Diagnosis of Familial Mediterranean Fever by a Molecular Genetics Method

Methods

Patients and DNA Samples

We obtained DNA samples from 107 persons. A total of 74 persons from 44 families gave specimens at the Sheba Medical Center in Tel Hashomer, Israel, as part of the project to identify the familial Mediterranean fever gene by positional cloning. Forty-three of these families were of non-Ashkenazi Jewish ancestry, and one family was Druze. Of the 74 family members, 19 had familial Mediterranean fever according to established clinical criteria [16], 48 were unaffected but were familial Mediterranean fever carriers according to haplotype analysis, and 7 were unaffected and were noncarriers according to haplotype analysis. Two persons (1 affected person and 1 asymptomatic carrier) from 1 Armenian-American family also gave blood as part of the same positional cloning project at the Cedars-Sinai Medical Center. Thirty-one additional persons gave or sent samples to the National Institutes of Health for genetic testing after the familial Mediterranean fever gene had been identified. Of these 31 persons, 27 had undiagnosed fever syndromes and 4 were unaffected family members. By DNA sequencing, 1 person was an M694V homozygote, 2 were M694V/M680I compound heterozygotes, 6 were symptomatic but had only one copy of the three mutations, and 4 were asymptomatic heterozygote carriers. The remaining 18 persons were negative for all three mutations by DNA sequencing.

Informed consent of the participants in the study was obtained after approval by the human experimentation committee at each institution. Standard techniques were used to extract DNA from whole blood or from Epstein-Barr virus-transformed lymphocytes [17].

Mutation Detection by the Amplification Refractory Mutation System

The amplification refractory mutation system (ARMS) assay comprises two complementary reactions, each conducted with the same substrate DNA. One reaction includes an ARMS primer specific for the normal DNA sequence and cannot amplify mutant DNA at a given locus. The second reaction includes a mutant-specific primer and cannot amplify normal DNA. The same common primer is used in both reactions [17, 18].

The lack of PCR products according to use of a specific mutation primer set in patients suspected of carrying the mutation for familial Mediterranean fever suggests that the patient in question is not carrying the mutation being probed. However, an appropriate internal PCR control should be run to show that the DNA is amplifiable. Therefore, the complementary reaction with the normal primer set serves as an internal control for PCR amplification and allows discrimination of homozygotes from heterozygotes.

Mutations were assessed by amplifying the genomic DNA template with three sets of normal and mutant-specific ARMS primers designed to selectively amplify the normal or altered sequence of each of the three MEFV mutations. Each set of primers consisted of three oligonucleotides. For mutation M680I, the sequences were 5′-TTAGACTTGGAAACAAGTGGGAGAGGCTGC-3′ (common), 5′-ATTATCACCACCCAGTAGCCATTCTCTGGCGACAGAGCG-3′ (mutant), and 5′-ATTATCACCACCCAGTAGCCATTCTCTGGCGACAGAGCC-3′ (normal); for M694V, they were 5′-TGACAGCTGTATCATTGTTCTGGGCTCTCCG-3′ (common), 5′-TCGGGGGAACGCTGGACGCCTGGTACTCATTTTCCTTCCC-3′ (mutant), and 5′-TCGGGGGAACGCTGGACGCCTGGTACTCATTTTCCTTCCT-3′ (normal); and for V726A, they were 5′-TGGAGGTTGGAGACAAGACAGCATGGATCC-3′ (common), 5′-TGGGATCTGGCTGTCACATTGTAAAAGGAGATGCTTCCTG-3′ (mutant), and 5′-TGGGATCTGGCTGTCACATTGTAAAAGGAGATGCTTCCTA-3′ (normal).

Each DNA sample was tested for the three mutations. The PCR amplification was performed in a final volume of 25 µL containing 100 ng of purified genomic DNA, 0.04 U of Ampli Taq Gold (Perkin-Elmer, Branchburg, New Jersey) and its 1x PCR buffer (contains 15 mmol of MgCl₂ per L), 0.2 mmol of deoxynucleoside 5′-triphosphate mix per L (Gibco BRL, Gaithersburg, Maryland), and 1 pmol of each primer. Amplification conditions were kept the same for all of the ARMS tests, and the procedure was carried out as follows. The reaction was heated to 94°C for 9 minutes for denaturation, followed by 35 cycles with denaturation at 94°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 30 seconds. Final extension was done for 10 minutes at 72°C.

The amplified products were separated by electrophoresis on a 2% agarose gel. Ethidium bromide staining of the agarose gel was used to detect the amplified fragments.

Results

Samples of DNA from persons affected by familial Mediterranean fever, their relatives, and normal controls were screened to determine mutation status. The results of a representative assay for each mutation are shown in Figure 1.

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Figure 1. M680I. Middle. M694V. V726A. For each mutation, the ARMS assay consists of two ARMS reactions specific for the normal or mutant sequences per person. Samples from each study participant were analyzed for each mutation by performing the two reactions. Order of loading was kept from left to right for each reaction. Genotypes (“normal” for homozygous normal, “affected” for homozygous familial Mediterranean fever [FMF], and “carrier” for heterozygous familial Mediterranean fever) were inferred from the combined results of both reactions. Water was added as a negative control. The approximate size of the amplified fragments was 200 base pairs (bp); bands migrating anodal to the specifically amplified bands are the residual primers. Detection of three MEFV mutations by amplification refractory mutation system (ARMS) assay. Top.Bottom.

A summary of the results obtained for the study group is shown in the (Table 1). For 82 samples that were studied, all three mutations were found in accordance with mutation status. None of these mutations was identified in the remaining 25 samples, as predicted by previous sequencing analysis. The most frequent mutation in our predominantly non-Ashkenazi Jewish panel was M694V. All persons with the M680I mutation were of Armenian ancestry, whereas the V726A mutation was found in Armenians, Ashkenazi Jews, and Iraqi Jews. No false-positive or false-negative results were obtained by using the three sets of primers for each sample, indicating a sensitivity and specificity of 100% for this assay.

Discussion

We describe a simple, rapid, and highly reliable method for establishing the molecular diagnosis of familial Mediterranean fever, a disease primarily diagnosed on a clinical basis [3-7]. The results were validated by analyzing previously genotyped samples and proved to be totally accurate. For 107 samples that were independently genotyped by automatic sequencing as part of the project to identify the familial Mediterranean fever gene by positional cloning [14], no false-positive or false-negative results were obtained with the ARMS assay.

The ARMS assay used for molecular diagnosis in our study allows accurate detection of haplotypes with mutations involving single-base changes or small deletions [18, 19]. We developed a single ARMS assay for detection of the three most common MEFV point mutations described during the cloning of the familial Mediterranean fever gene [14, 15]. In addition to the reliability of the ARMS assay, several practical considerations associated with this assay may be of interest. First, genomic DNA or, alternatively, crude cell lysate of leukocytes is used as a source of template DNA. Second, it is not necessary to prepare high-quality DNA suitable for restriction enzyme digestion. Finally, the use of radioactive materials is not required. Moreover, additional mutations could be studied by using the same method; the three disease-associated mutations discussed here do not account for all familial Mediterranean fever carrier chromosomes, and at least eight additional mutations are under study ([20]; Aksentijevich et al. In preparation).

It seems that about 70% of carrier chromosomes from non-Ashkenazi Jews in Israel bear the M694V or V726A mutation. A population study that should clarify this issue is already under way. In a recent study of Turkish familial Mediterranean fever [21], the three mutations presented here accounted for 29 of 34 disease alleles. The occurrence of these mutations in the United States may be somewhat lower [20]. In persons with suggestive clinical and family history but no documented common mutation, an ARMS assay with additional sets of primers designed for other mutations or complete sequencing of the MEFV gene is indicated.

Accurate diagnosis of familial Mediterranean fever is important not only for genetic consultation and avoidance of unnecessary colchicine therapy but also for prevention of avoidable laparotomies due to misdiagnosed acute abdomen. Moreover, some patients develop amyloidosis early in the course of the attacks of familial Mediterranean fever (type II) [6], and proper genetic consultation may suggest early introduction of colchicine in these cases. The rapid, cost-effective ($150 according to Hadassah Hospital estimates), and highly reliable ARMS assay allows fast detection of the familial Mediterranean fever mutations prevalent in Israel. It enables early molecular confirmation of suspected cases of familial Mediterranean fever, thus preventing undesirable abdominal surgery and unnecessary colchicine therapy. In addition, it enables rapid typing of patients with familial Mediterranean fever and their relatives, thereby allowing proper genetic and therapeutic consultation, especially when early amyloidosis is suspected.

Drs. Aksentijevich, Deng, and Kastner: Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892.