- Email: [email protected]
Clinical and Molecular Aspects of Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean (Quebec, Canada)
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
S. R. Rivard, et al.
Clinical and Molecular Aspects of Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean (Quebec, Canada) Submitted 01/25/00 (communicated by Ernest Beutler, M.D., 01/25/00)
Sylvain R. Rivard,1 Catherine Mura,2 Herve´ Simard,3 Raynald Simard,3 Doria Grimard,4 Ge´rald Le Gac,2 Odile Raguenes,2 Claude Fe´rec,2 and Marc De Braekeleer5,6 ABSTRACT: We report the clinical, biochemical, and genetic characteristics of 13 hemochromatosis patients from Saguenay–Lac-Saint-Jean in whom the first symptoms appeared before age 30. Although the mean age at onset of the first symptoms was 21.5 years, their mean age at diagnosis was 23.8 years; the diagnosis was particularly delayed among women. Seventy-seven percent of the patients had hypogonadotrophic hypogonadism and 69% heart failure and/or cardiac arrhythmias. Genetic analysis of the HFE gene revealed heterozygosity for the C282Y mutation in 2 patients and for the S65C mutation in 2 others and homozygosity for the H63D mutation in 1 patient. The remaining 8 patients had no identified mutation in the HFE gene, although sequencing of all seven codons and intron– exon junctions was performed (5 patients). All 13 patients fulfill the clinical criteria of juvenile hemochromatosis and represent the largest cluster thus far reported. © 2000 Academic Press Keywords: juvenile hemochromatosis; mutation analysis; Quebec.
INTRODUCTION
been named juvenile hemochromatosis or hemochromatosis type 2. This autosomal recessive disorder is not associated with the HFE gene, neither linked to markers on 6p (14,15). The juvenile hemochromatosis locus has recently been identified on the long arm of chromosome 1, in a 4 cM interval between markers D1S442 and D1S2347 (16). Several hereditary disorders have been shown to have a high frequency in Saguenay–Lac-SaintJean, a geographically isolated region located 200 kilometers northeast of Quebec City and surrounded by tracks of unsettled land and forests (17,18). At the beginning of the 1990s, we conducted a clinical, biochemical and genetic study on 30 families with hereditary hemochromatosis in the region (2,19 –22). The values of the mean inbreeding and kin-
Hereditary hemochromatosis, or hemochromatosis type 1, is a common autosomal recessive disorder of iron metabolism that affects mostly males after age 40 (1,2). The HFE gene responsible for hereditary hemochromatosis has been mapped to the short arm of chromosome 6 and three mutations c.845G6A, c.187C6G, and c.193A6T (C282Y, H63D, S65C) have now been identified (3–5). Some patients under 30 years of age presenting symptoms and clinical signs due to iron overload have also been reported (6 –13). They usually develop hypogonadotrophic hypogonadism in their 20s and heart failure and/or cardiac arrhythmias in their 30s (7,10). This severe form of hemochromatosis affecting sexes equally has
Reprint requests to: Professor Marc De Braekeleer, Anthropologie et de De´mographie Ge´ne´tiques, Universite´ Victor Segalen Bordeaux2, 3ter, Place de la Victoire, F-33076 Bordeaux cedex, France. Fax: 05 56 92 55 64. E-mail: [email protected]. 1 De´partement des Sciences Fondamentales, Universite´ du Que´bec a` Chicoutimi, Canada. 2 Etablissement de Transfusion Sanguine de Bretagne Occidentale, Brest, France. 3 De´partement d’He´matologie–Oncologie, Complexe Hospitalier de la Sagamie, Chicoutimi, Que´bec, Canada. 4 De´partement de Biologie Me´dicale, Complexe Hospitalier de la Sagamie, Chicoutimi, Que´bec, Canada. 5 Laboratoire d’Anthropologie et de De´mographie Ge´ne´tiques, Universite´ Victor Segalen Bordeaux2, France. 6 Institut National d’E´tudes De´mographiques, Paris, France. 1079-9796/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved
10
S. R. Rivard, et al.
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
ship coefficients were much higher than those found in other hereditary hemochromatosis populations or in subpopulations with different autosomal recessive disorders in Saguenay–Lac-SaintJean (18,23). Seven consanguineous marriages were identified; they included one marriage between first-degree cousins, one between first-degree cousins once removed and three between second-degree cousins. The mean age at diagnosis of the hereditary hemochromatosis probands was much lower than usually reported (36 years versus 50 years) and was associated with a greater severity at a young age. Our results suggested that the high variability in the phenotype was the result, not only, of mutation variability, but also, of the action of modifying genes and/or other genes involved in iron metabolism (22). We report here the clinical and molecular features of 13 patients from Saguenay–Lac-SaintJean with iron overload in whom the first symptoms appeared before age 30.
dideoxynucleotide chain termination method, as previously described (4,5). RESULTS The 13 patients under study were distributed in 10 families (Table 1). There were seven males and six females. The mean age at diagnosis was 23.8 years (standard deviation SD: 7.7) although the mean age at onset of the first symptoms was 21.5 years (SD: 6.2). Ten patients (76.9%) had hypogonadotrophic hypogonadism, including one with hypothyroidism. Seven patients (53.8%) had cardiac arrhythmias and 5 (38.5%) congestive heart failure; in total, 9 (69.2%) had some heart problems. Liver cirrhosis and insulin-dependent diabetes were present in 2 (15.4%) and 4 (30.8%) patients, respectively. The biochemical data for each patient is presented in Table 1. The mean values for serum iron, transferrin saturation and serum ferritin were, respectively, of 48 mol/liter (SD: 6.4), 1 Fmol (SD: 0.1), and 3026 g/liter (SD: 2182). The genotype at the HFE locus was determined for each patient (Table 1). Two patients were heterozygous for the C282Y mutation, including one who had an affected sister (patient # 1012) with none of the three mutations. Both brothers in family #5 were heterozygous for the S65C mutation. One patient was homozygous for the H63D mutation. None of the three HFE mutations that were searched for was identified in the remaining 8 patients. The HFE gene of five patients belonging to five different families with no identified mutations was sequenced; no new mutation was found.
PATIENTS AND METHODS All the 13 hemochromatosis patients in whom the first symptoms appeared before age 30 and followed on a regular basis for phlebotomies at the department of hematology of the Complexe Hospitalier de la Sagamie were invited to participate at the present study. The diagnosis was made on the basis of clinical history, physical examination and persistently raised iron indices (transferrin saturation and serum ferritin). Liver biopsy was performed in, at least, one affected member of each sibship. The clinical and biochemical data were extracted from their files kept at the department of hematology. DNA was extracted by standard methods from the blood of these 13 patients. All three substitutions (C282Y, H63D, and S65C) were analyzed as previously described (4,5). All seven coding exons and exon-intron junctions of the HFE gene were sequenced after specific amplification of the relevant region by PCR. The PCRs were carried out under standard conditions and the amplified products were sequenced by the termination
DISCUSSION Juvenile hemochromatosis is a rare disorder that has usually been reported as “case reports” since the mid-1960s. Some patients with clinical signs suggestive of juvenile hemochromatosis have recently been included in the mutation analysis of the HFE gene of hemochromatosis patients. For example, Ryan et al. reported a 16year-old female with hypogonadotrophic hypogo11
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
S. R. Rivard, et al.
TABLE 1 Clinical, Biochemical, and Genetic Characteristics of 13 Patients with Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean Patient no. Family no. Sex Age (years) Age at diagnosis (years) Genotype HFE Age at first symptoms (years) Serum iron (mol/liter) Transferrin saturation (fmol) Serum ferritin (g/liter) Cardiac arrhythmia Heart failure Hypopituitarism Hypogonadotropic Hypogonadism Hypothyroidism Liver cirrhosis Hepatomegaly Arthralgia Skin hyperpigmentation Diabetes Splenomegaly Abdominal pain Nausea
1057 56 F 39 33 C282Y/ ND 27
1017 1012 1020 48 27 27 M F F 24 39 42 18 28 24 ND/ ND/ C282Y/ ND ND ND 18 26 19
1025 1028 1035 1052 18 8 8 5 F M M M 67 50 44 31 32 25 22 9 ND/ ND/ ND/ S65C/ ND ND ND ND 29 25 22 9
46 1 ⬎2500 ⫹
37 1 6000 ⫹ ⫹
54 1 1070 ⫹
47 1 4996
42 0.93 6882
⫹
⫹
⫹
⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹
⫹ ⫹
54 0.96 268 ⫹ ⫹⫹
61 0.95 573
53 0.96 2432 ⫹
46 1.28 4188
?
⫹
⫹
⫹
⫹
⫹
⫹ ⫹
⫹
⫹ ⫹ ⫹
⫹
⫹
⫹
⫹
51 0.97 739
1019 1018 1059 1060 2 12 57 58 F M F M 45 42 26 34 25 25 25 33 ND/ H63D/ ND/ ND/ ND H63D ND ND 25 24 21 ⫾25 43 0.94 4500 ⫹ ⫹⫹
⫹ ⫹
43 0.85 1517
1058 5 M 32 10 S65C/ ND 10
47 1.07 3681 ⫹
⫹ ⫹
⫹
⫹
⫹ ⫹
⫹ ⫹
⫹
Note. Normal values: serum iron, 9 –29 mol/liter; transferrin saturation, 0.20 – 0.45 fmol; serum ferritin, 30 – 425 g/liter (men), 10 –300 g/liter (women).
nadism (8). The UK hemochromatosis consortium reported a 29 year-old male with hypogonadotrophic hypogonadism and heart failure and a 16year-old male identified during family screening following the diagnosis of hemochromatosis in his 21-year-old brother who had heart failure (9). All three patients were negative for the C282Y and H63D mutations (8,9). To the best of our knowledge, it is thus far the largest series of juvenile hemochromatosis patients reported from a same region. All the patients in our series, but two brothers diagnosed at age 9 and 10, had heart problems and/or hypogonadotrophic hypogonadism. In a review of the literature on 52 patients with hemochromatosis diagnosed before age 30, Lamon et al. reported that 58.5% had cardiomyopathy and 64.2% hypogonadism (10). In our series, 69.2% had heart problems (arrhythmia and/or failure) and 76.9% hypogonadism, which is higher than in their literature review. Diabetes was present in 33.9%
and liver involvement in 83% of the patients, compared to 30.8% and 53.8% respectively among the patients from Saguenay–Lac-SaintJean. The differences observed between both series could be the result of a smaller number of patients in the Saguenay–Lac-Saint-Jean series or of a more homogeneous group. Indeed, several autosomal recessive disorders have acquired a high frequency by founder effect and genetic drift in Saguenay–Lac-Saint-Jean (18,24,25). Molecular studies have shown that, in many of these disorders, a sole mutation was responsible for the majority of the cases identified in the region. As already noticed by several authors, both sexes are equally affected by juvenile hemochromatosis (6,7,10). However, because females are less likely to have symptoms due to hemochromatosis type 1, the diagnosis might be delayed. In our series, 5 of the 6 women were diagnosed at least two years after the onset of the first symp12
S. R. Rivard, et al.
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
toms (mean difference: 3.2 years, SD: 1.9) whereas 6 of the 7 men were diagnosed less than a year after the first symptoms (mean difference: 0.2 years, SD: 0.4). Therefore, serum iron indices should be included in the biochemical workout of all young women with cardiac arrhythmia and/or unexplained secondary amenorrhea. Mutation analysis of the HFE gene showed that the majority of cases with juvenile hemochromatosis had no identified mutation (8,9,14,15,26,27). A few patients with juvenile hemochromatosis, including some in this series, were heterozygous for the C282Y mutation of the HFE gene (6). It is likely that the presence of the C282Y mutation is due to its high frequency rate in the general population although we cannot exclude that the mutation participated in the phenotype. The same conclusion applies for both brothers in family #5 found to be heterozygous for the rare mutation S65C. Its role in the phenotype of typical hereditary hemochromatosis is still debated but is definitely not associated with a severe phenotype (5). One patient was homozygous for the H63D mutation. This genotype is associated with a mild hereditary hemochromatosis phenotype and has even been found in asymptomatic patients (4). Given the high frequency of the H63D mutation in Saguenay–Lac-Saint-Jean (28), the homozygosity observed in our patient is presumably due to chance alone. It is highly unlikely that it is responsible for the severe phenotype, although it may have contributed to it in some undefined way. We are now contacting the families to realize a linkage analysis that will allow us to determine whether juvenile hemochromatosis in Saguenay– Lac-Saint-Jean is associated or not with the locus on chromosome 1q described by Roetto et al. (16).
de Cycles Supe´rieurs et de la Recherche at the Universite´ du Que´bec a` Chicoutimi.
ACKNOWLEDGMENTS
13.
REFERENCES 1.
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The authors thank Mrs. Marielle Gagnon and Mr. Jocelyn Tremblay for their invaluable help. This research was supported by INSERM grants from CRI9607 and the Association de Transfusion Sanguine et de Bioge´ne´tique Gae¨tan Saleun and a grant from the De´canat des Etudes
15.
13
Bothwell TH, Charlton RW, Motulsky AG. Hemochromatosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease, New York: McGraw-Hill, pp 1433–1462, 1989. De Braekeleer M. A prevalence and fertility study of hemochromatosis in Saguenay–Lac-Saint-Jean. Ann Hum Biol 20:501–505, 1993. Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 13:399 – 408, 1996. Mura C, Nousham JB, Verger P, et al. Phenotype– genotype correlation in haemochromatosis subjects. Hum Genet 101:271–276, 1997. Mura C, Raguene`s O, Fe´rec C. HFE mutations analysis in 711 hemochromatosis probands: Evidence for S65C implication in mild form of hemochromatosis. Blood 93:2502–2505, 1999. Kelly AL, Rhodes DA, Roland JM, Schofield P, Cox TM. Hereditary juvenile hemochromatosis: A genetically heterogeneous life-threatening iron-storage disease. Quart J Med 91:607– 618, 1998. Cazzola M, Ascari E, Barosi G, et al. Juvenile idiopathic haemochromatosis: A life-threatening disorder presenting as hypogonadotropic hypogonadism. Hum Genet 65:149 –154, 1983. Ryan E, O’Keane C, Crowe J. Hemochromatosis in Ireland and HFE. Blood Cells Mol Dis 24:428 – 432, 1998. The UK Haemochromatosis Consortium. A simple genetic test identifies 90% of UK patients with haemochromatosis. Gut 41:841– 844, 1997. Lamon JM, Marynick SP, Rosenblatt R, Donnelly S. Idiopathic hemochromatosis in a young female. A case study and review of the syndrome in young people. Gastroenterology 76:178 –183, 1979. Felts JH, Nelson JR, Herndon CN, Spurr CL. Hemochromatosis in two young sisters. Ann Intern Med 67:117–123, 1967. Kaikov Y, Wadsworth LD, Hassall E, Dimmick JE, Rogers PCJ. Primary hemochromatosis in children: Report of three newly diagnosed cases and review of the pediatric literature. Pediatrics 90:37– 42, 1992. Case records of the Massachusetts General Hospital. Case 25-1983. N Engl J Med 308:1521–1529, 1983. Camaschella C, Roetto A, Cicilano M, et al. Juvenile and adult hemochromatoses are distinct genetic disorders. Eur J Hum Genet 5:371–375, 1997. Camaschella C, Fargion S, Sampietro M, et al. Inherited HFE-unrelated hemochromatosis in Italian families. Hepatology 29:1563–1564, 1999.
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Roetto A, Totaro A, Cazzola M, et al. Juvenile hemochromatosis locus maps to chromosome 1q. Am J Hum Genet 64:1388 –1393, 1999. De Braekeleer M. Hereditary disorders in Saguenay– Lac-St-Jean. Hum Hered 41:141–146, 1991. De Braekeleer M. Inbreeding, kinship and surnames in hereditary disorders: The experience in Saguenay–LacSaint-Jean (Quebec). Coll Antropol 19:289 –304, 1995. De Braekeleer M, Vigneault A, Roy R, Simard H. Hereditary Hemochromatosis and HLA Antigens in Saguenay–Lac-Saint-Jean (Quebec, Canada), Universite´ du Que´bec a` Chicoutimi, Chicoutimi, Laboratoire de Recherches en E´pide´miologie Ge´ne´tique, 1992. De Braekeleer M, St-Pierre C, Vigneault A, Simard H, de Medicis E. Hemochromatosis and pyruvate kinase deficiency. Report of a case and literature review. Ann Hematol 62:188 –189, 1991. De Braekeleer M, Vigneault A, Simard H. Population genetics of hereditary hemochromatosis in Saguenay– Lac-St-Jean (Quebec, Canada). Ann Ge´ne´t 35:202– 207, 1992. De Braekeleer M, Simard H. A clinical and biochemical study of hereditary hemochromatosis in
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Saguenay–Lac-Saint-Jean (Quebec). Coll Antropol 19:337–343, 1995. De Braekeleer M, Gauthier S. Autosomal recessive disorders in Saguenay–Lac-Saint-Jean, Quebec: A study of inbreeding. Ann Hum Genet 60:51–56, 1996. De Braekeleer M, Dao T-N. Hereditary disorders in the French Canadian population of Quebec. I. In search of founders. Hum Biol 66:205–223, 1994. De Braekeleer M, Dao T-N. Hereditary disorders in the French Canadian population of Quebec. II. The contribution of Perche. Hum Biol 66:225–249, 1994. Pinson S, Yaouanq J, Jouanolle AM, Turlin B, Plauchu H. Non-C282Y familial iron overload: Evidence for locus heterogeneity in haemochromatosis. J Med Genet 35:954 –956, 1998. Cazzola M, Cerani P, Rovati A, Iannone A, Claudiani G, Bergamaschi G. Juvenile genetic hemochromatosis is clinically and genetically distinct from the classical HLA-related disorder. Blood 92:2979 –2981, 1998. Rivard SR, Mura C, Simard H, et al. Mutation analysis in the HFE gene in patients with hereditary hemochromatosis in Saguenay–Lac-Saint-Jean (Quebec, Canada). Br J Haematol, in press, 2000.
S. R. Rivard, et al.
Clinical and Molecular Aspects of Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean (Quebec, Canada) Submitted 01/25/00 (communicated by Ernest Beutler, M.D., 01/25/00)
Sylvain R. Rivard,1 Catherine Mura,2 Herve´ Simard,3 Raynald Simard,3 Doria Grimard,4 Ge´rald Le Gac,2 Odile Raguenes,2 Claude Fe´rec,2 and Marc De Braekeleer5,6 ABSTRACT: We report the clinical, biochemical, and genetic characteristics of 13 hemochromatosis patients from Saguenay–Lac-Saint-Jean in whom the first symptoms appeared before age 30. Although the mean age at onset of the first symptoms was 21.5 years, their mean age at diagnosis was 23.8 years; the diagnosis was particularly delayed among women. Seventy-seven percent of the patients had hypogonadotrophic hypogonadism and 69% heart failure and/or cardiac arrhythmias. Genetic analysis of the HFE gene revealed heterozygosity for the C282Y mutation in 2 patients and for the S65C mutation in 2 others and homozygosity for the H63D mutation in 1 patient. The remaining 8 patients had no identified mutation in the HFE gene, although sequencing of all seven codons and intron– exon junctions was performed (5 patients). All 13 patients fulfill the clinical criteria of juvenile hemochromatosis and represent the largest cluster thus far reported. © 2000 Academic Press Keywords: juvenile hemochromatosis; mutation analysis; Quebec.
INTRODUCTION
been named juvenile hemochromatosis or hemochromatosis type 2. This autosomal recessive disorder is not associated with the HFE gene, neither linked to markers on 6p (14,15). The juvenile hemochromatosis locus has recently been identified on the long arm of chromosome 1, in a 4 cM interval between markers D1S442 and D1S2347 (16). Several hereditary disorders have been shown to have a high frequency in Saguenay–Lac-SaintJean, a geographically isolated region located 200 kilometers northeast of Quebec City and surrounded by tracks of unsettled land and forests (17,18). At the beginning of the 1990s, we conducted a clinical, biochemical and genetic study on 30 families with hereditary hemochromatosis in the region (2,19 –22). The values of the mean inbreeding and kin-
Hereditary hemochromatosis, or hemochromatosis type 1, is a common autosomal recessive disorder of iron metabolism that affects mostly males after age 40 (1,2). The HFE gene responsible for hereditary hemochromatosis has been mapped to the short arm of chromosome 6 and three mutations c.845G6A, c.187C6G, and c.193A6T (C282Y, H63D, S65C) have now been identified (3–5). Some patients under 30 years of age presenting symptoms and clinical signs due to iron overload have also been reported (6 –13). They usually develop hypogonadotrophic hypogonadism in their 20s and heart failure and/or cardiac arrhythmias in their 30s (7,10). This severe form of hemochromatosis affecting sexes equally has
Reprint requests to: Professor Marc De Braekeleer, Anthropologie et de De´mographie Ge´ne´tiques, Universite´ Victor Segalen Bordeaux2, 3ter, Place de la Victoire, F-33076 Bordeaux cedex, France. Fax: 05 56 92 55 64. E-mail: [email protected]. 1 De´partement des Sciences Fondamentales, Universite´ du Que´bec a` Chicoutimi, Canada. 2 Etablissement de Transfusion Sanguine de Bretagne Occidentale, Brest, France. 3 De´partement d’He´matologie–Oncologie, Complexe Hospitalier de la Sagamie, Chicoutimi, Que´bec, Canada. 4 De´partement de Biologie Me´dicale, Complexe Hospitalier de la Sagamie, Chicoutimi, Que´bec, Canada. 5 Laboratoire d’Anthropologie et de De´mographie Ge´ne´tiques, Universite´ Victor Segalen Bordeaux2, France. 6 Institut National d’E´tudes De´mographiques, Paris, France. 1079-9796/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved
10
S. R. Rivard, et al.
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
ship coefficients were much higher than those found in other hereditary hemochromatosis populations or in subpopulations with different autosomal recessive disorders in Saguenay–Lac-SaintJean (18,23). Seven consanguineous marriages were identified; they included one marriage between first-degree cousins, one between first-degree cousins once removed and three between second-degree cousins. The mean age at diagnosis of the hereditary hemochromatosis probands was much lower than usually reported (36 years versus 50 years) and was associated with a greater severity at a young age. Our results suggested that the high variability in the phenotype was the result, not only, of mutation variability, but also, of the action of modifying genes and/or other genes involved in iron metabolism (22). We report here the clinical and molecular features of 13 patients from Saguenay–Lac-SaintJean with iron overload in whom the first symptoms appeared before age 30.
dideoxynucleotide chain termination method, as previously described (4,5). RESULTS The 13 patients under study were distributed in 10 families (Table 1). There were seven males and six females. The mean age at diagnosis was 23.8 years (standard deviation SD: 7.7) although the mean age at onset of the first symptoms was 21.5 years (SD: 6.2). Ten patients (76.9%) had hypogonadotrophic hypogonadism, including one with hypothyroidism. Seven patients (53.8%) had cardiac arrhythmias and 5 (38.5%) congestive heart failure; in total, 9 (69.2%) had some heart problems. Liver cirrhosis and insulin-dependent diabetes were present in 2 (15.4%) and 4 (30.8%) patients, respectively. The biochemical data for each patient is presented in Table 1. The mean values for serum iron, transferrin saturation and serum ferritin were, respectively, of 48 mol/liter (SD: 6.4), 1 Fmol (SD: 0.1), and 3026 g/liter (SD: 2182). The genotype at the HFE locus was determined for each patient (Table 1). Two patients were heterozygous for the C282Y mutation, including one who had an affected sister (patient # 1012) with none of the three mutations. Both brothers in family #5 were heterozygous for the S65C mutation. One patient was homozygous for the H63D mutation. None of the three HFE mutations that were searched for was identified in the remaining 8 patients. The HFE gene of five patients belonging to five different families with no identified mutations was sequenced; no new mutation was found.
PATIENTS AND METHODS All the 13 hemochromatosis patients in whom the first symptoms appeared before age 30 and followed on a regular basis for phlebotomies at the department of hematology of the Complexe Hospitalier de la Sagamie were invited to participate at the present study. The diagnosis was made on the basis of clinical history, physical examination and persistently raised iron indices (transferrin saturation and serum ferritin). Liver biopsy was performed in, at least, one affected member of each sibship. The clinical and biochemical data were extracted from their files kept at the department of hematology. DNA was extracted by standard methods from the blood of these 13 patients. All three substitutions (C282Y, H63D, and S65C) were analyzed as previously described (4,5). All seven coding exons and exon-intron junctions of the HFE gene were sequenced after specific amplification of the relevant region by PCR. The PCRs were carried out under standard conditions and the amplified products were sequenced by the termination
DISCUSSION Juvenile hemochromatosis is a rare disorder that has usually been reported as “case reports” since the mid-1960s. Some patients with clinical signs suggestive of juvenile hemochromatosis have recently been included in the mutation analysis of the HFE gene of hemochromatosis patients. For example, Ryan et al. reported a 16year-old female with hypogonadotrophic hypogo11
Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
S. R. Rivard, et al.
TABLE 1 Clinical, Biochemical, and Genetic Characteristics of 13 Patients with Juvenile Hemochromatosis in Saguenay–Lac-Saint-Jean Patient no. Family no. Sex Age (years) Age at diagnosis (years) Genotype HFE Age at first symptoms (years) Serum iron (mol/liter) Transferrin saturation (fmol) Serum ferritin (g/liter) Cardiac arrhythmia Heart failure Hypopituitarism Hypogonadotropic Hypogonadism Hypothyroidism Liver cirrhosis Hepatomegaly Arthralgia Skin hyperpigmentation Diabetes Splenomegaly Abdominal pain Nausea
1057 56 F 39 33 C282Y/ ND 27
1017 1012 1020 48 27 27 M F F 24 39 42 18 28 24 ND/ ND/ C282Y/ ND ND ND 18 26 19
1025 1028 1035 1052 18 8 8 5 F M M M 67 50 44 31 32 25 22 9 ND/ ND/ ND/ S65C/ ND ND ND ND 29 25 22 9
46 1 ⬎2500 ⫹
37 1 6000 ⫹ ⫹
54 1 1070 ⫹
47 1 4996
42 0.93 6882
⫹
⫹
⫹
⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹
⫹
⫹ ⫹
54 0.96 268 ⫹ ⫹⫹
61 0.95 573
53 0.96 2432 ⫹
46 1.28 4188
?
⫹
⫹
⫹
⫹
⫹
⫹ ⫹
⫹
⫹ ⫹ ⫹
⫹
⫹
⫹
⫹
51 0.97 739
1019 1018 1059 1060 2 12 57 58 F M F M 45 42 26 34 25 25 25 33 ND/ H63D/ ND/ ND/ ND H63D ND ND 25 24 21 ⫾25 43 0.94 4500 ⫹ ⫹⫹
⫹ ⫹
43 0.85 1517
1058 5 M 32 10 S65C/ ND 10
47 1.07 3681 ⫹
⫹ ⫹
⫹
⫹
⫹ ⫹
⫹ ⫹
⫹
Note. Normal values: serum iron, 9 –29 mol/liter; transferrin saturation, 0.20 – 0.45 fmol; serum ferritin, 30 – 425 g/liter (men), 10 –300 g/liter (women).
nadism (8). The UK hemochromatosis consortium reported a 29 year-old male with hypogonadotrophic hypogonadism and heart failure and a 16year-old male identified during family screening following the diagnosis of hemochromatosis in his 21-year-old brother who had heart failure (9). All three patients were negative for the C282Y and H63D mutations (8,9). To the best of our knowledge, it is thus far the largest series of juvenile hemochromatosis patients reported from a same region. All the patients in our series, but two brothers diagnosed at age 9 and 10, had heart problems and/or hypogonadotrophic hypogonadism. In a review of the literature on 52 patients with hemochromatosis diagnosed before age 30, Lamon et al. reported that 58.5% had cardiomyopathy and 64.2% hypogonadism (10). In our series, 69.2% had heart problems (arrhythmia and/or failure) and 76.9% hypogonadism, which is higher than in their literature review. Diabetes was present in 33.9%
and liver involvement in 83% of the patients, compared to 30.8% and 53.8% respectively among the patients from Saguenay–Lac-SaintJean. The differences observed between both series could be the result of a smaller number of patients in the Saguenay–Lac-Saint-Jean series or of a more homogeneous group. Indeed, several autosomal recessive disorders have acquired a high frequency by founder effect and genetic drift in Saguenay–Lac-Saint-Jean (18,24,25). Molecular studies have shown that, in many of these disorders, a sole mutation was responsible for the majority of the cases identified in the region. As already noticed by several authors, both sexes are equally affected by juvenile hemochromatosis (6,7,10). However, because females are less likely to have symptoms due to hemochromatosis type 1, the diagnosis might be delayed. In our series, 5 of the 6 women were diagnosed at least two years after the onset of the first symp12
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Blood Cells, Molecules, and Diseases (2000) 26(2) January 31: 10 –14 doi:10.1006/bcmd.2000.0271, available online at http://www.idealibrary.com on
toms (mean difference: 3.2 years, SD: 1.9) whereas 6 of the 7 men were diagnosed less than a year after the first symptoms (mean difference: 0.2 years, SD: 0.4). Therefore, serum iron indices should be included in the biochemical workout of all young women with cardiac arrhythmia and/or unexplained secondary amenorrhea. Mutation analysis of the HFE gene showed that the majority of cases with juvenile hemochromatosis had no identified mutation (8,9,14,15,26,27). A few patients with juvenile hemochromatosis, including some in this series, were heterozygous for the C282Y mutation of the HFE gene (6). It is likely that the presence of the C282Y mutation is due to its high frequency rate in the general population although we cannot exclude that the mutation participated in the phenotype. The same conclusion applies for both brothers in family #5 found to be heterozygous for the rare mutation S65C. Its role in the phenotype of typical hereditary hemochromatosis is still debated but is definitely not associated with a severe phenotype (5). One patient was homozygous for the H63D mutation. This genotype is associated with a mild hereditary hemochromatosis phenotype and has even been found in asymptomatic patients (4). Given the high frequency of the H63D mutation in Saguenay–Lac-Saint-Jean (28), the homozygosity observed in our patient is presumably due to chance alone. It is highly unlikely that it is responsible for the severe phenotype, although it may have contributed to it in some undefined way. We are now contacting the families to realize a linkage analysis that will allow us to determine whether juvenile hemochromatosis in Saguenay– Lac-Saint-Jean is associated or not with the locus on chromosome 1q described by Roetto et al. (16).
de Cycles Supe´rieurs et de la Recherche at the Universite´ du Que´bec a` Chicoutimi.
ACKNOWLEDGMENTS
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The authors thank Mrs. Marielle Gagnon and Mr. Jocelyn Tremblay for their invaluable help. This research was supported by INSERM grants from CRI9607 and the Association de Transfusion Sanguine et de Bioge´ne´tique Gae¨tan Saleun and a grant from the De´canat des Etudes
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