Phenotype 2 Familial Mediterranean Fever: Evaluation of 22 Case Series and Review of the Literature on Phenotype 2 FMF

Abstract

Familial Mediterranean fever (FMF) is an autosomal recessive autoimmune disorder characterized by recurrent bouts of fever and serosal inflammation. FMF may be complicated by AA-type amyloidosis, worsening the prognosis, with associated renal failure in some patients. Complication rate varies with race, being as high as 60% in Turks and as low as 2% in Armenians. In a few cases of patients with FMF (phenotype 2), amyloid nephropathy may be the presenting manifestation. This study included 420 patients who were admitted to the Nephrology and Rheumatology Departments of Atatürk Education and Research Hospital with unexplained proteinuria/nephrotic syndrome. The initial screening test for amyloidosis was the presence of significant proteinuria (300 mg/24 h). All MEFV gene exons were screened for causative mutations by direct DNA sequencing to check for any mutations. There were 22 phenotype 2 FMF patients with 27 allelic variants. The most prevalent allelic variants were M694V (10/27, 37%) and E148Q (7/27, 26%). Phenotype 2 FMF is not as rare as it was thought before; this should be kept in mind for all patients with unexplained proteinuria and/or acute phase response in high-risk ethnic groups for FMF.

• familial Mediterranean fever
• MEFV gene mutation
• phenotype 2
• proteinuria
• amyloidosis

INTRODUCTION

Familial Mediterranean fever (FMF) is an autosomal recessive genetic disease, characterized by serositis, arthritis, and fever.¹ The disease may affect some ethnic groups, especially Turks, Arabs, Armenians, and Jews.² Pyrin or marenostrin encoded by MEFV gene has been suggested to play a role in inflammation.³ The relationship between FMF and its lethal complication secondary amyloidosis was established about 60 years ago. The drug colchicine has been shown to prevent development of amyloidosis. However, in untreated or noncompliant patients, amyloidosis may develop. In a large series of patients from Turkey, amyloidosis was reported to be present in 12.9% of the patients.⁴

FMF may be complicated by AA-type amyloidosis (AA), worsening the prognosis, with associated renal failure in some patients. Complication rate varies with race, being as high as 60% in Turks and as low as 2% in Armenians.⁵

In a few cases of patients with FMF (phenotype 2), amyloid nephropathy may be the presenting manifestation. These patients present with amyloid nephropathy before or without development of other symptoms of FMF. In this study, we examined 22 patients with phenotype 2 FMF,⁶ and discussed the literature.

MATERIALS AND METHODS

This study included 420 patients who were admitted to the Nephrology and Rheumatolology Departments of Atatürk Education and Research Hospital with unexplained proteinuria/nephrotic syndrome from January 2010 to July 2012. Forty-five of them had end-stage renal disease (ESRD). Patients were asked for a history of recurrent attacks of abdominal and/or pleural pain, fever, arthritis of the hip, knee or ankle, erysipelas like erythema, appendicitis, and renal disease. Patients who had clinical diagnosis of FMF, fulfilling the criteria for diagnosis of FMF,⁷ before development of amyloidosis were excluded.

To exclude the other etiologies of nephrotic syndrome and severe proteinuria, anticyclic citrullinated peptide, serum complement C3 and C4, antinuclear antibody, antidouble stranded DNA, antiglomerular basement membrane antibody, p-antineutrophil cytoplasmic antibody (ANCA), c-ANCA, anti-Ro, anti-La, and immunoglobulin G, A, M rheumatoid factor were evaluated; ophthalmic and funduscopic eye investigations were performed. None of the patients had diabetes mellitus, nor renal stone disease, and only one of them had mild hypertension—without eye involvement—as a comorbidity.

For genetic testing, 5 ml of blood was drawn from patients into tubes containing ethylenediaminetetraacetic acid (EDTA). DNAs were extracted using a commercial kit (QIAamp DNA mini kit; Qiagen, Hilden, Germany). In this study, sequencing was performed by using a 48-well capillary machine (MegaBACE, Amersham, UK) for the analysis of the whole MEFV coding sequence. All MEFV gene exons were screened for causative mutations by direct DNA sequencing in order to detect any mutations. We have applied this technique to approximately 150 different mutations and polymorphisms including single nucleotide polymorphisms in different exons of the MEFV gene. The sequencing data were analyzed with Sequencher 4.6 (Gene Codes, Ann Arbor, MI, USA). A hundred percent of these nucleotide alterations were distinguished by this method.

The 24-h urinary protein and microalbumin, serum albumin (ALB), creatinine, C-reactive protein (CRP), fibrinogen (FIB), urea, and erythrocyte sedimentation rates (ESR) were noted. The initial screening test for amyloidosis was the presence of significant proteinuria (300 mg/24 h). If this was present, duodenal–rectal biopsies (Bxs) were performed and if proteinuria was >1000 mg/day renal Bx was performed. The renal, duodenal, and rectal Bx specimens were stained with crystal violet for amyloidosis A (AA) with Lieb’s method. Then the slides were washed and rinsed with water. Duration was adjusted according to the control tissue sample properties. Congo Red staining was performed with Bennhold’s method.⁸

RESULTS

Twelve of the patients were male and 10 were female (M/F, 1.2). The mean age of the patients was 45 ± 9.2 years. The mean onset of the disease age was 0.11 years. Six of the patients underwent hemodialysis (HD) (3–48 months), the 5th, the 7th, the 8th, the 10th, the 21st, and the 22nd patients were under HD treatment for almost 3, 20, 22, 15, 48, and 12 months, respectively, after they were diagnosed as phenotype 2 FMF. Two patients (13th and 18th patients) had renal transplantation (Tx) (26–60 months) and were diagnosed after the renal Tx. One of the patients (20th patient) was already under continuous ambulatory peritoneal dialysis (CAPD) treatment for 24 months. The remaining patients were still not under any dialysis treatment. Two had family history for FMF, one had family history for renal disease, and none had family history for any other rheumatic diseases. The mean urea was 58.3 ± 29.3 mg/dL (normal range,10–48.5), creatinine was 2.4 ± 1.7 mg/dL (normal range, 0.7–1.2), ALB was 3.8 ± 0.5 g/dL (normal range, 3.5–5.2), ESR was 53.2 ± 34.5 mm/h (normal range, 0–20), CRP was 4.5 ± 8.7 mg/dL (normal range, 0–0.8), FIB was 433.9 ± 159.1 mg/dL (normal range, 150–400), and 24-h proteinuria and microalbuminuria were 2281.9 ± 1754.9 (normal range, 0–140) and 1383.2 ± 1401.9 mg/24 h (normal range, 0–30), respectively (Tables 1 and 2; 2).

Table 1.  Laboratory parameters in 22 patients with phenotype 2 FMF.

Patient no.	Urea (mg/dL)	Creatinine (mg/dL)	ALB (g/dL)	Proteinuria (mg/24 h)	Microalbuminura (mg/24 h)	CRP (mg/dL)	ESR (mm/h)	FIB (mg/dL)
1	24	0.9	4	3114	1106	0.14	3	292
2	25	0.8	4.1	329	255	0.5	6	290
3	31	0.8	4	2240	1856	6.2	50	290
4	61	1.5	3.8	4479	2607	0.19	60	303
5	97	4.2	3	1847	639	11.2	81	425
6	30	0.6	4.4	322	36	0.8	22	414
7	36	1.9	3.8	1539	1107	3	96	436
8	95	4.4	3.6	998	538	0.2	76	538
9	27	0.7	3.5	2537	2320	0.2	20	298
10	93	4.5	3.2	350	59	0.3	10	252
11	51	1.4	3.9	810	562	4.1	45	332
12	121	6.3	3.3	1456	909	2.1	40	313
13	36	1.8	4	4536	1536	36.2	110	565
14	76	2.2	4.4	2489	1813	1.73	101	731
15	58	1.9	4.5	732	601	0.4	11	315
16	29	0.7	4.6	3106	952	0.65	88	519
17	39	0.7	4	877	738	0.2	67	380
18	44	1.6	3.8	3201	1538	1.5	11	518
19	54	1.5	4.1	1505	472	1	44	475
20	76	4.1	4.4	2105	892	6.5	97	901
21	93	4.3	3.4	7524	6288	21.9	64	525
22	88	5.1	3.2	4106	3606	1.4	68	435

Table 2.  Distribution of MEFV variants, AA stain of biopsy specimen in 22 patients with phenotype 2 FMF.

Patient no.	Gender	Age (years)	Duodenal Bx	Renal Bx	Rectal Bx	MEFV variants
1	Female	46	N/A	AA(−)	N/A	A744S Heterozygous
2	Male	48	AA(−)	N/A	N/A	P369S Heterozygous
3	Female	39	AA(−)	N/A	N/A	A744S Heterozygous
4	Female	51	AA(+)	N/A	N/A	E148Q Heterozygous
5	Male	64	N/A	AA(+)	N/A	M694V/V726A
6	Male	27	AA(−)	N/A	N/A	M694V/E148Q
7	Male	50	AA(−)	N/A	N/A	E148Q Heterozygous
8	Female	42	N/A	N/A	AA(−)	P369S Heterozygous
9	Female	54	AA(−)	N/A	N/A	M694V Heterozygous
10	Male	26	AA(−)	N/A	N/A	A744S Heterozygous
11	Female	57	AA(−)	N/A	N/A	E148Q Heterozygous
12	Male	40	N/A	AA(+)	N/A	M694V Heterozygous
13	Male	37	AA(−)	AA(−)	AA(−)	E148Q/P369S
14	Female	44	N/A	AA(+)	N/A	M694V Heterozygous
15	Male	51	AA(−)	N/A	N/A	V726A Heterozygous
16	Female	49	N/A	N/A	AA(+)	A744S Heterozygous
17	Female	48	N/A	N/A	AA(−)	M694V Heterozygous
18	Male	38	N/A	AA(−)	N/A	E148Q Heterozygous
19	Female	46	AA(−)	N/A	N/A	E148Q Heterozygous
20	Male	33	N/A	AA(+)	N/A	M694V Heterozygous
21	Male	50	AA(+)	AA(+)	N/A	M694V/M680I
22	Male	50	N/A	N/A	AA(+)	M694V Homozygous

Note: N/A, not available.

The distribution of alleles according to disease status was shown in Table 2. There were 22 phenotype 2 FMF patients with 27 allelic variants. Five of the patients had heterozygous M694V and E148Q mutation, four had heterozygous A744S mutation, two patients had heterozygous P369S mutation, and the remaining patients had M694V/V726A mutation, M694V/M680I mutation, M694V/E148Q mutation, E148Q/P369S mutation, heterozygous V726A mutation, and homozygous M694V mutation. The most prevalent allelic variants were M694V (10/27, 37%) and E148Q (7/27, 26%) (Table 2).

Eight patients had amyloidosis demonstrated by renal and/or rectal and/or duodenal Bxs (Table 2).

DISCUSSION

In this study, we have shown that phenotype 2 FMF is not as rare as expected.

Reliable data presenting the epidemiology of systemic amyloidosis do not exist in the literature. In studies, which also include autopsy series, prevalence of amyloidosis was estimated to be 1/60,000.^9,10 Rheumatoid arthritis and the other chronic inflammatory diseases are the main causes of AA-type amyloidosis in western countries, although chronic infectious diseases are still the leading cause of AA-type amyloidosis in developing countries.^11–14 FMF is the main cause of systemic amyloidosis in Mediterranean basin countries in which Turks, non-Ashkenazi Jews, Armenians, and Arabs mostly live compared with other countries. In Turkey, FMF plays an important role in the etiology of systemic and renal amyloidosis and ESRD, especially in early ages.¹⁵

The most frequent mutation seen in FMF is M694V which is observed in 51% of the patients. V726A and M680I mutations are less frequently seen. Amyloidosis can be the only manifestation of the disease process in patients with phenotype 2 FMF.⁶ This kind of presentation with amyloidosis is rare. Risk groups include those with delay in diagnosis and positive family history for amyloidosis.⁴ In this study, M694V and E148Q were the most frequently detected mutations.

Cases with renal involvement usually present with nephrotic syndrome/edema, with renal dysfunction, and even with ESRD.¹¹ In a large series, AA-type amyloidosis patients were admitted to the hospital, of whom 97% had proteinuria, 11% had ESRD; in the progress, 33% of them developed ESRD and 44% died.¹² In this study, six patients were on HD, one patient was on CAPD, and two patients had renal Tx.

Congo Red is still the gold standard method for the diagnosis of amyloidosis, but it is a difficult technique for the diagnosis of amyloidosis.⁸ In a study, immunohistochemical analysis was reported as a helpful method to evaluate the overlooked or trace deposits of amyloidosis.¹⁶ In this study, Congo Red method (Liebs and Bennhold’s methods) for amyloid A staining was used for the evaluation of amount, localization, and type of glomerular accumulation.

In literature, there are some reports evaluating FMF related amyloidosis, but there are only a few reports concerning the phenotype 2 FMF. Three patients with similar features were described by Blum et al.¹⁷ In 1967, Sohar and colleagues reported eight cases of phenotype 2 FMF. In five patients, typical attacks appeared after the development of amyloidosis. The other three patients remained asymptomatic.¹⁸

In the study of Livneh et al, among 178 patients, 30 had amyloidosis. Twenty-seven of them were homozygous for M694V mutation. One patient was homozygous for both V726A and E148Q mutations. Amyloidosis was more common in patients homozygous for M694V mutation compared with patients with other mutations. Amyloidosis was the only manifestation of the disease (phenotype 2) in three patients homozygous for M694V mutation.¹⁹

Melikoglu et al. evaluated 461 relatives of 13 FMF patients with amyloidosis and six juvenile rheumatoid arthritis patients and found proteinuria in two cases whose rectal Bxs were negative for amyloidosis and concluded that phenotype 2 FMF is quite a rare disease.²⁰

Tunca et al. evaluated 2838 patients, and only nine of them were reported as phenotype 2 FMF.⁴ Genetic analysis was performed in four patients. Two patients were found to be homozygous for M694V, one was heterozygous, and no genetic mutation was detected in one patient.

M694V is one of the most common mutations in all ethnic groups and is associated with more severe symptoms and the development of amyloidosis.²¹ There are contradictory result between M694V homozygosity and development of amyloidosis.⁴ The development of amyloidosis cannot only be explained by genetic factors, since environmental factors may also affect it.⁴ Furthermore, amyloidosis progresses independently from the disease itself; that is, severity of the disease and amyloidosis development may not be concordant. In this study, we have demonstrated that besides the M694V mutation, other rarely seen or clinically nonsignificant mutations (E148Q, A744S, and P369S) may also cause development of phenotype 2 FMF associated amyloidosis.

In high-risk ethnic groups, any atypical presentation such as unexplained proteinuria/ESRD or acute phase response should be investigated for the possibility of underlying MEFV gene mutation carriage. Genetic screening should be recommended even in asymptomatic patients for FMF attack. Patients with nephrotic syndrome in high-risk ethnic groups for FMF should be investigated for amyloidosis with renal, rectal, duodenal, or skin biopsies since early treatment may prevent or delay disease progression.⁶

As a conclusion, phenotype 2 FMF is not as rare as it was thought before; this should be kept in mind for all patients with unexplained proteinuria and/or acute phase response in high-risk ethnic groups for FMF. Early diagnosis leads to early treatment of the disease and prevents or delays progression.

ACKNOWLEDGMENT

The authors thank Mr. Ferhat Bas for his technical support.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for content and writing of this paper.