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AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis
Hepatology Research 26 (2003) 154 /156 www.elsevier.com/locate/ihepcom
AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis Ai Hattori a, Shinnya Wakusawa a, Hisao Hayashi a,*, Ai Harashima a, Fujiko Sanae a, Miwa Kawanaka b, Gohtaro Yamada b, Motoyashi Yano c, Kenntaro Yoshioka c a
Department of Medicine, Faculty of Pharmaceutical Sciences of Hokuriku University, Ho 3, Kanagawa-machi, Kanazawa, Ishikawa 920-1181, Japan b Center of Liver Diseases, Kawasaki Hospital, Kawasaki Medical School, Nakayama-shita 2-1-80, Okayama 700-0821, Japan c Department of Third Internal Medicine, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan Received 9 September 2002; received in revised form 6 January 2003; accepted 31 January 2003
Abstract The majority of Caucasian patients with hemochromatosis are homozygous for C282Y mutation of the HFE gene. In contrast to its high prevalence in Caucasians, hemochromatosis is a rare disorder in Japan. This may be due to the low prevalence of the C282Y mutation of the HFE gene in Japanese. Recent reports suggest that the mutations of transferrin receptor 2 (TfR2) gene may be involved in non-HFE hemochromatosis. Therefore, we investigated the TfR2 gene of 6 sporadic and 5 familiar cases of Japanese hemochromatosis. Three siblings in one family were found to be homozygous for an AVAQ 594-597 deletion. All three had severe iron deposits in the hepatocytes and bile ducts, but none was affected by diabetes mellitus. This mutation was not detected in 100 control individuals. Further study was undertaken to investigate whether the large deletion of the TfR2 gene is the mutation responsible for some of the Japanese hemochromatosis cases. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cirrhosis; Diabetes; Iron; Liver
1. Introduction Recent identification of the key proteins involved in iron metabolism provided insight into hereditary hemochromatosis. The majority of Caucasian patients with hemochromatosis inherit this disease by transmission of the C282Y mutation of the HFE gene [1]. In contrast to its high prevalence among Caucasians, hemochromatosis is a rare disorder in Japan [2]. This may be due to the low prevalence of C282Y mutation of the HFE gene in Japanese [3]. After identification of HFE hemochromatosis, several subtypes of non-HFE hemochromatosis have been reported. These include juvenile hemochromatosis, and hemochromatosis with mutant genes in transferrin receptor 2 (TfR2) [4], ferroportin1 [5] and IRE of H ferritin subunit [6]. In 2000, the first paper on mutation of TfR2 gene was reported from Italy [4].
* Corresponding author. Tel./fax: /81-76-229-6203. E-mail address: [email protected] (H. Hayashi).
Subsequent case reports from other countries suggest that the hepatic transferrin receptor, TfR2 [7], may be involved in some non-HFE hemochromatosis. In this paper, we present a Japanese family with hemochromatosis showing a large deletion of the TfR2 gene.
2. Materials and methods The clinical features and results of HFE genetic testing in the 11 patients studied here were reported previously [3]. All 11 patients are residents of the main island of Japan. DNA was extracted from the peripheral blood cells of each patient after informed consent was obtained. Subsequent analysis demonstrated that all patients were negative for C282Y and H63D mutations. Five regions (exons 2, 4, 6, 16 and 17) of the TfR2 gene where mutations have been reported to be responsible for non-HFE hemochromatosis were amplified by PCR, followed by direct sequencing. The primers used for the five exons are listed in Table 1. When mutation was
1386-6346/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-6346(03)00086-X
A. Hattori et al. / Hepatology Research 26 (2003) 154 /156 Table 1 Sequences of TfR2 primers used in the polymerase chain reaction reactions [8] Exon 2 Exon 4 Exon 6 Exon 16 Exon 17
F: 5?-TCACTGACCTCATTATTGCC-3? R: 5?-AAGGCTGGCGGGTGGCAAGA-3? F: 5?-ACGTCTCTGGCATCCTTCCCT-3? R: 5?GGTGAGCGCCCCGAGCCGCG-3? F: 5?-GAGCCCTTTCCTGGGTCTCGAA-3? R: 5?-CCTGAACGATTCTCACTGGC-3? F: 5?-CCCAGCGTCCACCCTGTCCTGGC-3? R: 5?-CTGGATTGCCAGAGAGGACC-3? F: 5?-GGGTCCTGGCCCCTGCCGCCAG-3? R: 5?-AGGTCCAGGAGGTGGAG-3?
detected in patients, its frequency was analyzed in 100 control individuals.
3. Results Three of the 11 patients studied were homozygous for the AVAQ 594-597 deletion reported from Italy [4] (Fig. 1). These 3 patients were siblings with hepatic iron overload and elevated serum CA19-9 as described in detail elsewhere [11]. The proband was a 50-year-old man, with cirrhosis and severe iron deposits in the periportal hepatocytes and proliferated bile ducts. Oral glucose tolerance test was normal. Pigmentation was not reported. Subsequent screening tests in this family demonstrated that two siblings had severe iron deposits in the hepatocytes and bile ducts. A 47-year-old brother was generally pigmented but free of diabetes. A 55-yearold sister was free of both pigmentation and diabetes.
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This mutation was not found in two siblings of another family and 6 cases of sporadic hemochromatosis. One hundred control individuals also did not have this mutation. There were no other mutations found in the regions of the TfR2 gene that we examined.
4. Discussion In the literature, five mutations of the TfR2 gene have been reported in patients with non-HFE hemochromatosis [4,8 /10]. TfR2-linked hemochromatosis has been found not only in Caucasians but also in African Americans [12], suggesting a worldwide distribution of this minor type of hemochromatosis. The first family study of our patients suggested a diagnosis of familiar, non-HLA linked hemochromatosis [11]. The current gene analysis demonstrated that three siblings with clinically mild hemochromatosis exhibited a homozygous AVAQ 594-597 deletion of the TfR2 gene. This mutation was considered to be rare in the general population, since sequence analysis of 100 control individuals did not detect the mutation. Genetic analysis for family members other than the affected individuals is not yet complete so that the causative relation between the large deletion and hemochromatosis is not yet clear. However, the original paper from Italy [4] showed a close relationship between homozygosity of AVAQ 594597 deletion and hemochromatosis, indicating that the mutation is responsible for the iron overload disorder with recessive inheritance. As far as the genetic background is concerned, a few papers have reported on Japanese hemochromatosis.
Fig. 1. Direct sequence of exon 16 in the TfR2 gene. The figure shows three homozygotes of the AVAQ 594-597 deletion and normal control of exon 16 of the TfR2 gene.
156
A. Hattori et al. / Hepatology Research 26 (2003) 154 /156
One patient, with the C282Y mutation of the HFE gene, was reported by Sohda et al. [2]. One family, with four affected members, with a novel mutation of the H chain of ferritin was reported by Kato et al. [6]. Although this disorder has been listed in a monograph as the fourth leading cause of hemochromatosis [5], no other family with this disorder has been identified. Further study is required to clarify the genetic background of hemochromatosis in Japan, since neither the C282Y mutation of the HFE gene nor mutations in the TfR2 gene, including the AVAQ 594-597 deletion, are found in the majority of Japanese patients with hemochromatosis.
References [1] Feder JN, Gnirke A, Thomas W, et al. A novel MHC class 1-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399 /408. [2] Sohda T, Okubo R, Kamimura S, Ohkawara T. Hemochromatosis with HFE gene mutation in a Japanese patient. Am J Gastroenterol 2001;96:2487 /8. [3] Shiono Y, Ikeda R, Hayashi H, et al. C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan. Intern Med 2001;40:852 /6.
[4] Camaschella C, Roetto A, Cali A, et al. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat Genet 2000;25:14 /5. [5] Njajou OT, Vaessen N, Joosse M, et al. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat Genet 2001;28:213 /4. [6] Kato J, Fujikawa K, Kanda M, et al. A mutation, in the ironresponsive element of H ferritin mRNA, causing autosomal dominant iron overload. Am J Hum Genet 2001;69:191 /7. [7] Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptorlike family. J Biol Chem 1999;274:20826 /32. [8] Roetto A, Totaro A, Piperno A, et al. New mutation inactivating transferrin receptor 2 in hemochromatosis type 3. Blood 2001;97:2555 /60. [9] Mattman A, Huntsman D, Lockitch G, et al. Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation. Blood 2002;100:1075 /7. [10] Girelli D, Bozzini C, Roetto A, et al. Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene. Gastroenterology 2002;122:1295 /302. [11] Kawanaka M, Kinoyama S, Niiyama G, et al. A case of idiopathic hemochromatosis which occurred in three siblings with high level of serum CA 19-9. Nippon Shokakibyo Gakkai Zasshi 1998;95:910 /5 (In Japanese, abstract in English). [12] Barton EH, West PA, Rivers CA, Barton JC, Acton RT. Transferrin receptor-2 (TFR2) mutation Y250X in Alabama Caucasian and African American subjects with and without primary iron overload. Blood Cells Mol Dis 2001;27:279 /84.
AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis Ai Hattori a, Shinnya Wakusawa a, Hisao Hayashi a,*, Ai Harashima a, Fujiko Sanae a, Miwa Kawanaka b, Gohtaro Yamada b, Motoyashi Yano c, Kenntaro Yoshioka c a
Department of Medicine, Faculty of Pharmaceutical Sciences of Hokuriku University, Ho 3, Kanagawa-machi, Kanazawa, Ishikawa 920-1181, Japan b Center of Liver Diseases, Kawasaki Hospital, Kawasaki Medical School, Nakayama-shita 2-1-80, Okayama 700-0821, Japan c Department of Third Internal Medicine, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan Received 9 September 2002; received in revised form 6 January 2003; accepted 31 January 2003
Abstract The majority of Caucasian patients with hemochromatosis are homozygous for C282Y mutation of the HFE gene. In contrast to its high prevalence in Caucasians, hemochromatosis is a rare disorder in Japan. This may be due to the low prevalence of the C282Y mutation of the HFE gene in Japanese. Recent reports suggest that the mutations of transferrin receptor 2 (TfR2) gene may be involved in non-HFE hemochromatosis. Therefore, we investigated the TfR2 gene of 6 sporadic and 5 familiar cases of Japanese hemochromatosis. Three siblings in one family were found to be homozygous for an AVAQ 594-597 deletion. All three had severe iron deposits in the hepatocytes and bile ducts, but none was affected by diabetes mellitus. This mutation was not detected in 100 control individuals. Further study was undertaken to investigate whether the large deletion of the TfR2 gene is the mutation responsible for some of the Japanese hemochromatosis cases. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cirrhosis; Diabetes; Iron; Liver
1. Introduction Recent identification of the key proteins involved in iron metabolism provided insight into hereditary hemochromatosis. The majority of Caucasian patients with hemochromatosis inherit this disease by transmission of the C282Y mutation of the HFE gene [1]. In contrast to its high prevalence among Caucasians, hemochromatosis is a rare disorder in Japan [2]. This may be due to the low prevalence of C282Y mutation of the HFE gene in Japanese [3]. After identification of HFE hemochromatosis, several subtypes of non-HFE hemochromatosis have been reported. These include juvenile hemochromatosis, and hemochromatosis with mutant genes in transferrin receptor 2 (TfR2) [4], ferroportin1 [5] and IRE of H ferritin subunit [6]. In 2000, the first paper on mutation of TfR2 gene was reported from Italy [4].
* Corresponding author. Tel./fax: /81-76-229-6203. E-mail address: [email protected] (H. Hayashi).
Subsequent case reports from other countries suggest that the hepatic transferrin receptor, TfR2 [7], may be involved in some non-HFE hemochromatosis. In this paper, we present a Japanese family with hemochromatosis showing a large deletion of the TfR2 gene.
2. Materials and methods The clinical features and results of HFE genetic testing in the 11 patients studied here were reported previously [3]. All 11 patients are residents of the main island of Japan. DNA was extracted from the peripheral blood cells of each patient after informed consent was obtained. Subsequent analysis demonstrated that all patients were negative for C282Y and H63D mutations. Five regions (exons 2, 4, 6, 16 and 17) of the TfR2 gene where mutations have been reported to be responsible for non-HFE hemochromatosis were amplified by PCR, followed by direct sequencing. The primers used for the five exons are listed in Table 1. When mutation was
1386-6346/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-6346(03)00086-X
A. Hattori et al. / Hepatology Research 26 (2003) 154 /156 Table 1 Sequences of TfR2 primers used in the polymerase chain reaction reactions [8] Exon 2 Exon 4 Exon 6 Exon 16 Exon 17
F: 5?-TCACTGACCTCATTATTGCC-3? R: 5?-AAGGCTGGCGGGTGGCAAGA-3? F: 5?-ACGTCTCTGGCATCCTTCCCT-3? R: 5?GGTGAGCGCCCCGAGCCGCG-3? F: 5?-GAGCCCTTTCCTGGGTCTCGAA-3? R: 5?-CCTGAACGATTCTCACTGGC-3? F: 5?-CCCAGCGTCCACCCTGTCCTGGC-3? R: 5?-CTGGATTGCCAGAGAGGACC-3? F: 5?-GGGTCCTGGCCCCTGCCGCCAG-3? R: 5?-AGGTCCAGGAGGTGGAG-3?
detected in patients, its frequency was analyzed in 100 control individuals.
3. Results Three of the 11 patients studied were homozygous for the AVAQ 594-597 deletion reported from Italy [4] (Fig. 1). These 3 patients were siblings with hepatic iron overload and elevated serum CA19-9 as described in detail elsewhere [11]. The proband was a 50-year-old man, with cirrhosis and severe iron deposits in the periportal hepatocytes and proliferated bile ducts. Oral glucose tolerance test was normal. Pigmentation was not reported. Subsequent screening tests in this family demonstrated that two siblings had severe iron deposits in the hepatocytes and bile ducts. A 47-year-old brother was generally pigmented but free of diabetes. A 55-yearold sister was free of both pigmentation and diabetes.
155
This mutation was not found in two siblings of another family and 6 cases of sporadic hemochromatosis. One hundred control individuals also did not have this mutation. There were no other mutations found in the regions of the TfR2 gene that we examined.
4. Discussion In the literature, five mutations of the TfR2 gene have been reported in patients with non-HFE hemochromatosis [4,8 /10]. TfR2-linked hemochromatosis has been found not only in Caucasians but also in African Americans [12], suggesting a worldwide distribution of this minor type of hemochromatosis. The first family study of our patients suggested a diagnosis of familiar, non-HLA linked hemochromatosis [11]. The current gene analysis demonstrated that three siblings with clinically mild hemochromatosis exhibited a homozygous AVAQ 594-597 deletion of the TfR2 gene. This mutation was considered to be rare in the general population, since sequence analysis of 100 control individuals did not detect the mutation. Genetic analysis for family members other than the affected individuals is not yet complete so that the causative relation between the large deletion and hemochromatosis is not yet clear. However, the original paper from Italy [4] showed a close relationship between homozygosity of AVAQ 594597 deletion and hemochromatosis, indicating that the mutation is responsible for the iron overload disorder with recessive inheritance. As far as the genetic background is concerned, a few papers have reported on Japanese hemochromatosis.
Fig. 1. Direct sequence of exon 16 in the TfR2 gene. The figure shows three homozygotes of the AVAQ 594-597 deletion and normal control of exon 16 of the TfR2 gene.
156
A. Hattori et al. / Hepatology Research 26 (2003) 154 /156
One patient, with the C282Y mutation of the HFE gene, was reported by Sohda et al. [2]. One family, with four affected members, with a novel mutation of the H chain of ferritin was reported by Kato et al. [6]. Although this disorder has been listed in a monograph as the fourth leading cause of hemochromatosis [5], no other family with this disorder has been identified. Further study is required to clarify the genetic background of hemochromatosis in Japan, since neither the C282Y mutation of the HFE gene nor mutations in the TfR2 gene, including the AVAQ 594-597 deletion, are found in the majority of Japanese patients with hemochromatosis.
References [1] Feder JN, Gnirke A, Thomas W, et al. A novel MHC class 1-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399 /408. [2] Sohda T, Okubo R, Kamimura S, Ohkawara T. Hemochromatosis with HFE gene mutation in a Japanese patient. Am J Gastroenterol 2001;96:2487 /8. [3] Shiono Y, Ikeda R, Hayashi H, et al. C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan. Intern Med 2001;40:852 /6.
[4] Camaschella C, Roetto A, Cali A, et al. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat Genet 2000;25:14 /5. [5] Njajou OT, Vaessen N, Joosse M, et al. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat Genet 2001;28:213 /4. [6] Kato J, Fujikawa K, Kanda M, et al. A mutation, in the ironresponsive element of H ferritin mRNA, causing autosomal dominant iron overload. Am J Hum Genet 2001;69:191 /7. [7] Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptorlike family. J Biol Chem 1999;274:20826 /32. [8] Roetto A, Totaro A, Piperno A, et al. New mutation inactivating transferrin receptor 2 in hemochromatosis type 3. Blood 2001;97:2555 /60. [9] Mattman A, Huntsman D, Lockitch G, et al. Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation. Blood 2002;100:1075 /7. [10] Girelli D, Bozzini C, Roetto A, et al. Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene. Gastroenterology 2002;122:1295 /302. [11] Kawanaka M, Kinoyama S, Niiyama G, et al. A case of idiopathic hemochromatosis which occurred in three siblings with high level of serum CA 19-9. Nippon Shokakibyo Gakkai Zasshi 1998;95:910 /5 (In Japanese, abstract in English). [12] Barton EH, West PA, Rivers CA, Barton JC, Acton RT. Transferrin receptor-2 (TFR2) mutation Y250X in Alabama Caucasian and African American subjects with and without primary iron overload. Blood Cells Mol Dis 2001;27:279 /84.