Abstract
Objective
FMF has traditionally been considered an autosomal recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only one demonstrable MEFV mutation. Here, an extensive search for a second MEFV mutation was performed in 46 patients clinically diagnosed with FMF and carrying only one high-penetrance FMF mutation.
Methods
MEFV and other candidate genes were sequenced by standard capillary electrophoresis. The entire 15 kb MEFV genomic region was re-sequenced in 10 patients using a hybridization-based chip technology. MEFV gene expression levels were determined by qRT-PCR and pyrin protein levels were examined by Western blotting.
Results
A second MEFV mutation was not identified in any of the screened patients. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between single and double variant patients; however, FMF patients of both types showed higher protein expression compared to controls and non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare variants in a small number of patients, suggesting the possibility of digenic inheritance.
Conclusion
Our data underscore the existence of a significant subset of FMF patients who are carriers of only one MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of most common mutations appears sufficient in the presence of clinical symptoms to diagnose FMF and initiate a trial of colchicine.
Introduction
Familial Mediterranean fever (FMF, OMIM 249100) is an autosomal recessive autoinflammatory disease characterized by episodic, self-limiting attacks of fever along with abdominal pain, pleurisy, arthritis, and a distinctive rash (1). Systemic amyloidosis is the most severe manifestation of the disease, commonly affecting the kidneys (11% of cases), and sometimes the adrenals, intestine, spleen, lung and testis (2). Of the known hereditary periodic fevers, FMF is the most prevalent and best characterized. FMF is common in Middle Eastern populations, including Sepharadi and Ashkenazi Jews, Turks, Armenians, and Arabs and is not uncommon in other Mediterranean populations such as Italians, Spanish, Portuguese, French, and Greeks. FMF cases have also been described in many other populations, including Northern Europeans and Japanese (1). The carrier frequency for MEFV mutations is quite high in the four classically affected populations, ranging from 37–39% in Armenians and Iraqi Jews, to 20% in Turks, North African and Ashkenazi Jews, and Arabs. The high prevalence of carriers in multiple Middle Eastern and Mediterranean populations suggests that heterozygosity may confer a selective advantage. Despite high carrier frequencies in these populations, the prevalence of FMF is less than expected, indicating that the disease is either underdiagnosed or that disease-associated mutations have reduced penetrance.
The gene responsible for FMF, designated MEFV, encodes a 781 amino acid protein known as pyrin (alternatively, marenostrin) (3, 4). Pyrin is primarily expressed in polymorphonuclear cells, cytokine-activated monocytes, dendritic cells, and synovial fibroblasts (5, 6). Pyrin affects the inflammatory response by regulating the processing of mature IL-1β, a potent pyrogenic cytokine. Depending on the experimental system employed, pyrin has been shown to act as both as an inhibitor and an activator of IL-1β processing (7–9).
To date, over 50 disease-associated mutations have been identified in MEFV, with the majority of mutations being missense changes and more than half clustering in exons 2 and 10 (10). A subset of MEFV mutations (usually E148Q in exon 2 and M680I, M694I, M694V, and V726A in exon 10) may account for as much as 80% of FMF cases in classically affected populations (11); however, it has been observed that a substantial number of patients with clinical FMF (up to 30% depending on the population) possess only one demonstrable mutation despite sequencing of the entire coding region (12–16). These single-variant patients often have a typical disease history and respond well to colchicine, the standard treatment for FMF.
One explanation for this phenomenon is a lack of sensitivity in screening techniques. The majority of FMF patients in classically affected populations are screened for a limited number of mutations, which account for a majority of carrier chromosomes in a given population. This approach typically targets only the most prevalent MEFV mutations in a specific population, thus rare or novel variants can be overlooked. Another possibility is that the second disease-associated mutation may reside in the non-coding (intronic) or regulatory regions of MEFV, possibly affecting mRNA expression or splicing. The entire genomic region encompassing the MEFV transcript is 15 kb in size, thus it is not practical for diagnostic sequencing using standard techniques. Although most disease-associated mutations are missense nucleotide changes, the possibility of genomic re-arrangements (e.g. deletions, or copy number variations) cannot be excluded as another mechanism of disease. However, a recent study using multiplex ligation-dependent probe amplification (MLPA) failed to identify any MEFV copy number variations (CNV) in a large cohort of 216 FMF patients, suggesting that MEFV CNVs do not contribute to FMF pathogenesis (17).
Two recent reports have raised the question of dominant inheritance in FMF. Booth et al. described a single mutation associated with the disease in 3 unrelated British patients (M694DEL) and the M694I-E148Q complex allele in two FMF families of Turkish and Indian ancestry (15). Complete MEFV sequencing failed to identify any coding region abnormality in the other allele in any of these cases. In addition, a three-generation Spanish family with 5 affected members presenting with severe disease and amyloidosis was found to transmit a novel MEFV mutation, H478Y, in a clearly dominant manner (16). The existence of the second common disease-associated MEFV mutation was ruled out by intragenic haplotype analysis of the affected members. Taken together, these data strongly suggest that a single-gene recessive model of inheritance is incapable of fully describing the broad spectrum of MEFV-associated phenotypes.
There is also evidence suggesting genetic heterogeneity of FMF in Turkish and Armenian populations. A subset of Turkish FMF families appeared to be unlinked to chromosome 16p13.3, where MEFV resides (18), and a divergence from Hardy Weinberg equilibrium with a relative excess of patients without FMF mutations has been described among Armenian patients from the Karabakh (14).
In previous studies, only a few groups have attempted to search for FMF mutations in the entire coding region of MEFV (12–16). In this study, we report an extensive search for the second MEFV mutation in 46 patients clinically diagnosed with FMF and carrying only one known high penetrance FMF-associated mutation.
Patients and Methods
Patients
Patients included in this study were clinically diagnosed with FMF. About half of all patients (28/46) were seen at the National Institutes of Health, Bethesda MD, thus only a subset of patient records was available for the retrospective study. For participation in the study all patients (or parents or legal guardians if the patient was a minor) provided written informed consent as approved by the NIAMS/NIDDK institutional review board.
Mutational Analysis
Genomic DNA was isolated from peripheral blood leukocytes using the Gentra Puregene Blood Kit (Qiagen, Valencia, CA). For 46 patients, fluorescent sequencing was performed on the coding regions and splice junctions of MEFV with BigDye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, CA) on an ABI 3100 Genetic Analyzer. The sequencing data were analyzed with Sequencher 4.6 (Gene Codes, Ann Arbor, MI). For 10 patients, a hybridization-based resequencing system (Hychip: Callida Genomics, Sunnyvale, CA) was used to analyze 15 kb of genomic sequence encompassing all 10 exons, all 9 introns, and 1 kb of upstream sequence likely to include the promoter region of MEFV.
Control DNA genotyping
The allele frequencies of novel variants were evaluated in a panel of 376 or 750 Caucasian control DNA samples from the North American Rheumatoid Arthritis Collection (NARAC) or in a panel of 382 Jewish control DNA samples from the New York Cancer Project (NYCP) panel using mass spectrometry (the Sequenom homogeneous MassExtend assay, Sequenom Inc., San Diego, CA).
Gene Expression Analysis
Total RNA was isolated from peripheral blood mononuclear cells (PBMCs) using Trizol Reagent (Invitrogen, Carlsbad, CA) and standard chloroform extraction. The PBMCs were processed within 1 hour from drawing the blood. cDNA was synthesized from 1 µg of total RNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). For allelic expression, 5 sets of primers were designed to produce overlapping amplicons covering the entire coding region of MEFV. These amplicons were then sequenced using the method described above. The relative expression of MEFV was measured by real-time RT-PCR using TaqMan Gene Expression Assays with the 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Primers (Assay ID: Hs00925528-g1) targeting exon junction 9–10 were used, and the expression level was compared to the level of beta-2-microglobulin (B2M), which served as an internal control. The primer set was validated for its efficiency using a standard serial dilution of cDNA.
Protein Study
Patients were all on a treatment when sampled. Patient granulocytes were purified from 10–20cc of whole blood using standard dextran sedimentation protocol. Purified cells were treated with DFP (Diisopropyl fluorosphosphate, Sigma) to prevent proteolysis. Total protein from the cell lysates was subjected to SDS-PAGE for Western blotting with an anti-pyrin polyclonal antibody. The intensity of the pyrin band was measured by densitometry (Bio-Rad GS800Scanner, Quantity One software) and normalized to an endogenous control, GAPDH.
Structural modeling
A structure for the CARD domain of ASC was modeled on the crystal structures of two other CARD domains; from the proteins RAIDD (Chou, 1998 Cell), and Apaf-1 (Vaughn 1999, J Mol Biol). A structural alignment was first generated using the FUGUE algorithm (Shi 2001 J Mol Biol) and then energy minimization was performed using an implementation of GROMOS in the program Swiss-Model (Guex 1997 Electrophoresis). Structural images were created using the program PyMOL (http://www.pymol.org)
Results
Clinical Features of Patients with One Mutation
Table 1 summarizes the clinical features of 28 patients for whom detailed clinical histories were available. The remaining patients were not evaluated at the National Institutes of Health, and the respective referring physicians made the clinical diagnosis of FMF. In most patients, genetic testing was done after the FMF clinical diagnosis was already established. Within this cohort of single-variant patients, a variability of phenotype and some inconsistency in responsiveness to colchicine treatment was observed. All of the patients met FMF clinical criteria based on the experiences of the Sheba Medical Center of Tel Hashomer in Israel (19), though most of them presented with an incomplete abdominal attack (abdominal pain without frank peritonitis) as the major criterion of the disease. We adopted a conservative approach in defining the abdominal attacks as incomplete, as no patients were seen at the NIH during an attack. Three patients did not report any abdominal pain while in attack, but presented with pleurisy and/or arthralgia. The onset of the disease was variable, ranging from 1 month to 40 years, and the duration of attacks was less than a week, with the majority of attacks lasting 2–3 days. Sixteen of the 28 patients reported arthralgia, and 12 patients developed skin rash. Colchicine response was either complete or partial in 84% of patients (21/25), with the exception of 4 patients carrying the delM694 mutation who did not respond at all. Information on colchine responsiveness was unavailable for 3 patients. Fourteen patients (56%) had a complete response, while 7 patients (28%) had periodic attacks of inflammation although less frequently while on treatment (Table 1).
Table1.
Clinical symptoms of FMF patients with only one identified MEFV mutation.
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|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M694V/? | 27* | Armenian | F | 30yo | 3–7d | N | N | Y | N | Y | Y | N | Y | complete |
| M694V/? | 108 | Armenian | M | 1mo | 3d | Y | N | N | N | N | U | N | Y | complete |
| M694V/? | 116* | Sephardic/Ashken Jewish |
F | childhood | 2–3d | Y | Y | Y | Y | N | N | N | N | partial |
| M694V/? | 139 | Sephardic Jewish | F | 12yo | U | Y | Y | Y | N | N | N | N | N | partial |
| M694V/? | 171 | Italian | F | 14yo | 2–3d | Y | Y | N | N | N | N | N | Y | complete |
| M694V/? | 234 | Greek | F | childhood | NA | Y | Y | N | N | Y | N | Y | Y | complete |
| M694V/? | 248* | Sephardic Jewish | F | 17yo | 3–5d | Y | Y | Y | N | Y | Y | N | Y | partial |
| M694V/? | 337 | mixed Euro/ Iraqi Jewish | M | 6y | 1–2d | Y | Y | Y | N | N | N | N | N | not known |
| M694V/? | 381 | Italian | F | 16yo | 4–7d | Y | Y | N | N | Y | N | N | N | infreq attack |
| M694V/? | 395* | Russian/AshkenaziJewis h |
M | 19yo | 1–2d | Y | Y | Y | N | N | N | N | N | complete |
| M694V/? | 817 | Italian | F | 17yo | NA | N | Y | Y | N | N | Y | N | N | complete |
| M694V/? | 1198 | Jordanian/English | M | 5yo | 2–3d | Y | Y | Y | N | N | N | Y | Y | partial |
| M694V/? | 1312 * |
Turkish | M | 20yo | <24h r |
Y | Y | N | N | Y | N | N | N | complete |
| M694V/? | 1313 | Jewish | M | childhood | 3–4d | Y | Y | N | N | Y | N | N | N | complete |
| M694V/? | 1454 | Italian/Irish/German | M | 12yo | 3–4d | Y | Y | Y | N | N | N | N | N | not known |
| M694V/? | 1630 * |
Armenian/ European | F | 13y | 2–3d | Y | Y | N | N | Y | N | N | Y | complete |
| delM694/? | 407* | mixed European | F | 1yo | 3–5d | Y | Y | Y | N | N | N | Y | N | none |
| delM694/? | 408 | mixed European | F | 10yo | 3–4d | Y | Y | Y | N | N | N | Y | Y | none |
| delM694/? | 1803 | German-Welsh | M | 30y | 1–2d | Y | Y | N | N | N | N | N | N | none |
| delM694/? | 1804 | German-Welsh-Hispanic | M | 1 mo | 3–4d | Y | Y | N | N | N | N | N | N | intolerant |
| V726A/? | 1044 | Jewish/Dutch | M | 4yo | 3–5d | Y | N | Y | N | Y | N | N | Y | partial |
| V726A/? | 785 | Ashkenazi Jewish | M | 8yo | 3–5d | Y | Y | N | N | Y | N | N | Y | complete |
| E148Q,V726A/ ? |
977* | Ashkenazi Jewish | F | 13yo | 5–7d | Y | Y | N | N | N | N | N | Y | complete |
| E148Q,V726A/ ? |
1532 | Sephardic Jewish | M | childhood | 2–3d | Y | Y | Y | N | N | N | N | Y | complete |
| E148Q,V726A/ ? |
1533 | Ashkenazi Jewish | M | childhood | 2–3d | Y | Y | Y | N | Y | Y | N | Y | complete |
| E148Q,V726A/ ? |
1587 | Ashkenazi Jewish | M | 40Y | 2–3d | N | Y | N | N | N | N | N | Y | complete |
| R653H/? | 569* | Lebanese/Irish | F | 1yo | 5d | Y | Y | N | N | Y | N | N | Y | partial |
| R761H/? | 1048 | mixed European, Jewish |
M | 1yo | 2–3d | Y | Y | N | N | N | N | N | Y | partial |
Denotes patients sequenced throughout the 15 kb genomic region encompassing MEFV
U Indicates that patients data was not available
- complete response: patients who respond to colchicine, but may have very infrequent attacks
- partial response: patients who are taking colchicine regularly but still have less frequent attacks
- intolerant: patients who can not tolerate colchicine due to side effects
Most of the 46 patients were carriers of M694V or V726A mutations. Included in this study were patients with apparently dominantly inherited disease and with uncommon ancestry. For example, patients 1803 and 1804 were a father and son with German-Welsh ancestry. Patients 407 and 408 were a mother and daughter of mixed European ancestry. All 4 of these patients were carriers for a single M694DEL mutation. Patients 367 and 742 were sibling carriers with the rare mutation R653H and were of mixed European ancestry. Although many of the patients included in this study were of Middle Eastern background, about half of the patients had an atypical ancestry, either European or mixed Middle Eastern/European. We have also identified the first reported African-American FMF patient, who possessed the common FMF mutation, M694V.
MEFV mutation screening
Utilizing standard sequencing, 46 FMF patients with one MEFV mutation were screened for a second disease-associated mutation in all 10 exons of MEFV, including exon-flanking regions. Thirty-one of these patients possessed a mutation affecting residue M694 (M694V, M694I, M694DEL), and six patients were carriers for another common FMF associated mutation, V726A. Of the remaining 9 patients, 3 had the R653H mutation, one patient each was a carrier for R761H and S702C, and four patients were carriers for the complex allele V726A-E148Q as shown by family studies. Additionally, the MEFV coding region was sequenced in 13 FMF-like patients who carry milder FMF-associated mutations/functional polymorphisms such as E148Q and K695R. No new mutation was identified in any of the patients analyzed.
A subset of 10 DNA samples was selected for additional sequencing of the entire 15kb MEFV genomic region using a hybridization-based chip technology (Callida Genomics, CA). This method is capable of detecting single nucleotide variations as well as small deletions or insertions (20). All but three of 46 patients were found to be heterozygous for at least one of the many known SNPs in the MEFV genomic region, arguing against the presence of large genomic deletions (Table 2). In addition, the MEFV genomic region was amplified through a set of overlapping PCR fragments, and based on the size of these fragments, no intragenic genomic deletions were identified. A novel heterozygous variant was identified in the putative regulatory region 1kb upstream of the start codon at position c.−888G>A in a North American patient (# 407). This heterozygous change was determined to be a SNP based on its allele frequency of 4% in a panel of 350 Caucasian controls and 3% in a panel of 378 Ashkenazi Jewish controls.
Table 2.
The intragenic MEFV haplotype analysis of FMF patients with positive family history
| IDs | Mutation/ Haplotype |
2.1 D102D |
2.2 G138G |
2.3 A165A |
2.4 R202Q |
3.1 R314R |
5.1 E474E |
5.2 Q476Q |
5.3 D510D |
Intron 6 c.1610+96C>T |
Intron 8 c.1759+8C>T |
Exon 9 P588P |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Med/A haplotype | C | G | A | A | C | G | A | T | C | A | A | |
| 116 | M694V | C | G | A | A | C | G | A | T | C | A | A |
| 116 | 2nd HAPLOTYPE | T | A | C | G | C | G | A | T | C | T | A |
| 139 | M694V | C | G | A | A | C | G | A | T | C | A | A |
| 139 | 2nd HAPLOTYPE | T | A | C | G | C | G | A | T | C | A | A |
| delM694 haplotype | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G | |
| 407 | delM694 | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 407 | 2nd HAPLOTYPE | T | G | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 408 | delM694 | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 408 | 2nd HAPLOTYPE | C | G | A | A | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 1803 | delM694 | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 1803 | 2nd HAPLOTYPE | C | G | A | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 1804 | delM694 | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| 1804 | 2nd HAPLOTYPE | T | A | C | G | C/T | A/G | A/G | C/T | C/T | A/T | A/G |
| R653H haplotype | T | A | C | G | C | G | A | C | C | A | A | |
| 742 | R653H | T | A | C | G | C | G | A | C | C | A | A |
| 742 | 2nd HAPLOTYPE | T | A | C | G | C | G | A | C | C | A | A |
| 367 | R653H | T | A | C | G | C | G | A | C | C | A | A |
| 367 | 2nd HAPLOTYPE | T | A | C | G | T | A | G | T | T | T | G |
Haplotype analysis
Haplotype analysis was performed with patients who had affected first-degree relatives. First, a haplotype associated with the disease-associated mutation within a family was deduced. For example in a Jewish family with M694V (patients 116,139), the A (also known as Med) haplotype, a previously reported founder haplotype associated with the M694V mutation, was identified (Table 2) (3, 21). The patients, a mother and daughter, shared the same second haplotype, which could be consistent with the existence of a common mutation on the second allele, or could simply reflect a conserved haplotype in the population. In a second family, two affected siblings carriers for the R653H mutation (patients 367,742) did not entirely share the same haplotype on the second chromosome. The intragenic haplotype analyses were done with other sporadic FMF patients who carry the M694V mutation, but none shared a common haplotype that could be associated with a mutation in the second allele. Interestingly, the first and only African-American patient with FMF described thus far has the same Med (A) haplotype associated with M694V as patients in other Mediterranean populations.
In 2 families with 4 patient carriers for M694DEL (patients 1803, 1804 and patients 407, 408), an unambiguous common intragenic M694DEL-associated haplotype was identified only at the 5 ‘end of the gene. The 5’ end haplotype (T-A-C-G) based on 4 SNPs within exon2 appears to be a relatively common haplotype in the general population (Table 2). All four patients were heterozygous for the same alleles in SNPs residing at 3’end of the gene. Under the recessive model of inheritance, there was no common haplotype within each family that could be associated with a putative mutation on the second allele.
MEFV Expression
Allelic expression was performed to examine if both MEFV transcripts were expressed in 8 FMF patients. The RNA was converted into cDNA and the entire transcript was sequenced with 5 overlapping amplicons. All patients were carriers for at least one heterozygous variant, indicating that both alleles were expressed. In addition, no transcript size variants were identified in any of the 8 patients.
qRT-PCR was then used to compare the expression levels of MEFV in a group of single variant patients to a group of double-variant patients and a group of healthy controls. MEFV expression levels were measured with primers directed against exon junction 9–10, and β2 microglobulin (B2M) levels were measured as an internal control. Due to the large degree of intra-group variability and the relatively small number of samples examined, a significant difference in the expression level of MEFV was not demonstrated in any of the three groups (Fig 1). In contrast to previous findings (22, 23), there was a trend for higher MEFV expression in patients compared to controls.
Figure 1.
Mean relative expression levels of MEFV (exons 9–10) comparing FMF patients with 1 MEFV mutation, patients with 2 mutations, and healthy controls. All results are relative to the mean of the healthy control group. No significant difference in expression level was observed between any of the groups. Values represent fold change and were generated using β2 microglobulin (B2M) as the endogenous control. Error bars indicate standard error of the mean.
Pyrin expression in FMF patients
To date, only one study has examined pyrin protein expression in the cells of FMF patients (24). To further investigate the observed trend of higher MEFV gene expression, Western blot analysis was performed on cell lysates isolated from PBMCs using a polyclonal antibody for pyrin. Due to the presence of multiple bands, these blots proved difficult to analyze. This experiment was then repeated with lysates isolated from polymorphonuclear cells (PMN) cells which highly express pyrin in its native state (Fig 2A). The WBs were clear and showed a trend of higher pyrin levels in FMF patients compared to healthy controls. Although there was variability among samples in each group, we did not observe a statistically significant difference between patients with one or two disease mutation. However, there was a significant difference in the pyrin levels between FMF patients and controls (p=0.007, Fig 2B). To investigate whether the higher pyrin expression is specific to FMF patients, we compared pyrin levels in FMF patients to non-FMF patients with active inflammation. We observed significantly higher pyrin expression in FMF patients in disease remission relative to patients with non-FMF related active inflammatory disease (inflammatory bowel disease and chronic granulomatous disease) and to controls (Fig 3 A, B).
Figure 2.
A. Western blots of granulocyte lysates from FMF patients with one or two MEFV mutations and healthy controls probed with Abs to pyrin or GAPDH. B. Densitometry analysis of pyrin bands. Values represent pyrin expression relative to GAPDH in each individual and are shown as pyrin’s percentage of GAPDH (pyrin/GAPDH×100). Values are normalized to the mean pyrin expression in the healthy control group within each individual blot. Patient groups exhibited significantly higher levels of pyrin as determined by a Mann-Whitney U test (P = 0.007). There was no significant difference in pyrin levels between patients with one or two MEFV mutations. Error bars indicate standard error of the mean.
Figure 3.
A. Western blots of granulocyte lysates from non-FMF patients with active inflammation (ID), FMF patients, and healthy controls probed with Abs to pyrin or GAPDH. B. Data analysis was done as described in Figure 2. FMF patients exhibited significantly higher levels of pyrin as determined by a Mann-Whitney U test (FMF vs. patients with active inflammation P=0.0286, FMF patients vs. controls P=0.0095). There was no significant difference in pyrin levels between patients with active inflammation and controls. Error bars indicate standard error of the mean.
Screening for mutations in other known autoinflammatory genes
Although these patients did not appear to present symptoms associated with other autoinflammatory diseases, the possibility that they may have mutations in other known autoinflammatory genes was considered. Fourteen patients were screened for the TRAPS-associated mutations in TNFRSF1A, and only one patient (1454) was found to carry the R92Q reduced penetrance mutation. The same 14 patients tested negative for the two most common HIDS-associated mutations (I268T and V377I).
Digenic model of inheritance
Oligogenic and digenic inheritance has recently been described in the genetics of several diseases that were initially characterized as monogenic disorders. Bardet-Biedl syndrome and deafness are examples of such diseases initially thought to be recessively inherited (25). Thus, we considered the possibility that FMF patients with only one MEFV mutation might have a second disease-associated mutation in a gene that acts in concert with MEFV to produce a disease phenotype. Likely candidates for this second mutation are genes encoding proteins known to interact with pyrin or genes that have some function in regulating the IL-1 β pathway.
Several genes encoding proteins fitting this description were chosen for analysis in 10 patients: ASC, SIVA, CASP1, PSTPIP1, POP1, and POP2. In a single FMF patient, a novel PSTPIP1 nucleotide variant was identified at position c. 540 G>A, which did not change the amino acid at position K181. Two novel missense substitutions were identified in the genes encoding ASC and SIVA, while the genes encoding CASP1, POP1, and POP2 were mutation negative. In exon 3 of SIVA, the novel substitution, 416 G>A, was identified at codon 120 replacing a valine with a methionine (V120M). We screened 374 Caucasian controls samples for this allele as well as 382 Jewish control samples to match the ancestry of the original carrier. Two carriers were identified in the Caucasian panel and 12 carriers in the Jewish panel, thus qualifying V120M as a novel polymorphism (Supplemental Table 1).
In contrast to most other genes associated with autoinflammatory pathways, ASC contains no known sequence variants. We identified a novel variant, W171X (513G>A), in a Jewish FMF patient, which introduces a premature stop codon that could delete the last 24 amino acids of the protein. At a structural level, a homology model for the CARD domain of ASC was generated and it could be observed that this premature stop codon removes the last two of the 6 helices in the CARD domain of ASC (Figure 4). Only a single carrier for W171X was identified in a panel of 371 Caucasian DNA samples and 2 carriers were found in 382 Jewish DNA samples. The combined allele frequency of W171X in both panels of control samples is 0.004 (Supplemental Table 1). We also screened a panel of 70 FMF patients with only one MEFV mutation, including milder FMF-associated mutations. In this cohort, one additional patient of Turkish background was identified as a carrier of W171X.
Figure 4.
A modeled structure for the CARD domain of ASC (residues 109–195). This homology model, based on the CARD domain structures of RAIDD and Apaf-1, describes the ASC CARD domain as a 6-helix structure adopting a Greek key fold. The mutation W171X would truncate the final two alpha-helices from the C-terminus, and these are highlighted in blue.
Discussion
In this study, we searched for a second disease-associated mutation in a panel of 46 FMF patients with only one documented MEFV mutation. To date, this is the most comprehensive search for a second disease-associated mutation. Two different sequencing techniques were utilized in this endeavor, and both failed to identify a second mutation in the MEFV gene.
MEFV transcript analysis established the presence of both alleles in a subset of 8 patients. In this same cohort of eight patients, relative gene expression analysis via qRT-PCR did not show a significant difference in the MEFV expression level among FMF patients with one or two mutations. These data contrast two previous studies showing that FMF patients with one or two MEFV mutations appear to express lower levels of MEFV when compared to healthy controls (22, 23). Like these previous studies, our experiments were carried out with total RNA derived from peripheral blood mononuclear cell (PBMCs); however, our study differs in the time frame in which samples were processed, the real-time PCR platform used, the reaction chemistry and primer sets utilized, and the manner in which the data were analyzed.
In order to study this question at the level of expressed protein, we examined pyrin levels in the granulocytes of FMF patients. Granulocytes are perhaps the most relevant cells to study in FMF patients since the symptoms of disease are caused by a massive influx of granulocytes into affected areas. This is the first study to investigate this question in unstimulated patient cells. Samples were collected from 13 FMF patients and 6 controls, and we observed no significant difference in pyrin expression between patients harboring one vs. two mutations. We did observe a significant increase (p=0.007) in pyrin expression in FMF patients compared to controls, consistent with our observed trend for increased mRNA expression in FMF patients. The experiment shown in Figure 3A and 3B suggests that the increase in pyrin expression is not merely a consequence of inflammation, and appears to be specific for FMF patients. This intriguing result should be corroborated with studies of pyrin expression in additional FMF patients and in patients with active disease.
There are multiple explanations for the apparent divergence from the typical paradigm of recessive inheritance seen in FMF. Against the hypothesis of pseudo-dominant inheritance, there is the fact that a number of FMF patients with a single MEFV mutation in this study have atypical ancestry, thus the possibility of having the second FMF-associated mutation in two successive generations is highly unlikely. Previous studies of FMF families with Spanish and British ancestry also failed to identify the common haplotype that should be associated with the transmission of the second MEFV mutation within a pedigree (15, 16). In the present study, we were unable to identify one or two common haplotypes co-segregating with the second disease-associated mutation both in familial and sporadic cases.
Although the possibility that these patients have other periodic fevers such as TRAPS, HIDS, or CAPS cannot be completely excluded, it is highly unlikely for several reasons: patients clinically appear to have FMF, significant numbers of patients respond to colchicine, and other periodic fevers are typically uncommon in patients from the Middle East. Nevertheless, we have screened 14 FMF patients for TRAPS and HIDS-associated mutations. One patient with mixed European ancestry was identified as a carrier of the R92Q mutation, which has a carrier frequency of 2–5% in Caucasians depending on the population. Our most recent data indicate that the R92Q carrier frequency in North American Caucasian control samples is 0.038 (Supplemental Table 1).
One remaining explanation is that having only one MEFV mutation may give rise to a FMF phenotype in the presence of one or more modifying alleles in other related genes, or other environmental factors like a stress. Asymptomatic carriers for one FMF mutation have biochemical evidence for subclinical inflammation (26, 27) and a recent study found a higher frequency of carriers for highly penetrant FMF mutations among patients with systemic inflammatory response syndrome (SIRS) and sepsis (28). Further supporting this hypothesis is the observation that patients who carry complex MEFV alleles appear to have more severe disease (29). Previous studies in FMF patients showed that the presence of modifying alleles in MICA and SAA are associated with a severe FMF phenotype and susceptibility to amyloidosis (30, 31). Therefore, modifying alleles could contribute to an inflammation dosage threshold, which is necessary to develop systemic inflammation and symptomatic FMF.
Although hereditary recurrent fevers are considered monogenic diseases, a few reports have recently described patients who are compound heterozygotes for mutations in two known recurrent fever genes (32–34). These patients were found to have two or more reduced penetrance mutations such as E148Q in MEFV, R92Q or P46L in TNFRSF1A, V377I in MVK, and V198M in CIAS1. In some cases, patients presented with symptoms of both diseases, or with a more severe disease, and their treatment was also compromised due to unknown gene interactions among mutations in the known recurrent fever genes (35). Considering that these variants have carrier frequencies close to or higher than 1% in control populations, it is likely that compound heterozygotes will be identified. Supplemental Table 1 summarizes the carrier frequencies for these variants generated in our lab using Caucasian and Jewish control DNA samples. Finally, as a major referral lab for patients with recurrent fevers we have tested samples from more than 1900 patients and the majority of them are negative for mutations in known recurrent fever genes, suggesting that there are additional recurrent fever genes to be identified. Thus, the interactions between mutations and modifying alleles among known and unknown recurrent fever genes could give rise to a range of inflammatory phenotypes.
Under the hypothesis of a digenic inheritence, we screened 6 candidate genes for mutations. Two missense variants were identified, one in SIVA and one in ASC. Although the SIVA variant appears to be a polymorphism, it could still have potential consequences in FMF pathogenesis. Both the variant in SIVA and ASC are currently under investigation. Although our candidate gene approach was limited, it is the first attempt to investigate polygenic inheritance in FMF. Ideally, this question should be interrogated using a genome wide association study (GWAS) similar to those conducted for complex diseases. This approach would require setting up a large international collaborative project, which would include FMF patients with only one highly penetrant MEFV mutation and the presence of the second-disease associated mutation completely ruled out.
This study has shown for the first time that pyrin expression appears higher in the granulocytes of FMF patients compared to controls. The relevance of this finding is difficult to interpret, as there is some controversy regarding the function of pyrin. Depending on the experimental model used, pyrin has been shown to both activate and inhibit the caspase-1/IL-1β signaling pathway (7–9, 36, 37). An explanation for why pyrin levels are higher in patient granulocytes would greatly depend on pyrin’s function in the cell and whether the mutations associated with FMF are gain or loss of function. Increased levels of pyrin could lead to an increase in the caspase-1/IL-1β signaling pathway and would explain the apparent dominant inheritance of FMF in some patients. Alternatively, FMF mutations could cause a loss of function and the observed increase in pyrin expression may be a compensatory mechanism to recover this deficit.
Pyrin likely regulates the NF-Kβ pathway, apoptosis, and possibly other aspects of inflammation independently of IL-1β, so it is reasonable to assume that pyrin’s role in modulating inflammation may be more complex than previously hypothesized. Given the high carrier frequency of FMF mutations and the less than expected prevalence of the disease, it seems possible that other alleles could modify inflammatory signals initiated by mutant pyrin. Thus, FMF may not be a simple monogenic inflammatory disease and the FMF phenotype may occur in patients with only one MEFV mutation in the presence of other permissive alleles or environmental factors.
Our study has two important messages for the practitioner. First, screening for the set of most common mutations seems to be sufficient in the presence of clinical symptoms to diagnose FMF and initiate a trial of colchicine. Second, our data underscore the need for continued referral of single-mutation cases to research laboratories actively investing potential modifier genes to facilitate the identification of new susceptibility loci.
Acknowledgments
We would like to thank all of the physicians who referred their patients to us for molecular diagnostic testing. We would also like to acknowledge Dr. Peter Gregersen for sharing the North American Rheumatoid Arthritis Collection and New York Cancer Project control samples and Dr. Doug Kuhns and Ms. Deborah Long Priel for their expertise and help in processing patient granulocytes.
This study was supported by the Intramural Research program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
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