AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 28:241-242 © 1992 MUNKSGAARD
Mapping of the Familial Mediterranean Fever Gene to Chromosome 16 LUIS GRUBERG, IVONA AKSENTIJEVICH, ELON PRAS, DANIEL L. KASTNER, AND MORDECHAI PRAS Department ofMedicine F, Sheba Medical Center (L.G.), Tel Hashomer 52621, Israel, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes ofHealth (lA., DL.K.), Bethesda, Maryland, and Heller Institute for Medical Research, Sheba Medical Center (M P.), Tel Hashomer 52621, Israel ABSTRACT: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever, synovitis, peritonitis, or pleurisy. Some patients eventually develop systemic amyloidosis. The biochemical cause of the disease is unknown. We have conducted a genome-wide search for the FMF locus using 125 different DNA markers and mapped the FMF gene to the short arm of chromosome 16. The study was performed on 35 Israeli families primarily of North African and Iraqi origin. For the five markers D16S82 (p41·1 Sael), D16S80 (24-1 Taql), DI6S84 (pCMM65 Taql), D16S83 (pEKMDA2·1 Rsal), and HBA (5'HVR Rsal) we obtained maximum lod scores of 2.72 (6 = 0.08), 10.34 (6 = 0.04),9.66 (6 = 0.050,9.35 (6 = 0.03), and 14.31 (6 = 0.08), respectively. Multipoint analysis with BBA and DI6S84 defined as a fixed loci gave a maximum lod score of 19.86 centromeric to D16S84. Crossovers defined by these markers place the FMF gene in an area of approximately 5 cM between D16S80 and DI6S84. Other genes mapped to this area (16pI3.3) inelude phosphodiesterase m (PDEIB), hydroxyacyl-glutathione hydrolase (HAGH), phosphoglycolate phosphatase (PGP), and the gene that causes adult polycystic kidney disease (PKD1). None of these genes bear an obvious pathophysiological relationship to FMF. Using additional markers from this region we hope to localize more precisely the FMF gene and to offer the possibility of prenatal diagnosis in selected cases. Our ultimate goal is to isolate and characterize the FMF gene. (Am J Reprod Immunol. 1992; 28:241-242.) Key words: Familial Mediterranean Fever, gene, chromosome INTRODUCTION
Familial Mediterranean fever, (FMF) is an autosomal recessive disease that affects primarily individuals of nonAshkenazi Jewish, Armenian, Turkish, and Arab origin.l? The gene frequency is very high (1:16) amongst Jews of Libyan origin and within the Armenian population in Los Angeles (1:14).5 The disease is characterized by intermittent attacks of high fever with severe abdominal pain, pleurisy, or arthritis. The arthritis generally affects the big joints of the lower extremities, having no sequelae, but 6%-8% of the patients go into a protracted course that may last weeks to years, and may lead to destructive changes at the hip joints. Amyloidosis is the most serious and fatal
Submitted for publication August 27,1992; accepted September I, 1992. Address reprint requests to Elon Pras, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.
feature of FMF; it affects mainly the kidneys and leads to end-stage renal disease." The Heller Institute at Tel Hashomer now sees more than 2,200 FMF patients. In 1972, the use of continuous colchicine treatment was described by Goldfinger," Since then we have proved its efficacy in preventing not only the attacks, but also the development of amyloidosis." We have followed very small children, some below the age of3, and closely monitored more than 500 pregnancies. In a collaborative study of the Heller Institute at Tel Hashomer, Israel, and the Genetic Unit of the Arthritis and Rheumatism Branch at the National Institutes of Health, Bethesda, Maryland, we have been involved in the search of the FMF gene, using reverse genetics techniques in our search to uncover the gene of FMF and the molecular and biological properties of its encoding proteins. MATERIALS AND METHODS
The first phase of the project began in Israel. We obtained blood samples from more than 350 FMF patients and their family members, most of North African, Iraqi, and Turkish origin. From 20 ml of heparinized blood, peripheral blood lymphocytes were immortalized with Epstein-Barr virus and cultured, producing an endless source of genetic material from each individual. The DNA was extracted and digested with restriction enzymes, electrophoresed and transferred to a nylon membrane, then hybridized with radioactive probes whose chromosomal location is known. The nylon membranes were exposed to X-ray film and the different bands revealed were read and interpreted. The data were loaded into a computer with the Linkage program? and a lod score derived. (Lod score is the logarithm to the base 10 of the odds in favor oflinkage to the odds against linkage.) A lod score of more than 3 means that the odds in favor oflinkage are 1000:1, which by convention is proof oflinkage, and a lod score of - 2 is considered evidence against linkage. RESULTS
In our search for the FMF gene, two different strategies were used: (1) Because the real cause of the disease is not yet known, and its principal feature is the inflammatory process, in the candidate gene approach we looked into the genes that are thought to be involved in the inflammatory reaction that we see in FMF, such as the genes for complements 3 and 5, complement receptors 1 and 2, decay accelerating factor, C4 binding protein, interleukins In and 113, interleukins 6 and 8, lipocortins I and II, serum amyloid A and dopamine l3-hydroxylase. lO None proved to be linked to FMF. (2) The second approach consisted in a methodological exclusion of the different chromosomes. We looked at 107 different probes that covered one-third of the human genome, (chromosomes 1,5, 10,9, 15, and 17)11 without success. However, probe number 108, known as pCMM65 (DI6S84), that maps
242
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to the telomeric region of the short arm of chromosome 16,12 gave us a positive lod score of 9.17 at a distance of - .04 cM. To confirm this result, we looked at another marker in this region that is associated with the hemoglobin (X complex that maps 5 cM telomeric to the first probe.P giving us a lod score of 14.45. Using multipoint linkage analysis we got a maximal lod score of 19.86. Thus the possibilit6. that the gene for FMF is located in this region is 10 2 :1, far more than the 103 : 1 that is required as proofoflinkage. 14 CONCLUSION
Description of the chromosomal location of the gene is the first step that ultimately will allow the identification of the gene itself, and in this way it should provide new insights into the pathophysiology not only of this disease, but also into the mechanisms of inflammation inman. We hope that in the near future the present mapping ofthe FMF gene to the short arm of chromosome 16 will enable a decisive laboratory diagnosis of equivocal FMF patients and will improve genetic counseling and prenatal diagnosis. REFERENCES 1. Sohar E, Gafni J, Pras M, Heller H. Familial Mediterranean fever. A survey of 470 cases and review of the literature. Am J Med. 1967; 43:227-253. 2. Schwabe AD, Peters RS. Familial Mediterranean reverin Armenians. Analysis of 100 cases. Medicine (Baltimore). 1974; 53:453-462.
3. Ozdemir AI, Sokmen C. Familial Mediterranean fever among Turkish people. Am J Gastroenterol. 1969; 51:311-316. 4. Barakat MH, Karnik AM, Majeed HWA, El-Sobki NI, Fenech FF. Familial Mediterranean fever (recurrent hereditary polyserositis) in Arabs. A study of175 patients and review of the literature. Quart J Med. 1986; 60:837-847. 5. Rogers DB, Shohat M, Petersen GM, Bickal J, Congleton J, Schwabe AD, Familial Mediterranean fever in Armenians: Autosomal recessive inheritance with high gene frequency. Am J Genetics. 1989; 34:168-172. 6. Meyerhoff J. Familial Mediterranean fever: Report of a large family, review ofthe literature, and discussion of the frequency of amyloidosis. Medicine (Baltimore). 1980; 59:66-77. 7. Goldfinger SE. Colchicine for familial Mediterranean fever. N Engl J Med. 1972; 287:1302. 8. Zemer D, Pras M, Sohar E, Colchicine in the prevention and treatment of the amyloidosis in familial Mediterranean fever. N Engl J Med. 1986; 314:1001-1005. 9. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Nat! Acad Sci USA. 1984; 81:3443-3446. 10. Gruberg L, Aksentijevich I, Balow J, Dean M, Kovo M, Pras M, Kastner DL. Exclusion of candidate genes in familial Mediterranean fever. Cytogen Cell Genet. 1991; 98:2113. (Abstr) 11. Aksentijevich I, Gruberg L, Balow JE Jr, Dean M, Pras M, Kastner DL. Linkage analysis in familial Mediterranean fever. Am J Hum Genet. 1991; 49 (4 Suppl):335. (Abstr) 12. Nakamura Y, Martin C, Krapcho K, O'Connell P, Leppert M, Lathrop GM, Isolation and mapping of a polymorphic DNA sequence (pCMM65) on chromosome 16 (D16S84). Nucleic Acids Res. 1988; 16:3122. 13. Jarman Ap, Higgs DR. A new hypervariable marker for the human Alpha-globin gene cluster. Am J Hum Genet. 1988; 43:249-256. 14. Pras E, Aksentijevich I, Gruberg L, Balow JE Jr, Prosen L, Dean M, Steinberg AD, Pras M, Kastner DL. Mapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16. N Engl J Med. 1992; 326:1509-1513.
Mapping of the Familial Mediterranean Fever Gene to Chromosome 16 LUIS GRUBERG, IVONA AKSENTIJEVICH, ELON PRAS, DANIEL L. KASTNER, AND MORDECHAI PRAS Department ofMedicine F, Sheba Medical Center (L.G.), Tel Hashomer 52621, Israel, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes ofHealth (lA., DL.K.), Bethesda, Maryland, and Heller Institute for Medical Research, Sheba Medical Center (M P.), Tel Hashomer 52621, Israel ABSTRACT: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever, synovitis, peritonitis, or pleurisy. Some patients eventually develop systemic amyloidosis. The biochemical cause of the disease is unknown. We have conducted a genome-wide search for the FMF locus using 125 different DNA markers and mapped the FMF gene to the short arm of chromosome 16. The study was performed on 35 Israeli families primarily of North African and Iraqi origin. For the five markers D16S82 (p41·1 Sael), D16S80 (24-1 Taql), DI6S84 (pCMM65 Taql), D16S83 (pEKMDA2·1 Rsal), and HBA (5'HVR Rsal) we obtained maximum lod scores of 2.72 (6 = 0.08), 10.34 (6 = 0.04),9.66 (6 = 0.050,9.35 (6 = 0.03), and 14.31 (6 = 0.08), respectively. Multipoint analysis with BBA and DI6S84 defined as a fixed loci gave a maximum lod score of 19.86 centromeric to D16S84. Crossovers defined by these markers place the FMF gene in an area of approximately 5 cM between D16S80 and DI6S84. Other genes mapped to this area (16pI3.3) inelude phosphodiesterase m (PDEIB), hydroxyacyl-glutathione hydrolase (HAGH), phosphoglycolate phosphatase (PGP), and the gene that causes adult polycystic kidney disease (PKD1). None of these genes bear an obvious pathophysiological relationship to FMF. Using additional markers from this region we hope to localize more precisely the FMF gene and to offer the possibility of prenatal diagnosis in selected cases. Our ultimate goal is to isolate and characterize the FMF gene. (Am J Reprod Immunol. 1992; 28:241-242.) Key words: Familial Mediterranean Fever, gene, chromosome INTRODUCTION
Familial Mediterranean fever, (FMF) is an autosomal recessive disease that affects primarily individuals of nonAshkenazi Jewish, Armenian, Turkish, and Arab origin.l? The gene frequency is very high (1:16) amongst Jews of Libyan origin and within the Armenian population in Los Angeles (1:14).5 The disease is characterized by intermittent attacks of high fever with severe abdominal pain, pleurisy, or arthritis. The arthritis generally affects the big joints of the lower extremities, having no sequelae, but 6%-8% of the patients go into a protracted course that may last weeks to years, and may lead to destructive changes at the hip joints. Amyloidosis is the most serious and fatal
Submitted for publication August 27,1992; accepted September I, 1992. Address reprint requests to Elon Pras, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.
feature of FMF; it affects mainly the kidneys and leads to end-stage renal disease." The Heller Institute at Tel Hashomer now sees more than 2,200 FMF patients. In 1972, the use of continuous colchicine treatment was described by Goldfinger," Since then we have proved its efficacy in preventing not only the attacks, but also the development of amyloidosis." We have followed very small children, some below the age of3, and closely monitored more than 500 pregnancies. In a collaborative study of the Heller Institute at Tel Hashomer, Israel, and the Genetic Unit of the Arthritis and Rheumatism Branch at the National Institutes of Health, Bethesda, Maryland, we have been involved in the search of the FMF gene, using reverse genetics techniques in our search to uncover the gene of FMF and the molecular and biological properties of its encoding proteins. MATERIALS AND METHODS
The first phase of the project began in Israel. We obtained blood samples from more than 350 FMF patients and their family members, most of North African, Iraqi, and Turkish origin. From 20 ml of heparinized blood, peripheral blood lymphocytes were immortalized with Epstein-Barr virus and cultured, producing an endless source of genetic material from each individual. The DNA was extracted and digested with restriction enzymes, electrophoresed and transferred to a nylon membrane, then hybridized with radioactive probes whose chromosomal location is known. The nylon membranes were exposed to X-ray film and the different bands revealed were read and interpreted. The data were loaded into a computer with the Linkage program? and a lod score derived. (Lod score is the logarithm to the base 10 of the odds in favor oflinkage to the odds against linkage.) A lod score of more than 3 means that the odds in favor oflinkage are 1000:1, which by convention is proof oflinkage, and a lod score of - 2 is considered evidence against linkage. RESULTS
In our search for the FMF gene, two different strategies were used: (1) Because the real cause of the disease is not yet known, and its principal feature is the inflammatory process, in the candidate gene approach we looked into the genes that are thought to be involved in the inflammatory reaction that we see in FMF, such as the genes for complements 3 and 5, complement receptors 1 and 2, decay accelerating factor, C4 binding protein, interleukins In and 113, interleukins 6 and 8, lipocortins I and II, serum amyloid A and dopamine l3-hydroxylase. lO None proved to be linked to FMF. (2) The second approach consisted in a methodological exclusion of the different chromosomes. We looked at 107 different probes that covered one-third of the human genome, (chromosomes 1,5, 10,9, 15, and 17)11 without success. However, probe number 108, known as pCMM65 (DI6S84), that maps
242
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to the telomeric region of the short arm of chromosome 16,12 gave us a positive lod score of 9.17 at a distance of - .04 cM. To confirm this result, we looked at another marker in this region that is associated with the hemoglobin (X complex that maps 5 cM telomeric to the first probe.P giving us a lod score of 14.45. Using multipoint linkage analysis we got a maximal lod score of 19.86. Thus the possibilit6. that the gene for FMF is located in this region is 10 2 :1, far more than the 103 : 1 that is required as proofoflinkage. 14 CONCLUSION
Description of the chromosomal location of the gene is the first step that ultimately will allow the identification of the gene itself, and in this way it should provide new insights into the pathophysiology not only of this disease, but also into the mechanisms of inflammation inman. We hope that in the near future the present mapping ofthe FMF gene to the short arm of chromosome 16 will enable a decisive laboratory diagnosis of equivocal FMF patients and will improve genetic counseling and prenatal diagnosis. REFERENCES 1. Sohar E, Gafni J, Pras M, Heller H. Familial Mediterranean fever. A survey of 470 cases and review of the literature. Am J Med. 1967; 43:227-253. 2. Schwabe AD, Peters RS. Familial Mediterranean reverin Armenians. Analysis of 100 cases. Medicine (Baltimore). 1974; 53:453-462.
3. Ozdemir AI, Sokmen C. Familial Mediterranean fever among Turkish people. Am J Gastroenterol. 1969; 51:311-316. 4. Barakat MH, Karnik AM, Majeed HWA, El-Sobki NI, Fenech FF. Familial Mediterranean fever (recurrent hereditary polyserositis) in Arabs. A study of175 patients and review of the literature. Quart J Med. 1986; 60:837-847. 5. Rogers DB, Shohat M, Petersen GM, Bickal J, Congleton J, Schwabe AD, Familial Mediterranean fever in Armenians: Autosomal recessive inheritance with high gene frequency. Am J Genetics. 1989; 34:168-172. 6. Meyerhoff J. Familial Mediterranean fever: Report of a large family, review ofthe literature, and discussion of the frequency of amyloidosis. Medicine (Baltimore). 1980; 59:66-77. 7. Goldfinger SE. Colchicine for familial Mediterranean fever. N Engl J Med. 1972; 287:1302. 8. Zemer D, Pras M, Sohar E, Colchicine in the prevention and treatment of the amyloidosis in familial Mediterranean fever. N Engl J Med. 1986; 314:1001-1005. 9. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Nat! Acad Sci USA. 1984; 81:3443-3446. 10. Gruberg L, Aksentijevich I, Balow J, Dean M, Kovo M, Pras M, Kastner DL. Exclusion of candidate genes in familial Mediterranean fever. Cytogen Cell Genet. 1991; 98:2113. (Abstr) 11. Aksentijevich I, Gruberg L, Balow JE Jr, Dean M, Pras M, Kastner DL. Linkage analysis in familial Mediterranean fever. Am J Hum Genet. 1991; 49 (4 Suppl):335. (Abstr) 12. Nakamura Y, Martin C, Krapcho K, O'Connell P, Leppert M, Lathrop GM, Isolation and mapping of a polymorphic DNA sequence (pCMM65) on chromosome 16 (D16S84). Nucleic Acids Res. 1988; 16:3122. 13. Jarman Ap, Higgs DR. A new hypervariable marker for the human Alpha-globin gene cluster. Am J Hum Genet. 1988; 43:249-256. 14. Pras E, Aksentijevich I, Gruberg L, Balow JE Jr, Prosen L, Dean M, Steinberg AD, Pras M, Kastner DL. Mapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16. N Engl J Med. 1992; 326:1509-1513.
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