sequencing in 17 individuals from 5 different families who were diagnosed according to
the clinical criteria, responded positively to colchicine treatment, but had no biallelic MEFV mutation.

Results
A disease-causing variant or a common affected cellular pathway that was shared in all index cases was not detected.
When cases were examined individually, two de novo variants were identified in the BIRC2 and BCL10 genes, both
of which play a role in inflammatory pathways. Functional studies are needed to confirm the physiopathological
relationship of these genes with FMF.

Conclusion
This study is one of the most extensive aetiological researches in FMF cases with monoallelic MEFV mutation.
We have shown that genotype-phenotype correlation in these cases may not be established by rare genetic variants
and discussed underlying causes. Clinical criteria with emphasis on colchicine response and family history should
be the main tool and genetic results should only be used for support in FMF diagnosis.

Key words
BCL10, BIRC2, familial Mediterranean fever, MEFV, whole exome sequencing

Clinical 2020
Clinical and Experimental Rheumatology and Experimental Rheumatology 2023; 41: 2017-2026.
Investigation of monoallelic mutant FMF / M. Kocabey et al.

Mehmet Kocabey, MD* Introduction sequencing with expression analysis in

Tufan Cankaya, MD, PhD Familial Mediterranean fever (FMF) is 20 cases and excluded the possibility of
Meral Torun Bayram, MD an autoinflammatory disease character- a loss of expression due to an unknown
Ayfer Ulgenalp, PhD
ised by self-limiting episodes of fever mutation or a mosaic mutation specific
Ahmet Okay Caglayan, MD**
Ozlem Giray Bozkaya, MD** and serous membrane inflammation. It to leukocytes (10). Deletions and dupli-
is believed to be a monogenic disease cations were also evaluated as possible
*Present affiliation of M. Kocabey:
Department of Medical Genetics, with an autosomal recessive inheritance underlying causes but no rearrange-
Hatay Training and Research Hospital, but there are several issues requiring an ments were found (10, 11). Haplotype
Antioch, Hatay, Turkey. explanation regarding MEFV-related analyses were conducted in affected
**These authors contributed equally. phenotype such as low penetrance, siblings, but different alleles were found
Please address correspondence to: difficulties in clinical interpretation of to be inherited in wild-type MEFV al-
Mehmet Kocabey, MEFV variants, the lack of a reliable leles (9, 10). Considering these results,
Department of Medical Genetics, genotype-phenotype correlation and explaining monoallelic FMF patients
Hatay Training and Research Hospital, patients with monoallelic mutations. by autosomal recessive inheritance of
Antioch 31175, Hatay, Turkey. FMF is most common in Eastern Medi- MEFV variants alone is not possible.
E-mail: mehmet_kocabey@[Link] terranean populations, but studies re- This prompted the definition of a sub-
ORCID ID: 0000-0001-9565-6907 port a wide range of disease prevalence. type of FMF with autosomal domi-
Received on November 8, 2022; accepted In Turkish population the prevalence, nant inheritance (OMIM#134610),
in revised form on January 27, 2023. as determined by clinical evaluation, is and also another clinical entity named
© Copyright Clinical and reported in the range of 0.027%–0.82% as Pyrin Associated Autoinflamma-
Experimental Rheumatology 2023.
(1-3). While the carrier rate as deter- tion with Neutrophilic Dermatosis
mined by genetic evaluation of MEFV (OMIM#608068) which is associ-
is reported in the range of 3.75%-27%. ated with dominant inheritance of two
(4, 5). The common point of all these specific MEFV mutations (p.S242R,
studies is that, contrary to expectations, p.E244K) (12-15). This also led to the
they have a lower prevalence com- hypothesis that MEFV variants have a
pared to high carrier rate. Either FMF dose-dependent phenotypic effect in
is largely underdiagnosed or MEFV FMF pathogenesis (16). Although these
variants alone are insufficient in deter- subtypes show that dominant inherit-
mining carrier state and other genetic/ ance is possible, they do not answer the
environmental factors affect disease main question. What causes the classi-
penetrance. cal FMF disease, in individuals carrying
There are more than 350 variants identi- common heterozygous mutations? Con-
fied within MEFV in the Infevers data- trary to the expectations from dose-de-
base (6). Variants can be found across pendent phenotypic effects, there are no
all parts of the gene (5’ UTR, 3’ UTR, clinical differences that can be clearly
all exons and introns) but only a limited distinguished between biallelic mutant
number of well-established mutations cases and monoallelic mutant cases (10,
(mostly exon 10 variants) are associated 17). Additionally, the vast majority of
with FMF. The rest of the variants are monoallelic mutant individuals carry-
generally classified as variants of uncer- ing known mutations are asymptomatic
tain significance (6). This high degree carriers.
of uncertainty is another aspect of FMF In this study we aimed to reveal rare
genetics and until date, an international- variants that acts in conjunction with
ly recognised genotype-phenotype cor- a single MEFV mutation in the patho-
relation that enables predicting disease genesis of FMF. We performed an ex-
outcome of MEFV variants is not estab- tensive search with whole-exome se-
lished (7). Apart from the difficulties of quencing (WES) in 5 unrelated index
missing penetrance and uncertain vari- patients and their first-degree relatives
ants, it is shown that around 20%–26% who are clinically diagnosed with FMF
of patients only have a single mutation and carrying only one MEFV mutation.
Funding: this study was funded by a (8). Many studies searched for a second
research grant from the Dokuz Eylul mutation. Booty et al. analysed the en- Material and methods
University Scientific Research. tire 15 Kb genomic region of MEFV in Patient selection
Projects Coordination Unit 10 cases, but they were unable to show All patients referred for FMF in Dokuz
(Project no.: [Link].009). a second mutation (9). Marek-Yagel Eylul University Paediatric Nephrol-
Competing interests: none declared. et al. performed complementary DNA ogy and Paediatric Genetics outpatient

2018 Clinical and Experimental Rheumatology 2023

 Investigation of monoallelic mutant FMF / M. Kocabey et al.

Table I. Phenotypic characteristics of the cohort.

Cases Gender Age Age at Abdominal Fever Arthralgia Arthritis Frequency of Episode
(year) onset pain episodes† duration
(Day)

Family I
I.1 (index) F 11 5 + + + - 1-2/year 5-7
I.2 (sister) F 19 - - - - - - -
I.3 (mother) F 45 6 + + + + 6-7/year 5-10

Family II
II.1 (index) M 12 8 + + - - 6/year 3
II.2 (mother) F 34 - - - - - - -
II.3 (father) M 35 - - - - - - -

Family III
III.1 (index) F 8 2 - + + + 4-5/year 1-2
III.2 (mother) F 35 18 + + + + 10-12/year 1-2
III.3 (father) M 37 - - - - - - -

Family IV
IV.1 (index) F 7 1 + + + - 4-6/year 2-3
IV.2 (mother) F 27 - - - - - - -
IV.3 (father) M 31 - - - - - - -

Family V
V.1 (index) M 4 1 - + - - 12/year 2-3
V.2 (dizygotic twin) M 4 - - - - - - -
V.3 (brother) M 8 2 - + + + 3-4/year 3-7
V.4 (mother) F 37 - - - - - - -
V.5 (father) M 38 - - - - - - -

Table I (continued). Phenotypic characteristics of the cohort.

Cases ELE Chest Diarrhoea Nausea Myalgia ESR/CRP Leukocyte Laparotomy Colchicine
pain / cons. /vomit

Family I
I.1 (index) - - + + + 41‡ / 0,3‡ 8300‡ - 1 mg/day
I.2 (sister) - - - - - - - - Healthy
I.3 (mother) - + - - - 13§ / 3,9§ 5400§ + 1,5 mg/ day

Family II
II.1 (index) + - - - - 3§ / 0,7§ 9900§ - 1 mg/ day
II.2 (mother) - - - - - - - - Healthy
II.3 (father) - - - - - - - - Healthy

Family III
III.1 (index) - - - - + 40‡ / 2,9‡ 5000§ - 1 mg/ day
III.2 (mother) - + + + - 30‡ / 11‡ 9900‡ - 1,5 mg/ day
III.3 (father) - - - - - - - -Healthy

Family IV
IV.1 (index) - - + + - 32‡ / 6,1‡ 3100‡ - 1 mg/ day
IV.2 (mother) - - - - - - - - Healthy
IV.3 (father) - - - - - - - - Healthy

Family V
V.1 (index) + - - - - 81‡ / 110,6‡ 12100‡ - 0,5 mg/ day
V.2 (dizygotic twin) - - - - - - - - Healthy
V.3 (brother) - - - - - 17‡ / 20,3‡ 15300‡ - 0,5 mg/ day
V.4 (mother) - - - - - - - - Healthy
V.5 (father) - - - - - - - - Healthy

M: male; F: female; ELE: erysipelas-like erythema; Cons: constipation; ESR: erythrocyte sedimentation rate (normal range: 0-15 mm/h); CRP: C-reactive
protein (normal range: 0-5 mg/L).
Leukocyte normal range: 4000-10000/µl.
†
The frequency of episodes reflects the frequency before treatment, and it is decreased in all cases after colchicine initiation.
‡
Laboratory values (ESR, CRP, leukocyte) taken during crises.
§
Basal values of patients in asymptomatic intervals when episodic data were not available.

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Investigation of monoallelic mutant FMF / M. Kocabey et al.

clinics in 2020 were retrospectively ducted on Illumina NextSeq550 system associate more frequent variants that
reviewed. MEFV mutation status was (Illumina, San Diego, CA, USA). Bio- could act through oligogenic/polygenic
determined after a two-step genetic informatic steps and data analysis were inheritance. Then, benign/likely benign
testing procedure as recommended in performed as stated below. variants in ClinVar were omitted from
the literature (18, 19). Patients with a the remaining rare exonic non-synon-
confirmatory genotype for any auto- Whole-exome sequencing ymous variants (missense, nonsense,
inflammatory disease as explained in and data analysis frameshift, canonical splice site, stop
Eurofever criteria were excluded (20). To generate exome capture libraries, loss, initiator codon variants, in-frame
Among the rest, priority was given to Human Core Exome Kit (Twist Bio- deletions, and in-frame insertions).
those who benefited from colchicine science, USA) that enriches 33 Mb of Therefore, we revealed rare variants
treatment, and to those with a family the human consensus coding sequence with likely phenotypic consequences.
history of FMF. A total of 17 individu- was used according to the manufactur- No candidate gene-based filtering was
als including 5 index cases, who were er’s protocols. The enriched DNA was applied. Instead in the second step, all
clinically diagnosed with FMF but only paired-end sequenced on HiSeq2000 remaining variants were searched for
have a single MEFV mutation, and Sequencer (Illumina, San Diego, CA, functional relevance with the MEFV
their first-degree relatives were select- USA). Sequence alignment and vari- gene.
ed. All individuals (or parents when the ant calling were performed against the Functional enrichment analyses was
patient was a minor) provided written reference human genome (hg19) using performed on the frequency filtered ex-
informed consent for molecular analy- SEQ programme v. 16.7 ([Link] onic non-synonymous variants by DA-
sis and for the publication of clinical [Link]/) (Genomize, Turkey). VID (27). In this analysis, we searched
findings. The study was performed in The SEQ algorithm works according whether any of our filtered variant is
line with the principles of the Declara- to the GATK recommendations and present in a gene involved in the ‘Nod-
tion of Helsinki. Approval was granted incorporates Burrows-Wheeler Aligner like receptor signalling pathway’ or
by the Ethics Committee of Dokuz Ey- for read mapping, FreeBayes for vari- not. We also searched for other cellular
lul University (date: 01.11.2018/No. ant calling, PCR dedup for removing pathways where the variant burden is
2018/28-25). duplicates and Indel Realignment to increased. To distinguish this, the list
remove alignment artifacts (21). SEQ of genes to which the filtered variants
MEFV mutation analysis also annotates variants using several belong was entered as ‘Official Gene-
Peripheral blood samples were collect- online resources such as Ensembl Vari- Symbols’. Homo sapiens was used as
ed from all individuals. Total genomic ant Effect Predictor v. 78, Clinvar, db- the reference genome. p<0.05 was con-
DNA were isolated using QIAamp® SNP v. 151 and dbNSFP v. 3.5 (22-25). sidered statistically significant.
DNA Blood Mini kit (Qiagen, Ger- GeneMANIA and STRING web appli-
many) via Qiacube® automated DNA Variant filtering and interpretation cations were also used for functional fil-
isolation device (Qiagen, Germany). Every patient’s data were analysed in- tering (28, 29). 13 genes that play an ac-
In 5 index patients, PCR amplification dividually and then cross examined for tive role in pyrin inflammasome activa-
with melting curve analysis for hot- joint variants. For every individual, a tion [MEFV, CASP1, IL18, IL1B, MVK,
spot mutations (p.E148Q, p.R202Q, two-step variant filtering strategy was PKN1, PKN2, PSTPIP1, PYCARD,
p.P369S, p.M680I, p.M694V, p.M694I, applied. At the first step, we excluded RHOA, RHOB, RHOC, YWHAB] were
p.V726A, p.A744S) was performed as variants with an allele frequency great- searched along with filtered rare vari-
a first step analysis. LightSNiP® FMF er than 0.1% or 1% in Gnomad version ant genes. The idea was that any physi-
kit (TIB Molbiol, Germany) were used 2.1.1 and in SEQ’s in-house database. opathologically important gene must
according to the manufacturer’s proto- SEQ contains around 2500 individu- play a biological part (co-localisation,
cols in Cobas® z480 Analyzer (Roche al’s exome sequencing data from Tur- physical interaction, pathway interac-
Diagnostics, Switzerland). As a second key (26). Two minor allele frequency tion, etc.) with aforementioned genes
step testing in index patients, target cap- thresholds were applied between the inside the cell. While determining the
ture and enrichment based next genera- different variant types. A less restric- functional interactions in the STRING
tion sequencing was performed to ana- tive 1% threshold was used for null program, high-confidence interactions
lyse the entire coding region and splice type variants since these mutations are with a minimum interaction score of
junctions (± 10 bp) of MEFV and 11 relatively few and have serious phe- 0.7 and above were taken as the basis.
other autoinflammatory disease related notypic consequences. For missense Known physical interactions and same
genes [TNFRSF1A, MEFV, MVK, IL- and in-frame variants 0.1% frequency pathways were also taken as basis on
1RN, LPIN2, NOD2, NLRP3, NLRP12, threshold is chosen because, the disease the GeneMANIA and other interactions
ADA2, PSTPIP1, TNFRSF11A, ELA- prevalence in our region is 0.1% and were not included.
NE]. Library preparation was carried monoallelic FMF patients are a smaller In silico data of the REVEL algorithm
out using autoinflammatory diseases cluster within all patients. Also, since were used for missense variants and
panel (Celemics, South Korea) and this study was conducted with five in- thresholds were decided in line with that
massively parallel sequencing was con- dex cases, it would not be possible to of Ioannidis et al. (30). REVEL scores

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 Investigation of monoallelic mutant FMF / M. Kocabey et al.

Fig. 1. Pedigrees showing index cases and their first-degree relatives. Age, MEFV mutation status and case numbers of participating family members are
shown below each individual. Benign and likely benign variants are not included.

between 0.5 and 0.75 were considered (Mean: 8.4). The mean age of the entire were uncovered by parental and sibling
as uncertain [variants of uncertain sig- study group was 23.6. The total num- analyses (Fig. 3). One heterozygous de
nificance (VUS)] and scores greater ber of individuals diagnosed with FMF novo variant was detected in BIRC2
than 0.75 were considered as support- was 8. One sibling and two parents gene in Case V.1, PRR21 gene in Case
ing evidence of pathogenicity. Also, from different families were receiving IV.1, PELP1 gene in Case III.1, BCL10
Gnomad database were used to obtain colchicine treatment. All the clinical and IMP4 genes in Case II.1. Since the
observed/expected (o/e) ratios of dif- data are summarised in Table I. MEFV father of Case I.1 was not available, de
ferent gene-variant type combinations. testing results of the members are novo variants were not evaluated in that
Since o/e scores reflect the ratio of the shown alongside pedigrees in Figure 1. case.
actual number of specific type variants The results of DAVID’s pathway en-
encountered in the general population Whole-exome sequencing richment analysis were as follows,
to the evolutionarily expected number The mean depth was 176.4 reads and • ‘Tight junction’ pathway (p=0.045)
of that type of variant, a score lower the mean target coverage in at least in Case I.1,
than 0.35 threshold is used to support 50x depth was 91.73%. 50x coverage • No statistically significant pathway
pathogenicity. was below 85% in only case V.5 (fa- in Case II.1,
After evaluation of functional predic- ther of the index patient in family V), • ‘Autoimmune thyroid disease’ path-
tive information in the second step but the coverage rose to 93.38% at 20x. way (p=0.031) in Case III.1,
and filtering, pathogenicity classifica- Therefore, it was deemed sufficient • ‘Gap junction’ (p=0.014), ‘In-
tion were determined according to the and evaluated accordingly. A mean of flammatory mediator regulation
American College of Medical Genetics 33,474 variants per index case was de- of TRP channels’ (p=0.02), ‘Neu-
and Genomics (ACMG) standards (31). tected (Fig. 2). Two-step filtering was roactive ligand-receptor interac-
Cross examination of joint variants be- performed on these variants. In the first tion’ (p=0.027), ‘Oocyte meiosis’
tween different families was performed step, variants were filtered separately (p=0.03) and ‘Calcium signalling’
and possible causal relationship in FMF according to variant types for their mi- (p=0.04) pathways in Case IV.1,
is discussed in light of current literature. nor allele frequencies (MAF) and Clin- • ‘Retinol metabolism’ (p=0.036) and
Var reports which left an average of 252 ‘Adherens junction’ (p=0.046) path-
Results variants per index case (Fig. 2). ways in Case V.1 were found to be
Patient information In the second step, variants without any enriched in variant load.
A total of 17 people, including 5 index functional relevance to pyrin inflam- All variants after second-step filter-
cases and their first-degree relatives, masome pathway in DAVID, Gene- ing [MEFV variants, pathogenic (P)
were included in the study. The age MANIA and STRING in silico analysis or likely pathogenic (LP) variants, de
of index cases was between 4 and 12 were filtered out and de novo variants novo variants, Nod-like receptor sig-

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Investigation of monoallelic mutant FMF / M. Kocabey et al.

II.1, SLC19A3 gene in case III.1, and

BIRC2 (also de novo), KRIT1, LZTR1,
and RPE65 genes in case V.1.
In total, 115 variants in 5 index cases
were manually curated. 7 of them were
previously known MEFV variants, 8
of them were rare variants in the same
gene across index cases (CACNA1H,
HRNR, MYH13, TTN), 5 of them
were de novo variants (BCL10, IMP4,
PELP1, PRR21, BIRC2) and 7 of them
were P/LP variants (ABCA13, XPA,
TACR3, SLC19A3, KRIT1, LZTR1,
RPE65). 88 different gene variants
were left which are identified as pos-
sibly related to the pyrin inflamma-
some pathway. Among them, CHUK
p.Leu354Ile found in case II.1 is dis-
cussed separately with the closely re-
lated de novo BCL10 variant. The re-
maining 87 variants were classified as
VUS (Supplementary Table S1).

Discussion
The strongest evidence to associate a
genetic variant with FMF would have
been the detection of the same variant
in many unrelated cases. Genes of the
overlapping variants detected in this
study (CACNA1H, HRNR, MYH13,
TTN) were not involved in inflammato-
ry pathways. No information regarding
possible FMF association was found in
the literature for these genes. All vari-
ants were classified as VUS. Therefore,
an association with FMF was not con-
sidered and the fact that these rare vari-
ants overlapping in two out of five cases
were considered to be coincidental. The
absence of any shared pathway in gene
Fig. 2. First-step variant filtering strategy.
Numbers in the lower right boxes are the average number of variants detected in an index case after enrichment analysis was also interpret-
every stage. The numbers for each individual case are reported in the Supplementary Figure S1. ed as the lack of other cellular pathways
MAF: minor allele frequency. *Population frequencies from Gnomad database. involved in the FMF phenotype.
De novo variants play an important role
nalling pathway variants and pyrin was seen that there were two shared in many diseases, but de novo inherit-
inflammasome pathway related vari- variants in the CACNA1H and HRNR ance is a strong indicator of pathogenic-
ants] are shown case by case in Sup- genes in cases I.1 and V.1, while there ity, only if it can be associated function-
plementary Table S1. Variant name in were two other shared variants in the ally with disease (31). The function of
accordance with the international no- MYH13 and TTN genes in cases II.1 PRR21 is unknown, PELP1 encodes an
menclature, position, zygosity, paren- and IV.1. oestrogen receptor co-activator and the
tal origin, in silico prediction score, o/e Pathogenicity classification was made IMP4 gene encodes a protein involved
score, rs number, in-house MAF and to the rest of the filtered variants ac- in ribosome biogenesis localised in the
pathogenicity criteria are included in cording to the ACMG criteria. Eight nucleolus. No functional relationship
Supplementary Table S1. variants were interpreted as P/LP (apart to the pyrin inflammasome pathway or
In cross-comparison, a variant that is from MEFV mutations). P/LP variants a known association with any disease
overlapping in all five families were were detected in ABCA13 and XPA was found for these genes. Therefore,
not found. In separate comparisons, it genes in case I.1, TACR3 gene in case de novo variants other than BCL10 and

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 Investigation of monoallelic mutant FMF / M. Kocabey et al.

of fever. V.5 is healthy. We interpreted

the frameshift BIRC2 variant as likely
pathogenic, but functional studies are
required to determine the physiopatho-
logical relationship with FMF.
BCL10 gene encodes a signal protein
involved in adaptive immune system
activation and regulation. When B or
T cell receptors are stimulated; BCL10
forms a complex called ‘CBM’ with
various CARD proteins and MALT1
protein. The main result of this com-
plex is NFκB activation. This happens
by ubiquitin-regulating protein recruit-
ment and NEMO (‘NFκB essential
modulator’) activation along with IKK
alpha (‘inhibitor of NFκB kinase’, also
Fig. 3. Second-step variant filtering strategy.
Numbers in the lower right boxes are the average number of variants detected in an index case after known as CHUK) and beta proteins.
every stage. The numbers for each individual case are reported in Supplementary Figure S1. When IKK proteins are activated, they
deactivate NFκB kinase and therefore,
BIRC2 were not considered to be asso- tions, UBA domain that is responsible active NFκB is released in the cytosol
ciated with FMF. for ubiquitin binding, CARD and RING (38). In 2012, Gringhuis et al. report-
BIRC2 gene (formerly CIAP1) is a domains that are responsible for E2 pro- ed that Dectin-1, a pattern recognition
member of the inhibitor of apoptosis tein interactions, respectively (Fig. 4) receptor protein such as pyrin, causes
protein family. It functions as an E3 (36). The frameshift variant coincides the formation of the CARD9-BCL10-
ubiquitin ligase and regulates many just before the CARD domain and leads MALT1 complex in response to ex-
substrates, including itself, by ubiqui- to a stop codon after 7 amino acids (Fig. tracellular fungal pathogens, which in
tination (32). It’s most relevant func- 4). The BIRC2 gene has an o/e score of turn increases IL-1 beta transcription
tion in terms of FMF is the inhibition of 0.22, and therefore haploinsufficiency and maturation (39). Therefore, it has
IL-1 beta formation with other inhibi- has a high risk of clinical disease. Mu- been shown that although the physi-
tors of apoptosis proteins (XIAP and tations associated with inhibitors of ological processes in which BCL10 is
CIAP2). This function occurs via Cas- apoptosis proteins have generally been involved are not directly related to the
pase-8 along with NLRP3 inflammas- reported in somatic form, so there is no Pyrin inflammasome pathway, it can
ome (33, 34). However, contrary to this clear information about germline muta- increase IL1-beta synthesis. In case
information, there are also publications tions (37). In family V, index case (V.1), II.1, we detected a de novo heterozy-
reporting that CIAP1 and CIAP2 are his older brother (V.3) and his father gous missense variant (p.Arg25Cys)
essential for effective caspase-1 activa- (V.5) has MEFV p.R761H variant in in the BCL10 gene and a paternally
tion and therefore IL-1 beta formation heterozygous state while BIRC2 variant inherited heterozygous missense vari-
(35). In Case V.1, a de novo heterozy- is only found in the index case. While ant (p.Leu354Ile) in the CHUK gene
gous p.Glu443GlyfsTer7 frameshift this is not compatible with segregation, together. Lymphoma occurs in somatic
variant was detected in the 6th exon their symptoms are also not the same. mutations of the CBM complex, par-
of the BIRC2 gene. BIRC2 protein has V.1 has erythematous eruptions around ticularly in translocations involving
6 different domains. From the N to C ankles and frequent shorter episodes of BCL10 (40). Whereas in biallelic ger-
terminus; three BIR domains, that are fever, whereas V.3 has arthralgia, arthri- mline mutations, immunodeficiency
responsible for protein-protein interac- tis, and less frequent longer episodes syndromes called CBM-opathies occur

Fig. 4. BIRC2 protein structure. Amino acid positions are given under each domain. The mutation detected in Case V.1 is marked with a star.
BIR: baculoviral IAP repeat domain; UBA: ubiquitin associated domain; CARD: caspase recruitment domain; RING: really interesting new gene domain.

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Investigation of monoallelic mutant FMF / M. Kocabey et al.

Table II. In silico predictions and allele frequencies*.

Prediction tool BCL10:c.73C>T:p.R25C CHUK:c.1060C>A:p.L354I

PROVEAN Damaging (-7,35) Tolerable (-0,95)

SIFT Damaging (0) Tolerable (0,145)
Polyphen2_HDIV Probably_damaging (1) Probably_damaging (0.983)
Polyphen2_HVAR Probably_damaging (0.998) Probably_damaging (0.656)
CADD Damaging (24.3) Damaging (24.7)
LRT Deleterious (0) Deleterious (0)
MetaSVM Tolerable (-0.79) Tolerable (-0.773)
MetaLR Tolerable (0.15) Tolerable (0.205)
MutationAssessor Medium (2.175) Medium (2.58)
VEST3 Damaging (0.977) Damaging (0.383)
MutationTaster Disease_causing (1) Disease_causing (1)
REVEL Damaging (0.68) Tolerable (0.119)
GERP++ Conserved (3.7) Conserved (5.93)
PhastCons Conserved (1) Conserved (1)
PhyloP Conserved (3.827) Conserved (4.957)
FATHMM Tolerable (1.31) Tolerable (0.37)
FATHMM_MKL Damaging (0.987) Damaging (0.981)
DANN Damaging (0.999) Damaging (0.994)
Eigen Damaging (0.351) Damaging (0.664)

Population databases Allele frequency Allele frequency

gnomAD_exome_ALL - -
gnomAD_genome_ALL - -
ExAC_ALL - -
1000genomes_ALL - -
ESP6500_ALL - -

*All information was obtained from VarCards database (42).

(41). Biallelic mutations of the CHUK Pyrin inflammasome pathway. No such patients without MEFV mutations are
have been associated with Cocoon syn- evidence was found (except for CHUK needed. Our cases do not follow a clear
drome (OMIM#613630), which is fatal p.Leu354Ile). Genetic counselling was Mendelian inheritance pattern and peri-
in the prenatal period. No clinical re- provided where required, but potential odic fever is commonly seen in autoin-
port of heterozygous mutations for both clinical consequences of these muta- flammation process. Deep phenotyping
gene (BCL10/CHUK) has been found in tions are irrelevant to the scope of this is required because to date, studies have
the literature. Also, o/e scores for both study. An important point is that 57 of not shown any distinguishing features
genes (CHUK:0.608-BCL10:0.648) the 95 (60%) variants were filtered be- between monoallelic mutant FMF cases
reflect that haploinsufficiency may cause of their association with RhoGT- and other FMF cases (10, 17). The small
be tolerant. In silico predictions were Pases and 14-3-3 chaperone proteins. number of cases is the most important
concordant as both variants were not This is a reflection of the large num- limitation of this study. If monoallelic
predicted as pathogenic (Table II) (42). ber of interactions these proteins make FMF cases follow a polygenic/multi-
We classified both variants as VUS, but within the cell. Therefore, it can be pre- factorial inheritance, this study is not
reserved judgement as only gain-of- dicted that a multitude of targets can suitable to uncover any related variant.
function type variants in these genes alter the Pyrin inflammasome pathway Finally, Pyrin inflammasome is in direct
can be associated with autoinflamma- via Rho proteins and 14-3-3 proteins. interaction with environmental factors,
tory symptoms. Also, case II.1 has no There can be several hypotheses ex- especially infectious agents, as a part of
other family member diagnosed with plaining why a causal genetic variant the innate immune system. Considering
FMF for comparison. Healthy mother in every family was not detected. It is the geographical distribution of the dis-
(II.2) carrying the heterozygous MEFV possible that FMF is caused by several ease, an environmental agent specific to
p.K695R mutation did not have BCL10 different genetic aetiologies in conjunc- the Eastern Mediterranean may prompt
or CHUK variant. Therefore, functional tion with MEFV mutations. Similarly, the symptoms in cases with monoallelic
research is needed to conclude whether these cases might be phenocopies of mutations independent from other ge-
these variants are pathogenic or not. more than one autoinflammatory dis- netic variants. Various infectious agents
At the end of second step filtering; be- ease, independent of MEFV. For ex- such as Clostridium difficile, Vibrio
sides MEFV variants, overlapping vari- ample, the de novo BCL10 and BIRC2 parahaemolyticus, Histophilus somni,
ants and de novo variants, 95 variants variants might be the sole aetiology of and Clostridium botulinum are known
remained (Supplementary Table S1). a distinct autoinflammatory phenotype to trigger pyrin inflammasome activa-
All these variants were classified and in their respective patients but in or- tion (43). Studies of epigenetic factors
searched for potential relationship with der to prove this, identification of new and environmental/microbiota related

2024 Clinical and Experimental Rheumatology 2023

 Investigation of monoallelic mutant FMF / M. Kocabey et al.

processes that can trigger attacks in only be used as support for diagnosis. [Link]
10. MAREK-YAGEL D, BERKUN Y, PADEH S et
monoallelic and biallelic cases will Clinical criteria with emphasis on col-
al.: Clinical disease among patients het-
have interesting results. chicine response and family history erozygous for familial Mediterranean fever.
This study is one the most extensive should be the main tool in FMF diag- Arthritis Rheum 2009; 60(6): 1862-6.
etiological search conducted in mono- nosis. Clarifying the genetics of FMF, [Link]
11. van GIJN ME, SOLER S, de la CHAPELLE C
allelic mutant FMF cases to date. In is critical for developing accurate diag- et al.: Search for copy number alterations
the past, only few genes are searched nosis, treatment, and follow-up algo- in the MEFV gene using multiplex ligation
as candidate genes (9, 44). Mainly rithms in the future. It is necessary to probe amplification, experience from three
other autoinflammatory disease genes collect a large amount of clinical and diagnostic centres. Eur J Hum Genet 2008;
16(11): 1404-6.
or pyrin inflammasome pathway genes genetic data and to interpret the infor- [Link]
were investigated. One of the strengths mation by combining it with modelling 12. MASTERS SL, LAGOU V, JÉRU I et al.: Fa-
of our study is that no gene filter was systems. We hope that the results of milial autoinflammation with neutrophilic
put in during bioinformatic analysis. this study will contribute to the exist- dermatosis reveals a regulatory mechanism
of pyrin activation. Sci Transl Med 2016;
The diagnostic potential of WES is ing knowledge in examining genotype- 8(332): 1-10.
higher than analysis based on a limited phenotype relationships. [Link]
number of selected genes, but this ap- 13. MOGHADDAS F, LLAMAS R, DE NARDO D et
proach has limitations. As the target References al.: A novel Pyrin-Associated Autoinflamma-
tion with Neutrophilic Dermatosis mutation
region expands, coverage and depth 1. COBANKARA V, FIDAN G, TURK T, ZENCIR
further defines 14-3-3 binding of pyrin and
M, COLAKOGLU M, OZEN S: The prevalence
may decrease. Even though an average of familial Mediterranean fever in the Turk- distinction to Familial Mediterranean Fever.
of 91.73% coverage at a minimum 50x ish province of Denizli: a field study with Ann Rheum Dis 2017; 76(12): 2085-94.
https://
reading depth was achieved, an impor- a zero patient design. Clin Exp Rheumatol
2004; 22 (Supp. 34): S27-30. [Link]/10.1136/annrheumdis-2017-211473
tant limitation of this study is the pos- 14. SARRABAY G, TOUITOU I: Dominant familial
2. ONEN F, SUMER H, TURKAY S, AKYUREK O,
sibility of missing overlapping variants TUNCA M, OZDOGAN H: Increased frequency Mediterranean fever. Rheumatology 2017;
due to unread exonic regions. Also, of familial Mediterranean fever in Central 56(2): 173-5. https://
[Link]/10.1093/rheumatology/kew203
causal variants may reside in genomic Anatolia, Turkey. Clin Exp Rheumatol 2004;
22 (Suppl. 34): S31-3. 15. McKUSICK-NATHANS INSTITUTE OF GENETIC
regions outside the scope of the re- 3. KISACIK B, YILDIRIM B, TASLIYURT T et al.: MEDICINE: Online Mendelian Inheritance in
search since epigenetic changes, intron- Increased frequency of familial Mediterra- Man, OMIM®. Johns Hopkins University
ic regions and intergenic DNA regions nean fever in northern Turkey: a population- (Baltimore, MD). https://
based study. Rheumatol Int 2009; 29(11): [Link]/. Accessed October 25, 2022.
are not covered. Recently, Umar et al. 16. TOUITOU I: Inheritance of autoinflammatory
1307-9.
performed whole genome sequenc- [Link] diseases: Shifting paradigms and nomencla-
ing in 50 patients who has zero, single 4. YIGIT S, BAGCI H, OZKAYA O, OZDAMAR K, ture. J Med Genet 2013; 50(6): 349-59. https://
and double mutations in MEFV gene CENGIZ K, AKPOLAT T: MEFV mutations in [Link]/10.1136/jmedgenet-2013-101577
patients with familial mediterranean fever 17. KONÉ-PAUT I, HENTGEN V, GUILLAUME-
(45). With a gene filtering approach CZITROM S, COMPEYROT-LACASSAGNE S,
in the black sea region of Turkey: Samsun
(MAF<1%, autoinflammatory disease Experience [corrected]. J Rheumatol 2008; TRAN TA, TOUITOU I: The clinical spectrum
related genes), they found a heterozy- 35(2): 106-113. of 94 patients carrying a single mutated
MEFV allele. Rheumatology 2009; 48(7):
gous deletion in exon 11 of IL1RL1 5. KORKMAZ DT, ATAK PG, CELIK Ç: Frequen-
cies of the common MEFV gene mutations 840-2.
gene which was present in 9 monoal- in Adiyaman, Southeast Anatolia, Turkey. [Link]
lelic mutant FMF cases. They then vali- Balkan J Med Genet 2015; 17(2): 67-71. 18. SHINAR Y, OBICI L, AKSENTIJEVICH I et
dated their results in 184 FMF patients [Link] al.: Guidelines for the genetic diagnosis of
6. van GIJN ME, CECCHERINI I, SHINAR Y et hereditary recurrent fevers. Ann Rheum Dis
and 218 controls. Same deletion was 2012; 71(10): 1599-605. https://
al.: New workflow for classification of ge-
found in 12%, 21%, 23% of zero, sin- netic variants’ pathogenicity applied to he- [Link]/10.1136/annrheumdis-2011-201271
gle and double MEFV mutation carri- reditary recurrent fevers by the International 19. GIANCANE G, HAAR NMT, WULFFRAAT N
ers, respectively. Notwithstanding this Study Group for Systemic Autoinflamma- et al.: Evidence-based recommendations for
tory Diseases (INSAID). J Med Genet 2018; genetic diagnosis of familial Mediterranean
important finding, they also interpreted fever. Ann Rheum Dis 2015; 74(4): 635-41.
55(8): 530-7. https://
that a single rare variant would not be [Link]/10.1136/jmedgenet-2017-105216 https://
sufficient to explain the pathophysiol- 7. GANGEMI S, MANTI S, PROCOPIO V et al.: [Link]/10.1136/annrheumdis-2014-206844
ogy of FMF and a multifactorial model Lack of clear and univocal genotype-pheno- 20. GATTORNO M, HOFER M, FEDERICI S et al.:
type correlation in familial Mediterranean fe- Classification criteria for autoinflamma-
should be investigated (45). ver patients: a systematic review. Clin Genet tory recurrent fevers. Ann Rheum Dis 2019;
2018; 94(1): 81-94. 78(8): 1025-32. https://
Conclusion [Link] [Link]/10.1136/annrheumdis-2019-215048
8. OZEN S: Changing concepts in familial 21. McKENNA A, HANNA M, BANKS E et al.:
In conclusion, we have shown that The Genome Analysis Toolkit: a MapReduce
Mediterranean fever: Is it possible to have
genotype-phenotype correlation in an autosomal-recessive disease with only framework for analyzing next-generation
these cases may not be established by one mutation? Arthritis Rheum 2009; 60(6): DNA sequencing data. Genome Res 2010;
rare genetic variants and discussed un- 1575-7. [Link] 20(9): 1297-303.
9. BOOTY MG, JAE JC, MASTERS SL et al.: [Link]
derlying causes. Due to the incomplete Familial Mediterranean fever with a single 22. LANDRUM MJ, LEE JM, BENSON M et al.:
understanding of FMF genetics, we MEFV mutation: Where is the second hit? ClinVar: improving access to variant inter-
recommend that genetic testing should Arthritis Rheum 2009; 60(6): 1851-61. pretations and supporting evidence. Nucleic

Clinical and Experimental Rheumatology 2023 2025

Investigation of monoallelic mutant FMF / M. Kocabey et al.

Acids Res 2018; 46(D1): D1062-D1067. ants. Am J Hum Genet 2016; 99(4): 877-85. G, FUNG S-Y, SHARMA M, TURVEY SE:
[Link] [Link] Germline CBM-opathies: From immuno-
23. LIU X, LI C, MOU C, DONG Y, TU Y: dbNSFP 31. RICHARDS S, AZIZ N, BALE S et al.: Stand- deficiency to atopy. J Allergy Clin Immunol
v4: a comprehensive database of transcript- ards and guidelines for the interpretation of 2019; 143(5): 1661-73.
specific functional predictions and annota- sequence variants: a joint consensus recom- [Link]
tions for human nonsynonymous and splice- mendation of the American College of Medi- 39. GRINGHUIS SI, KAPTEIN TM, WEVERS BA
site SNVs. Genome Med 2020; 12(1): 103. cal Genetics and Genomics and the Asso- et al.: Dectin-1 is an extracellular pathogen
[Link] ciation for Molecular Pathology. Genet Med sensor for the induction and processing of
24. CUNNINGHAM F, ALLEN JE, ALLEN J et al.: 2015; 17(5): 405-23. IL-1β via a noncanonical caspase-8 inflam-
Ensembl 2022. Nucleic Acids Res 2022; [Link] masome. Nat Immunol 2012; 13(3): 246-54.
50(D1): D988-D995. 32. YANG YL, LI XM: The IAP family: endoge- [Link]
[Link] nous caspase inhibitors with multiple biolog- 40. WILLIS TG, JADAYEL DM, DU MQ et al.:
25. SHERRY ST, WARD M, SIROTKIN K: dbSNP- ical activities. Cell Res 2000; 10(3): 169-77. Bcl10 is involved in t(1;14)(p22;q32) of
database for single nucleotide polymorphisms [Link] MALT B cell lymphoma and mutated in mul-
and other classes of minor genetic variation. 33. LAWLOR KE, FELTHAM R, YABAL M et al.: tiple tumor types. Cell 1999; 96(1): 35-45.
Genome Res 1999; 9(8): 677-679. http:// XIAP loss triggers RIPK3- and Caspase- https://
[Link]/pubmed/10447503 8-driven IL-1β activation and cell death as [Link]/10.1016/s0092-8674(00)80957-5
26. KARCZEWSKI KJ, FRANCIOLI LC, TIAO G a consequence of TLR-MyD88-induced 41. TORRES JM, MARTINEZ-BARRICARTE R,
et al.: The mutational constraint spectrum cIAP1-TRAF2 degradation. Cell Rep 2017; GARCÍA-GÓMEZ S et al.: Inherited BCL10
quantified from variation in 141,456 humans. 20(3): 668-82. deficiency impairs hematopoietic and non-
Nature 2020; 581(7809): 434-43. [Link] hematopoietic immunity. J Clin Invest 2014;
[Link] 34. VINCE JE, WONG WW-L, GENTLE I et al.: 124(12): 5239-5248.
27. DENNIS G, SHERMAN BT, HOSACK DA et al.: Inhibitor of apoptosis proteins limit RIP3 [Link]
DAVID: Database for Annotation, Visualiza- kinase-dependent interleukin-1 activation. 42. LI J, SHI L, ZHANG K et al.: VarCards: an inte-
tion, and Integrated Discovery. Genome Biol Immunity 2012; 36(2): 215-27. grated genetic and clinical database for cod-
2003; 4(5): P3. http:// [Link] ing variants in the human genome. Nucleic
[Link]/pubmed/12734009 35. LABBÉ K, McINTIRE CR, DOIRON K, LE- Acids Res 2018; 46(D1): D1039-D1048.
28. WARDE-FARLEY D, DONALDSON SL, COMES BLANC PM, SALEH M: Cellular inhibitors [Link]
O et al.: The GeneMANIA prediction server: of apoptosis proteins cIAP1 and cIAP2 are 43. XU H, YANG J, GAO W et al.: Innate immune
biological network integration for gene pri- required for efficient caspase-1 activation by sensing of bacterial modifications of Rho
oritization and predicting gene function. the inflammasome. Immunity 2011; 35(6): GTPases by the Pyrin inflammasome. Nature
Nucleic Acids Res 2010; 38(Web Server is- 897-907. 2014; 513(7517): 237-41.
sue): W214-20. [Link] [Link]
[Link] 36. LOPEZ J, JOHN SW, TENEV T et al.: CARD- 44. NEOCLEOUS V, BYROU S, TOUMBA M et al.:
29. SZKLARCZYK D, GABLE AL, NASTOU KC mediated autoinhibition of cIAP1’s E3 ligase Evidence of digenic inheritance in autoin-
et al.: The STRING database in 2021: cus- activity suppresses cell proliferation and flammation-associated genes. J Genet 2016;
tomizable protein-protein networks, and migration. Mol Cell 2011; 42(5): 569-83. 95(4): 761-6.
functional characterization of user-uploaded [Link] [Link]
gene/measurement sets. Nucleic Acids Res 37. CHANG Y-S, HUANG H-D, YEH K-T, CHANG 45. UMAR M, MEGARBANE A, SHAN J et al.:
2021; 49(D1): D605-D612. J-G: Identification of novel mutations in en- Genome sequencing unveils mutational land-
[Link] dometrial cancer patients by whole-exome scape of the familial Mediterranean fever:
30. IOANNIDIS NM, ROTHSTEIN JH, PEJAVER V et sequencing. Int J Oncol 2017; 50(5): 1778- Potential implications of IL33/ST2 signal-
al.: REVEL: an ensemble method for predict- 84. [Link] ling. J Cell Mol Med 2020; 24(19): 11294-
ing the pathogenicity of rare missense vari- 38. LU HY, BIGGS CM, BLANCHARD-ROHNER 306. [Link]

2026 Clinical and Experimental Rheumatology 2023