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ORIGINAL ARTICLE
Ahead of print publication  

The effects of epigenetic regulation on phenotypic expressivity in Turkish patients with familial Mediterranean fever


1 Department of Pediatrics, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
2 Department of Pediatrics, Division of Genetics, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
3 Department of Biochemistry, Izmir Bozyaka Training and Research Hospital, Izmir, Turkey
4 Department of Pediatrics, Division of Nephrology, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey
5 Department of Medical Genetics, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey

Correspondence Address:
Ozlem Giray Bozkaya,
Department of Pediatrics, Division of Pediatric Genetic Diseases, Faculty of Medicine, Dokuz Eylul University, Inciralti, Izmir 35340
Turkey
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/injr.injr_24_19

  Abstract 


Introduction: In this study, we aimed to characterize the effect of methylation on clinical diversity and gene expression levels in familial Mediterranean fever.
Materials and Methods: Forty children, who were diagnosed with FMF according to the Tel-Hashomer criteria, were included in the study. The control group consisted of 32 healthy children. Demographic data, results of molecular studies, physical examination findings, number of attacks, response to treatment were recorded. To test the possibility that methylation is responsible for different phenotypic reflections of mutations, we classified FMF patients with respect to MEFV mutations and studied the differences of MEFV mRNA, pyrin and methylation levels between different subgroups.
Results: MEFV mRNA expression levels were significantly lower in FMF patients, compared to control subjects (P = 0.049). Consistent with this observation, pyrin levels were significantly lower in FMF patients (P = 0.037). In addition, we found that MEFV methylation level was higher in FMF patients compared to control subjects (P = 0.376).
Conclusions: There was no evidence that differences in methylation levels could be responsible for variable effects of different mutations and variable clinics in the MEFV gene. Further studies on larger patient groups are necessary to identify the effects of different factors.

Keywords: Autoimmune, disease association studies, epigenetics, methylation, pediatrics



How to cite this URL:
Dogan E, Gursoy S, Bozkaya G, Camlar SA, Kilicarslan OA, Soylu A, Ulgenalp A, Kavukcu S, Bozkaya OG. The effects of epigenetic regulation on phenotypic expressivity in Turkish patients with familial Mediterranean fever. Indian J Rheumatol [Epub ahead of print] [cited 2019 Nov 20]. Available from: http://www.indianjrheumatol.com/preprintarticle.asp?id=269122




  Introduction Top


Familial  Mediterranean fever More Details (FMF) is an autosomal recessive hereditary disease, which is characterized by episodes of fever attacks accompanied by erysipelas-like erythema and peritonitis, arthritis, and pleurisy due to serous membrane inflammation. FMF is common in Mediterranean populations, such as Turks, Armenians, and non-Ashkenazi Jews.[1],[2] Symptoms of FMF usually manifest in the first decade. More than 80% of patients are diagnosed during childhood or adolescence, and only 5% of the patients are diagnosed during adulthood.[3] Patients are diagnosed clinically according to the Tel-Hashomer criteria. Tel-Hashomer criteria are used for FMF diagnosis. Definitive diagnosis is done when 2 major criteria or 1 major plus 2 minor criteria are met. On the other hand, a probable diagnosis of FMF is established when 1 major criterion and 1 minor criterion are met.[3]

“Mediterranean Fever” (MEFV) gene was defined on the short arm of chromosome 16 in 1991.[4]MEFV gene encodes pyrin/marenostrin, which plays a role in inflammation through the activation of caspase-1. Caspase-1 is responsible for activation of nuclear factor-kappa B and maturation of interleukin-1β.[5],[6] Several disease-causing mutations on the MEFV gene have been reported. The majority of reported mutations are on the exon 10, on the nucleotide sequence corresponding to 681st–761th amino acids. The five most common mutations are M694V, M694I, M680I, V726A, and E148Q, and these mutations are found in approximately 80% of patients who are admitted with clinical findings of FMF.[7],[8] Homozygous M694V mutation is associated with early age at onset.[1]

For autosomal recessive diseases, clinical symptoms emerge when both alleles are mutant. On the other hand, some studies have provided evidence that some patients, who are diagnosed with FMF and respond well to colchicine treatment, have only a single mutation while several patients do not have any MEFV mutations at all.[3],[9] Potential explanations for these observations include the presence of a novel mutation in regulatory regions that is not screened during routine genetic analysis, epigenetic factors, dysfunction of other genes regulating the gene's expression, polygenic or multigenic nature of the disease, and dysfunctions related to protein transport.

DNA methylation is the most common epigenetic mechanism, which predominantly occurs at cytosine residues on CpG islands.[10] Methylation causes transcriptional silencing through inhibition of elongation of RNA polymerase-2 transcription.[11] Different studies have reported that MEFV mRNA expression is lower in patients with MEFV mutations; yet, some studies suggest that MEFV mRNA expression is higher in patients with MEFV mutations.[12],[13]

It is known that different mutations on the MEFV gene can cause various clinical phenotypes. For instance, the clinical symptoms are more severe in patients with homozygous M694V mutation. In addition, despite having similar genotypes, even in case of individuals having the same parents and the same mutations, clinical symptoms of FMF patients can differ significantly. In this study, we investigated the relationship of this clinical diversity with differences in MEFV gene methylation levels and expression pattern.

After classifying patients according to the results of mutation analysis, we determined MEFV methylation patterns and expression levels and analyzed the effects of MEFV methylation levels on gene expression and clinical findings to elucidate the role that methylation plays.

Besides, differences in mRNA and protein levels were investigated to put forth the possibility that methylation is responsible for different phenotypic reflections of different mutations.


  Materials and Methods Top


Forty children (2–17 years), who were diagnosed with FMF according to the Tel-Hashomer criteria and tested for MEFV gene mutation, in a 6-month period, were included in the study. At the time of blood collection, none of the patients showed clinical symptoms of FMF. The control group consisted of 32 healthy children, who were admitted to the Healthy Child Outpatient Clinic for screening. The local ethics committee approved the study (2014/25-12), and written informed consent was obtained from all individuals involved.

Demographic data, results of molecular analysis, complaints, physical examination notes, number of attacks, response to treatment, complete blood count, and urine protein measurements were recorded.

Total RNA was isolated from peripheral blood samples using “Prime Perfect Pure RNA Blood Kit” (#2302110). High-Capacity cDNA Reverse Transcription Kit (ABI, #4368814) was used to reverse transcribe total RNA samples into cDNA. cDNA samples were used to analyze MEFV gene expression in real time-polymerase chain reaction.

To analyze MEFV methylation levels, “EZ DNA Methylation-Gold™ Kit” (Zymo Research) was used to perform bisulfite modification on DNA samples. Bisulfite method was used to synthesize two different DNAs (methylated and unmethylated) to measure DNA methylation level. Methylation analysis was carried out on exon 2 of the MEFV gene, as it is the richest region for CpG islands[14] [Figure 1].
Figure 1: Sequence analysis of exon 2 after bisulfite modification. The black arrows indicate CpG islands, in which methylated cytosine bases are not converted to thymine. The green arrows indicate unmethylated cytosine bases, which are converted to thymine on bisulfite modification

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For pyrin measurement with ELISA, venous blood samples were collected from FMF patients and controls after 10 h of fasting of patients and controls. Blood samples were collected into 8 ml vacuumed and gel separator tubes (Vacuette, Greiner Bio-One, Austria). Blood samples were allowed to coagulate for 30 min, and then, centrifuged at 3000 rpm for 10 min at room temperature. Serum samples were aliquoted and stored at −20°C until further analysis. A commercial sandwich ELISA kit (Sunred, Republic of China) was used to measure for serum pyrin levels, and spectrophotometric measurements were performed at 450 nm wavelength on a “Thermo Scientific Multiskan GO” ELISA plate reader. Spectrophotometric measurements of diluted standards were used to plot a standard curve, and pyrin concentrations (ng/ml) were calculated accordingly.

Patient groups

Due to the small sample size and wide distribution of mutations, we were unable to evaluate mutations individually. To compare the effect of methylation pattern on clinical findings we grouped the patients according to results of mutation analysis. Group A1: patients who have two or more disease-causing allele changes on the MEFV gene (n = 22; 55%) and Group A2: patients who have a disease-causing single allele change on the MEFV gene (n = 18; 45%). The mean age at onset was 6.3 ± 2.5 years in Group A1 and 7.0 ± 3.4 years in Group A2 (P = 0.513). The detailed clinical findings are summarized in [Table 1].
Table 1: Clinical findings of patients in Group A1 and Group A2

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As the clinical symptoms are more severe in patients with homozygous M694V mutation, we also classified FMF patients into two different subgroups. Group B1: patients carrying M694V mutation in at least one allele of MEFV gene (n = 22) and Group B2: patients who do not have any M694V mutation in any allele of MEFV gene (n = 18).

In addition, to test the minimalization effect of R202Q variation on clinical severity, we also classified patients as: Group C1: patients carrying M694V mutation in at least one allele of MEFV gene without R202Q variation (n = 9) and Group C2: patients carrying M694V mutation in at least one allele of MEFV gene together with R202Q mutation (n = 13). The clinical findings of these subgroups are summarized in [Table 2].
Table 2: Clinical findings of patients in Groups B1, B2, C1, and C2

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Statistical analysis

SPSS 15 (SPSS Inc., Chicago, IL, USA) software was used for statistical analysis. Data were presented as mean ± standard deviation. Chi-square test was used to compare ratios between groups. Fisher's exact Chi-square test was used for variables with small cell sizes (<5). Student's t-test was used to compare the mean values between two groups with a sample size >30. Mann–Whitney U-test was used to compare the mean values between two groups with a sample size <30. P < 0.05 was considered as statistically significant. Correlation analysis was done by the Pearson correlation test.


  Results Top


We analyzed 40 patients from different families and 32 controls. The mean age was 10.2 ± 3.6 years (2–17 years) in the study group and 12 ± 3.8 years (3–17 years) in the healthy control group. The mean age at onset of clinical symptoms was 6.6 ± 3.1 years (2–14) and the mean age at diagnosis was 7.3 ± 3.2 years (2–15 years). The mean number of attacks per year was 9.2 ± 5.8. Fourteen patients (35%) had a family history of FMF. Interestingly, the family history of FMF was the most common in mother and siblings. Five patients (12.5%) had a history of consanguineous marriage in their parents.

The two most common symptoms were abdominal pain (82.5%) and fever (82.5%). In the study group, the clinical details of the subgroups were summarized in [Table 1] and [Table 2].

Genotype–phenotype relationship

Mutation analysis showed a heterogeneous distribution of MEFV mutations. The results of mutation analysis with respect to allele frequencies are shown in [Table 3].
Table 3: Allele frequencies of familial Mediterranean fever patients

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Comparison of Group B1 and Group B2 showed that the mean number of attacks per year was higher in patients who did not have M694V mutation but was not statistically significant (P = 0.79). Similarly, comparison of Group C1 and Group C2 showed that the mean number of attacks per year was higher in Group C2, but this difference was not statistically significant (P = 0.18).

Comparison of MEFV gene expression patterns

When we compare MEFV gene expression levels among FMF patients (n = 40) and controls (n = 32), MEFV mRNA expression was significantly lower in the patient group (P = 0.049) [Figure 2]. The mean MEFV mRNA expression levels of the study and control groups were 0.008758 and 0.012388, respectively. However, there were no significant differences in MEFV mRNA expression levels among patient groups (Group A1 vs. A2, Group B1 vs. B2, and Group C1 vs. C2; P = 0.07; P = 0.73, and P = 0.51, respectively).
Figure 2: Expression level of Mediterranean fever gene in patient and control groups.RNA was isolated from peripheral leukocytes as described in materials and methods. Expression levels were measured by real time polymerase chain reaction. The expression levels of the familial Mediterranean fever patients and control groups were 0.008758 and 0.012388, respectively. The expression was significantly lower in familial Mediterranean fever patients compared to healthy controls

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Comparison of pyrin measurements

The mean pyrin levels of the study and control groups were 20.027 and 27.480 ng/ml, respectively. Pyrin levels were significantly lower in FMF patients, compared to controls (P = 0.037) [Figure 3]. When we compared pyrin levels among patient groups (Group A1 and A2, Group B1 and B2, Group C1 and C2), there were no significant differences between the groups (P = 0.93, P = 0.51, and P = 0.55, respectively).
Figure 3: Comparison of pyrin measurements in familial Mediterranean fever patients and control groups. ELISA kit was used to measure the serum pyrin levels. The details of analysis were described in material and method section. The mean pyrin levels of the study and control groups were 20,027 and 27,480 ng/ml, respectively. Pyrin levels were significantly lower in familial Mediterranean fever patients, compared to controls (P = 0.037)

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Comparison of MEFV gene methylation patterns

Next, we compared methylation levels between 40 FMF patients and 32 healthy children in the control group and found that methylation level was slightly higher in FMF patients; however, the results are not significant (P = 0.376) [Figure 4]. The mean methylation levels of the study and control groups were 70.1% and 69.3%, respectively. Furthermore, methylation level was not different in Groups A1 versus A2 (P = 0.37) and Group C1 versus C2 (P = 0.29).
Figure 4: Comparison of Mediterranean fever gene methylation patterns between patient and control group. Bisulfite method was performed to measure DNA methylation levels. When we compared the study and control groups, we detected that the methylation level was slightly higher in patient group; however, the results are not significant (P = 0.376)

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In addition, there was no correlation between MEFV mRNA levels and methylation levels. Groupwise comparisons did not show any correlation between methylation levels and MEFV mRNA levels.

Similarly, we did not determine a correlation between pyrin levels and methylation levels. When all groups were evaluated individually, there was no correlation between methylation levels and pyrin levels.

There was no significant difference in methylation levels between patients with and without joint involvement (P = 0.08). Similarly, there was no significant difference in methylation levels between patients with myalgia and patients without myalgia (P = 0.82).


  Discussion Top


Considering methylation as a potential factor, we aimed to determine the effects of methylation on the development of the disease, clinical diversity, and gene expression in FMF. For this purpose, we analyzed the MEFV mRNA expression, methylation pattern, and pyrin levels in FMF patients and healthy controls.

FMF can present itself with various phenotypic findings and has genotypic diversity. The disease has an autosomal recessive inheritance and has a prevalence of 1/1000 in Turkey. Family history of FMF has been reported in 18%–41% of FMF patients.[15] In this study, 35% of the patients had a family history of FMF. The Turkish FMF study group carried out a multicenter study involving 35 centers and 2.838 cases and determined that the mean age at onset is 9.6 years and the mean age at diagnosis is 16.4 years.[1] In this study, the mean age at onset of symptoms was 6.6 years and the mean age at diagnosis was 7.3 years.

High fever is generally seen during FMF attacks and lasts for 12–72 h. On the other hand, fever is relatively low during mild attacks and in patients who use colchicine and may not be noticed. In our study, recurrent high fever was seen in 33 of 40 patients (82.5%). According to the Turkish FMF study group's study in 2005, the incidence of recurrent high fever in FMF patients is 92.5%.[1]

Abdominal pain is one of the most common symptoms in FMF. Clinical condition and laboratory findings are consistent with acute peritonitis. In this study, abdominal pain was found in 33 of 40 patients (82.5%). According to the Turkish Study Group data, abdominal pain has an incidence of 93.7% in FMF patients.[1] Similarly, the authors carried out a study in 1558 patients and reported that the incidence of abdominal pain is 95%.[16]

Joint pain and arthritis are other common features of FMF. Arthritis is classically monoarticular, and knee, elbow, or hip involvement are the most common options. In this study, joint pain was seen in 20 of 40 patients (50%). While joint pain has an incidence of approximately 75% in Jews of North African origin, this rate is <50% in other FMF populations.[17] The authors reported that the incidence of joint pain ranges between 27% and 70%.[18] Unilateral or bilateral chest pain usually results from pleuritis and is reported in 25%–50% of the patients. Symptoms usually last for 24–72 h and are commonly unilateral. On the other hand, pericarditis is a rare feature of FMF and is difficult to diagnose unless it is complicated with tamponade.[19],[20] In this study, chest pain was seen in 10 of 40 patients (25%). The frequency of skin-related findings ranges between 12% and 43% in different studies.[21] Among these findings, erysipelas-like erythema is the most common one. We have noticed that 5 of our 40 patients (12.5%) had erysipelas-like erythema.

M694V, M680I, M694I, E148Q, and V726A were the five most common mutations, most of which were located on exon 10. Previous studies have shown that M694V mutation is common among Turkish people and Jewish people living in the Mediterranean region.[1],[15] According to the Turkish Study Group's study in 1090 patients, M680I and V726A are the second and third most common mutations in Turkish patients.[1] In this study, we determined that M694V and R202Q were the most common variations.

DNA methylation which was the most common epigenetic mechanism occurs at the cytosine bases in CpG islands present in the gene promoter. Epigenetic mechanisms such as histone modifications, methylation, and microRNAs (miRNAs) may play a role in the pathogenesis of FMF.[22] miRNAs are small, noncoding RNAs that regulate gene expression at the posttranscriptional level by degrading mRNA or blocking its transcription. Methylation levels were slightly higher in FMF patients, compared to controls (P = 0.376). The lack of a significant difference in methylation levels between FMF patients and healthy controls suggests that other factors besides methylation are also effective in disease development and clinical diversity among patients. Previously, Kirectepe et al. compared methylation levels between 30 FMF patients and 21 controls and similarly found that the methylation level is slightly higher in patients with FMF.[14]

In the literature, the researchers showed that MEFV mRNA expression is significantly lower in FMF patients, compared to healthy controls.[12] When we compared MEFV gene expression levels between FMF patients and controls, MEFV gene expression was significantly lower in FMF patients (P = 0.049).

The clinical symptoms are more severe in patients who have M694V mutation whereas patients with E148Q and/or R202Q variations have milder symptoms.[23],[24]MEFV mRNA levels were slightly higher in patients in Group B2 (P = 0.070). Previous studies have shown a correlation between the type of MEFV mutation and MEFV mRNA expression level. MEFV mRNA expression levels are lower in patients carrying M694V mutation, especially those with a homozygous mutation. MEFV mRNA expression is higher if patients have E148Q variation, which is associated with mild clinical outcome. However, a comparison of patients with any MEFV mutation with controls indicates that MEFV mRNA expression levels are lower in FMF patients.[12]

Genetic variations in the regulatory region of the MEFV gene or other genes may affect MEFV gene expression and create similar FMF phenotypes. Reduced levels of MEFV methylation, without any MEFV gene mutations, can lead to the FMF phenotype. A significant correlation between MEFV gene expression and disease severity is found in patients who have M694V mutation in one allele or in patients who have homozygous M694V mutations.[25] In this study, we did not determine any significant difference due to the small number of patients with homozygous M694V mutation. Furthermore, this correlation is not seen in patients who have both M694V mutation and other mutations with milder clinical symptoms (e.g., V726A, E148Q, and R202Q). Taken together, these findings are considered as the protective effects of these mutations.[22]

Several hypotheses have been suggested to play a role in the etiology of FMF. Pyrin protein is known to have the most significant role in FMF pathogenesis. Pyrin is considered to inhibit the inflammatory response by enhancing the inhibition of pro-inflammatory molecules and transcription of anti-inflammatory proteins. Mutations on the MEFV gene lead to the production of abnormal pyrin protein, which in turn prevents efficient inhibition of inflammation. This results in a clinical condition with elevated and dysregulated inflammation. In our study, we determined a significant reduction in pyrin levels in FMF patients, compared to healthy controls (P = 0.037). Most studies have shown that pyrin protein levels are generally lower in FMF patients; on the other hand, there are reports indicating lower pyrin levels in controls. Booty et al. determined that pyrin levels are higher in FMF patients. In addition, the authors did not find any correlation between having a single mutation or two mutations and pyrin levels. Comparison of FMF patients, healthy controls, and patients with non-FMF inflammation showed that pyrin levels are higher in FMF patients.[25]

Previous studies have shown that colchicine treatment is not correlated with MEFV mRNA expression levels. In this study, we were unable to make such a comparison, as all patients were on colchicine treatment.


  Conclusion Top


It is clear that different mutations in the MEFV gene lead to different clinical outcomes related to disease severity. At the same time, it is known that different individuals with a given mutation can exhibit considerably diverse clinical findings. Taken together, we did not find any correlation between methylation rates and MEFV gene mutations, MEFV mRNA levels, pyrin levels, and clinical findings. Regulation of gene expression is a considerably complex process, which involves various unknown factors. Further studies on larger patient groups are necessary to identify the effects of different factors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Sedivá A, Horváth R, Maňásek V, Gregorová A, Plevová P, Horáčková M, et al. Cluster of patients with familial Mediterranean fever and heterozygous carriers of mutations in MEFV gene in the czech republic. Clin Genet 2014;86:564-9.  Back to cited text no. 15
    
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[PUBMED]  [Full text]  
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