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.
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
). 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 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
). 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
) 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
). 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
). 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
). 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.