- Email: [email protected]
Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene
European Journal of Medical Genetics xxx (2017) 1e4
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene Khushnooda Ramzan a, Faiqa Imtiaz a, Hamad I. Al-Ashgar b, Moeenaldeen AlSayed c, d, Raashda A. Sulaiman c, d, * a
Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Department of Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia d College of Medicine, Alfaisal University, Riyadh, Saudi Arabia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2016 Received in revised form 9 March 2017 Accepted 11 March 2017 Available online xxx
Juvenile hemochromatosis is a rare but the most severe form of hereditary hemochromatosis which develops due to mutations in the HJV or HAMP genes. It presents in the early adulthood mainly as cardiomyopathy, hypogonadism and liver fibrosis. Unlike hereditary hemochromatosis due to HFE mutation, hepatocellular carcinoma is not known to be associated with juvenile hemochromatosis. Here, we report a patient of Arab ancestry who presented with severe cardiomyopathy. Sequence analysis of the HJV gene followed by homozygosity mapping, identified a previously undescribed homozygous missense variation in exon 3 (c.497A > G; p.H166R) in both the proband and his clinically asymptomatic brother. The former, later developed hepatocellular carcinoma. To the best of our knowledge, neither the mutation identified in our patient, nor a case of juvenile hemochromatosis with hepatocellular carcinoma has been reported before. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Juvenile hemochromatosis HJV/HFE2 mutation Cardiomyopathy Hypogonadism Hepatocellular carcinoma
1. Introduction Juvenile hemochromatosis (JH) is an autosomal recessive disorder, characterized by the excessive intestinal iron absorption leading to iron overload and multiple organ damage. It is caused by a mutation either in HJV (also known as HFE2; NM_213653.03; OMIM 602390), encoding a protein-hemojuvelin; or a mutation in HAMP (NM_02117503; OMIM 613313) which encodes a peptide hepcidin. Hemojuvelin is a transcriptional regulator of hepcidin which plays a key role in regulating iron metabolism. Its deficiency leads to unregulated activity of ferroportin-a transmembrane iron exporter, thereby increasing iron absorption from the enterocytes into the circulation (Roetto et al., 2003). Early onset of severe iron overload in the first three decades of life differentiates JH from the more common HFE related hereditary hemochromatosis (HH). Both males and females are equally affected. Presenting clinical features include hypogonadotropic
* Corresponding author. Department of Medical Genetics, MBC 75, King Faisal Specialist Hospital and Research Centre, P.O. Box No 3354, Riyadh 11211, Saudi Arabia. E-mail address: [email protected] (R.A. Sulaiman).
hypogonadism, cardiomyopathy, glucose intolerance and liver fibrosis (De Gobbi et al., 2002). Cardiac disease is the main cause of death which usually occurs before the fourth decade of life (Santos et al., 2012). Hepatocellular carcinoma (HCC) has not been previously described in association with JH. Although HH is a common genetic disorder found in Caucasians (Adams et al., 2005), it has been rarely reported in people of Asian or Middle Eastern heritage. We report JH in two Arab patients due to a unique homozygous mutation in HJV gene, identified by homozygosity mapping and targeted gene sequencing. 2. Patient data 2.1. Patient 1 A 21-year-old male (IV:3; Fig. 1A), presented to a local hospital with history of progressive breathlessness, and bilateral leg edema for one month following a flu like illness. He was in cardiac failure. An echocardiogram confirmed dilated cardiomyopathy and severe left ventricular dysfunction with ejection fraction of 15 percent. Coronary angiography was unremarkable. He was treated conservatively before being referred to our hospital. There was no history
http://dx.doi.org/10.1016/j.ejmg.2017.03.011 1769-7212/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
2
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
of alcohol or any illicit drug intake, blood transfusion or jaundice. His parents were related and his mother had died of an unknown cause in younger age. He had one sister and two brothers who were apparently healthy. He was noticed to have bilateral gynecomastia, sparse facial, axillary, pubic hair growth (Tanner stage 3e4), and small testicles (6 ml size). He had poor libido. Blood test results showed high iron indices, disturbed liver function and hypogonadotrophic hypogonadism (Table 1). His abdominal ultrasound scan showed normal size of the liver with diffuse fatty infiltration and no focal lesion. Magnetic resonance imaging (MRI) of pituitary gland was unremarkable. Based on the clinical features and biochemical profile, he was suspected to have hereditary hemochromatosis. Subsequent molecular testing confirmed juvenile hemochromatosis. Liver biopsy was not done as hepatic fibro scan revealed minimal fibrosis (stage F1). Therapeutic venesection was initiated. A year later, the patient developed complete heart block requiring insertion of permanent pace maker. He was re-admitted after 18 months, with the history of abdominal pain, nausea, vomiting and diarrhea for three days following a meal at a restaurant. He was in severe cardiac failure with markedly disturbed liver function (ALT: 1108 U/L, bilirubin: 68 mmol/L INR: 3.3) and acute kidney injury. He was treated conservatively. Liver enzymes, INR and renal function gradually improved to base line levels over the next two weeks. However, abdominal ultrasound revealed a focal hypoechoic lesion measuring 2.1 1 1.6 cm in the right lobe of the liver. Dynamic computerized tomogram (CT) showed a 2 cm lesion in the right lobe of the liver with intense arterial uptake of contrast followed by corresponding washout in the venous-delayed phases confirming the diagnosis of HCC. No stigmata of hepatic cirrhosis were seen on the CT examination. Alpha feto-protein level was raised while serum ferritin concentration was 3872 mg/L and transferrin saturation was 94%. In view of
severe congestive heart failure, instead of radiofrequency ablation, he underwent arterial chemo-embolization of the tumor. He was then commenced on iron-chelation therapy with deferiprone (75 mg/kg/day orally) and deferoxamine (40 mg/kg/day as intravenous infusion). Deferoxamine was stopped after four weeks and he was discharged home on deferiprone and deferasirox. This combination therapy led to a remarkable improvement in his iron indices and liver enzymes levels as serum ferritin was reduced to 295 mg/L in four months. Iron chelating agents were then stopped and regular phlebotomy was resumed, removing 250 ml blood every two weeks. His latest ferritin level was 71.9 mg/L, serum iron 7.9 mmol/L, and a transferrin saturation of 12%. A repeat CT abdomen, four months after chemoembolization showed stable residual hyper vascular mass in liver. He is currently on the waiting list for a liver and heart transplant. 2.2. Patient 2 A 32-year-old male (IV:1; Fig. 1A), elder brother of case 1 was diagnosed based on family screening. He was asymptomatic. Physical examination was unremarkable. His blood test results are shown in Table 1. Abdominal ultrasound scan revealed normal size of liver with slightly echogenic texture. Spleen size was 11.5 cm. Hepatic fibro-scan confirmed significant fibrosis (stage F3) though his echocardiogram was normal and he had no hypogonadism. He was started on regular phlebotomy and is currently under surveillance for HCC. 3. Molecular genetic analysis Genomic DNA was isolated from leukocytes in whole blood using the Gentra DNA Extraction Kit (Qiagen, Germantown,
Fig. 1. Pedigree and molecular genetic analysis of HJV mutation. (A) Family pedigree. Open symbols unaffected; filled black symbols affected; double line consanguineous event. The genotypes are shown below each individual. (B) Homozygosity mapping of HH in the family showing homozygosity block between two affected individuals (IV:1 and IV:3) on chromosome 1q21.1 harboring HJV gene. (C) Sanger sequence chromatograms of HJV showing the normal control, carrier, and affected individuals. The arrow indicates a single base A > G transition in exon 3 of HJV that causes a substitution of histidine to arginine at codon 166 (p.H166R). Mutation name is based on full length HJV transcript (NM_213653) and encoded protein (NP_998818). (D) Multiple-sequence alignment in HJV from different species revealed that codon 166 (indicated by arrow), where the mutation (p.H166R) occurred, was located within a highly conserved region.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
3
Table 1 Comparison of the phenotype including laboratory parameters of the two siblings.
Presentation Hepatic involvement Hypogonadism Hemoglobin g/L (135e180) Iron mmol/L (6e27) Ferritin mg/L (30e400) Transferrin saturation % (8e43) ALT U/L (10e45) ALP U/L (50e116) FSH IU/L (1.5e12.4) LH IU/L (1.7e8.6) Prolactin mg/L (4e18.4) Testosterone nmol/L (9.9e27.8) TSH mU/L (0.27e4.2) Free T4 pmol/L (12e22) Hemoglobin A1C % (<65) Hepatitis A,B,C antibodies & autoimmune serology
Patient 1 (IV:3; 21 years old)
Patient 2 (IV:1; 32 years old)
Cardiac failure Minimal fibrosis Present 156 53.5 7722 95 94 126 0.3 0.6 13.4 0.35 1.9 13.5 60 Negative
Asymptomatic Advanced fibrosis Absent 153 46 9685 94 166 110 2.1 2.9 10.3 9.7 2.7 13.5 62 Negative
Maryland, USA). Initially, the molecular genetic analysis for the entire HFE gene (exon 1e6), which is most commonly associated with HH was performed but no apparent alteration in the HFE gene was detected. Genome-wide single nucleotide polymorphism genotyping was performed on the genomic DNA from the available individuals of the family (Fig. 1A): affected individuals (IV:1 and IV:3), their unaffected father (III:1) and sibling (IV:4) using Axiom® CEU Human Array (Affymetrix Santa Clara, CA, USA) according to the manufacturer's protocol. Considering the consanguinity of our enrolled family with an autosomal recessive mode of inheritance, analysis for runs of homozygosity (ROH) was performed by AutoSNPa software (Carr et al., 2006) to examine all the genes known to play a role in the development of the different subtypes of HH. The two affected siblings had a shared ROH consistent with the HJV on chromosome chr1q21, which was not present in unaffected members of the family (Fig. 1B) and was thus prioritized for sequencing. Three coding exons and flanking introns of HJV gene (transcript NM_213653.3) were PCR amplified by primer pairs designed by using Primer 3 v.0.4.0 program (http://bioinfo.ut.ee/primer3-0.4.0/ ). PCR reactions were typically performed in a 25 ml reaction volume containing standard reagents and 20 ng of genomic DNA (primer sequences and conditions are available on request). PCR amplicons were then purified and sequenced with ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3730 automated DNA Analyzer (Applied Biosystems, Inc., Foster City, CA, USA). Sequence analysis was performed using Lasergene (DNA Star Inc., Madison, WI, USA) software package, and then compared to the reference gene bank sequences (HJV; NM_213653). The only sequence variant identified in the affected individuals was the homozygous single base pair substitution c.497A > G in exon 3 of the HJV gene resulting in a missense variant at amino acid position 166 (p.H166R) (Fig. 1C). The unaffected father and brother were identified as heterozygous carriers and the unaffected sister was homozygous for the wild type allele (Fig. 1A). Prediction of pathogenicity for the variant identified in the study was performed by web-based interactive soft wares. The identified p.H166R substitution was scored “as probably damaging” by Poly-Phen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), “as deleterious” by Provean (http://provean.jcvi.org/index.php), “damaging” by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/www/ SIFT_enst_submit.html) and “as disease causing” by Mutation Taster (http://www.mutationtaster.org/). Project HOPE (Venselaar et al., 2010) (http://www.cmbi.ru.nl/hope/home); was used to predict the possible structural changes in the mutant p.H166R; HJV
protein (Q6ZVN8) by building the structure using template PDB: 4BQ8. Replacement of smaller neutral residue histidine with the bigger positively charged arginine might lead to bumps and repulsion of ligands or other residues with the same charge in its vicinity. According to the PISA-database (http://www.ebi.ac.uk/ pdbe/pisa/), the mutated residue is involved in a multimer contact and is located very close to a residue that makes a cysteine bond. The mutant 166R residue will affect possible rearrangements of surrounding residues disturbing the multimeric interactions and cysteine bond, thus may result in creating structural instability and loss of external interactions. In addition, the full coding regions of SLC40A1 (NM_014585.5) Njajou, O. T., (Njajou et al., 2001), TFR2 (NM_003227.3) (Camaschella et al., 2000), HAMP (NM_021175.3) (Roetto et al., 2003), and BMP6 (NM_001718.5) (Meynard et al., 2009) genes, which are associated with hereditary iron overload were also directly sequenced and found to be clear of any pathogenic variation. (The identified variant in this study has been submitted to the ClinVar database at NCBI archives (ID: SUB2472478; http://www. ncbi.nlm.nih.gov/clinvar/). 4. Discussion To date, there is no report of JH or HFE related HH from Saudi Arabia where autosomal recessive diseases are fairly prevalent due to high consanguinity rate. A study on 540 DNA samples derived from Saudi newborn subjects, demonstrated an extremely low incidence of < 0.001 for HFE p.C282Y mutation, and 0.177 for HFE p.H63D mutation (Alsmadi et al., 2006). This study does not exclude the presence of other novel HFE as well as non-HFE mutations in this population. Adams et al. (2005) have described highest levels of serum ferritin and transferrin saturation seen in Asians in their study, despite the lowest prevalence of p.C282Y homozygotes. Novel mutations in HJV, HAMP, and SLC40A1 genes were reported in eight Asian families who were studied for the primary iron overload (Lok et al., 2009). This suggests that non HFEhemochromatosis may not be as rare in Asia and the Middle East as was previously thought. Forty seven pathogenic mutations in HJV have been identified in the different populations (Human Gene Mutation Database; http:// www.hgmd.cf.ac.uk). In this case report, using a combination of homozygosity mapping and targeted gene sequencing, we identified a unique homozygous variation in HJV causing JH in two brothers born from a consanguineous union. The HJV p.H166R
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
4
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
amino acid change is predicted to be pathogenic by in-silico analyses. Protein sequence alignment of HJV orthologs using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) demonstrated that the histidine residue is highly conserved across species (Fig. 1D). In addition, the mutant variant segregated completely with the disease phenotype in the family, further strengthening the correlation of this mutation with the phenotype. The identified variant is not present in the dbSNP database (build 138; http:// www.ncbi.nlm.nih.gov/SNP/) 1000 Genome database (http:// www.1000genomes.org/), or in 200 controls from the same ethnic group. Hypogonadotrophic hypogonadism and cardiomyopathy are frequently the presenting features of JH (De Gobbi et al., 2002). Cardiomyocytes and the cells of the endocrine system have abundant mitochondria and less antioxidant (Lesnefsky et al., 2001). These organs are therefore, more susceptible to iron induced oxidative stress. Excess intracellular iron in HH, is catalyzed to form highly reactive oxygen species (ROS) and free radical hydroxyl ions which damage the cells leading to cell death and fibrosis. However, Lok and colleagues reported a lack of cardiomyopathy in the families with JH in their study (Lok et al., 2009). Our index case presented with severe dilated cardiomyopathy. He was then discovered to have hypogonadism and early hepatic fibrosis prior to developing HCC in the absence of cirrhosis. In contrast, his asymptomatic elder brother with the same HJV mutation had advanced liver fibrosis but to date, he has not developed cardiomyopathy, hypogonadism or HCC. No acquired risk factor for iron overload related organ damage was identified. The discordant phenotype of the two affected brothers suggests that there may be local environmental or other genetic factors, modifying organ specific damage in JH. Recent research is unravelling the role of associated modifying genes in modulating phenotype expression in HH. Exome sequencing of DNA from HFE p.C282Y homozygote males with or without iron overload, revealed a GNPAT gene variant associated with a high-iron phenotype (McLaren et al., 2015). An earlier case report of significant iron overload associated with heterozygous mutation in HJV with wild type HFE, TFR2, HAMP and FPN1, also suggests that other genetic variant might have contributed to the pathogenesis of JH (Pelusi et al., 2014). JH should be suspected in any young patient with unexplained cardiac failure, hypogonadism or cirrhosis. Screening with plasma transferrin-iron saturation and serum ferritin confirms systemic iron overload. In the absence of secondary causes of iron overload, patients under 30 years of age should be first tested for the HJV gene mutation which accounts for more than 90% cases of JH. HCC is a well-known complication of HFE related HH, especially in patients with liver cirrhosis (Harrison and Bacon, 2005). Interestingly, a recent meta-analysis of 36 studies to investigate the association of two common HFE mutations with the risk of cancer, demonstrated increased risk of HCC, breast cancer, colorectal cancer in HH due to HFE p.C282Y mutation while p.H63D mutation was not associated with significantly increased cancer risk (Lv et al., 2016). This study also showed higher risk of cancer in Asian population as compared to the Caucasians. This suggests that in addition to iron induced oxidative damage, other factors such as genegene and gene-environmental interactions might be playing significant role in cancer development by causing activation of oncogene or inactivation of cancer suppressor gene. To the best of our knowledge this is the first case report of HCC developing in a young patient of JH with a novel HJV mutation, who did not have hepatic cirrhosis or exposure to viral hepatitis. The exact etiology of cancer and the molecular mechanism of how certain mutations increase the cancer risk, remain to be elucidated.
Conflict of interest Authors declare no conflict of interest relevant to this manuscript.
Acknowledgements The authors wish to thank the family for the necessary medical data and blood samples. We also thank the Genotyping and Sequencing Core Facilities at the Department of genetics, KFSH&RC, for their technical help. We are also grateful to our colleagues in the departments of cardiology, endocrinology and intervention radiology for their contribution in the management of index case.
References Adams, P.C., Reboussin, D.M., Barton, J.C., McLaren, C.E., Eckfeldt, J.H., McLaren, G.D., Dawkins, F.W., Acton, R.T., Harris, E.L., Gordeuk, V.R., Leiendecker-Foster, C., Speechley, M., Snively, B.M., Holup, J.L., Thomson, E., Sholinsky, P., 2005. Hemochromatosis and iron-overload screening in a racially diverse population. N. Engl. J. Med. 352 (17), 1769e1778. Alsmadi, O.A., Al-Kayal, F., Al-Hamed, M., Meyer, B.F., 2006. Frequency of common HFE variants in the Saudi population: a high throughput molecular beaconbased study. BMC Med. Genet. 7, 43. Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., Gasparini, P., 2000. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat. Genet. 25 (1), 14e15. Carr, I.M., Flintoff, K.J., Taylor, G.R., Markham, A.F., Bonthron, D.T., 2006. Interactive visual analysis of SNP data for rapid autozygosity mapping in consanguineous families. Hum. Mutat. 27 (10), 1041e1046. De Gobbi, M., Roetto, A., Piperno, A., Mariani, R., Alberti, F., Papanikolaou, G., Politou, M., Lockitch, G., Girelli, D., Fargion, S., Cox, T.M., Gasparini, P., Cazzola, M., Camaschella, C., 2002. Natural history of juvenile haemochromatosis. Br. J. Haematol. 117 (4), 973e979. Harrison, S.A., Bacon, B.R., 2005. Relation of hemochromatosis with hepatocellular carcinoma: epidemiology, natural history, pathophysiology, screening, treatment, and prevention. Med. Clin. N. Am. 89 (2), 391e409. Lesnefsky, E.J., Moghaddas, S., Tandler, B., Kerner, J., Hoppel, C.L., 2001. Mitochondrial dysfunction in cardiac disease: ischemiaereperfusion, aging, and heart failure. J. Mol. Cell Cardiol. 33 (6), 1065e1089. Lok, C.Y., Merryweather-Clarke, A.T., Viprakasit, V., Chinthammitr, Y., Srichairatanakool, S., Limwongse, C., Oleesky, D., Robins, A.J., Hudson, J., Wai, P., Premawardhena, A., de Silva, H.J., Dassanayake, A., McKeown, C., Jackson, M., Gama, R., Khan, N., Newman, W., Banait, G., Chilton, A., Wilson-Morkeh, I., Weatherall, D.J., Robson, K.J., 2009. Iron overload in the Asian community. Blood 114 (1), 20e25. Lv, Y.F., Chang, X., Hua, R.X., Yan, G.N., Meng, G., Liao, X.Y., Zhang, X., Guo, Q.N., 2016. The risk of new-onset cancer associated with HFE C282Y and H63D mutations: evidence from 87,028 participants. J. Cell Mol. Med. 20 (7), 1219e1233. McLaren, C.E., Emond, M.J., Subramaniam, V.N., Phatak, P.D., Barton, J.C., Adams, P.C., Goh, J.B., McDonald, C.J., Powell, L.W., Gurrin, L.C., Allen, K.J., Nickerson, D.A., Louie, T., Ramm, G.A., Anderson, G.J., McLaren, G.D., 2015. Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload. Hepatology 62 (2), 429e439. Meynard, D., Kautz, L., Darnaud, V., Canonne-Hergaux, F., Coppin, H., Roth, M.P., 2009. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat. Genet. 41 (4), 478e481. Njajou, O.T., Vaessen, N., Joosse, M., Berghuis, B., van Dongen, J.W., Breuning, M.H., Snijders, P.J., Rutten, W.P., Sandkuijl, L.A., Oostra, B.A., van Duijn, C.M., Heutink, P., 2001. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat. Genet. 28 (3), 213e214. Pelusi, S., Rametta, R., Della Corte, C., Congia, R., Dongiovanni, P., Pulixi, E.A., Fargion, S., Fracanzani, A.L., Nobili, V., Valenti, L., 2014. Juvenile hemochromatosis associated with heterozygosity for novel hemojuvelin mutations and with unknown cofactors. Ann. Hepatol. 13 (5), 568e571. Roetto, A., Papanikolaou, G., Politou, M., Alberti, F., Girelli, D., Christakis, J., Loukopoulos, D., Camaschella, C., 2003. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat. Genet. 33 (1), 21e22. Santos, P.C., Dinardo, C.L., Cancado, R.D., Schettert, I.T., Krieger, J.E., Pereira, A.C., 2012. Non-HFE hemochromatosis. Rev. Bras. Hematol. Hemoter. 34 (4), 311e316. Venselaar, H., Te Beek, T.A., Kuipers, R.K., Hekkelman, M.L., Vriend, G., 2010. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinform. 11, 548.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene Khushnooda Ramzan a, Faiqa Imtiaz a, Hamad I. Al-Ashgar b, Moeenaldeen AlSayed c, d, Raashda A. Sulaiman c, d, * a
Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Department of Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia d College of Medicine, Alfaisal University, Riyadh, Saudi Arabia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2016 Received in revised form 9 March 2017 Accepted 11 March 2017 Available online xxx
Juvenile hemochromatosis is a rare but the most severe form of hereditary hemochromatosis which develops due to mutations in the HJV or HAMP genes. It presents in the early adulthood mainly as cardiomyopathy, hypogonadism and liver fibrosis. Unlike hereditary hemochromatosis due to HFE mutation, hepatocellular carcinoma is not known to be associated with juvenile hemochromatosis. Here, we report a patient of Arab ancestry who presented with severe cardiomyopathy. Sequence analysis of the HJV gene followed by homozygosity mapping, identified a previously undescribed homozygous missense variation in exon 3 (c.497A > G; p.H166R) in both the proband and his clinically asymptomatic brother. The former, later developed hepatocellular carcinoma. To the best of our knowledge, neither the mutation identified in our patient, nor a case of juvenile hemochromatosis with hepatocellular carcinoma has been reported before. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Juvenile hemochromatosis HJV/HFE2 mutation Cardiomyopathy Hypogonadism Hepatocellular carcinoma
1. Introduction Juvenile hemochromatosis (JH) is an autosomal recessive disorder, characterized by the excessive intestinal iron absorption leading to iron overload and multiple organ damage. It is caused by a mutation either in HJV (also known as HFE2; NM_213653.03; OMIM 602390), encoding a protein-hemojuvelin; or a mutation in HAMP (NM_02117503; OMIM 613313) which encodes a peptide hepcidin. Hemojuvelin is a transcriptional regulator of hepcidin which plays a key role in regulating iron metabolism. Its deficiency leads to unregulated activity of ferroportin-a transmembrane iron exporter, thereby increasing iron absorption from the enterocytes into the circulation (Roetto et al., 2003). Early onset of severe iron overload in the first three decades of life differentiates JH from the more common HFE related hereditary hemochromatosis (HH). Both males and females are equally affected. Presenting clinical features include hypogonadotropic
* Corresponding author. Department of Medical Genetics, MBC 75, King Faisal Specialist Hospital and Research Centre, P.O. Box No 3354, Riyadh 11211, Saudi Arabia. E-mail address: [email protected] (R.A. Sulaiman).
hypogonadism, cardiomyopathy, glucose intolerance and liver fibrosis (De Gobbi et al., 2002). Cardiac disease is the main cause of death which usually occurs before the fourth decade of life (Santos et al., 2012). Hepatocellular carcinoma (HCC) has not been previously described in association with JH. Although HH is a common genetic disorder found in Caucasians (Adams et al., 2005), it has been rarely reported in people of Asian or Middle Eastern heritage. We report JH in two Arab patients due to a unique homozygous mutation in HJV gene, identified by homozygosity mapping and targeted gene sequencing. 2. Patient data 2.1. Patient 1 A 21-year-old male (IV:3; Fig. 1A), presented to a local hospital with history of progressive breathlessness, and bilateral leg edema for one month following a flu like illness. He was in cardiac failure. An echocardiogram confirmed dilated cardiomyopathy and severe left ventricular dysfunction with ejection fraction of 15 percent. Coronary angiography was unremarkable. He was treated conservatively before being referred to our hospital. There was no history
http://dx.doi.org/10.1016/j.ejmg.2017.03.011 1769-7212/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
2
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
of alcohol or any illicit drug intake, blood transfusion or jaundice. His parents were related and his mother had died of an unknown cause in younger age. He had one sister and two brothers who were apparently healthy. He was noticed to have bilateral gynecomastia, sparse facial, axillary, pubic hair growth (Tanner stage 3e4), and small testicles (6 ml size). He had poor libido. Blood test results showed high iron indices, disturbed liver function and hypogonadotrophic hypogonadism (Table 1). His abdominal ultrasound scan showed normal size of the liver with diffuse fatty infiltration and no focal lesion. Magnetic resonance imaging (MRI) of pituitary gland was unremarkable. Based on the clinical features and biochemical profile, he was suspected to have hereditary hemochromatosis. Subsequent molecular testing confirmed juvenile hemochromatosis. Liver biopsy was not done as hepatic fibro scan revealed minimal fibrosis (stage F1). Therapeutic venesection was initiated. A year later, the patient developed complete heart block requiring insertion of permanent pace maker. He was re-admitted after 18 months, with the history of abdominal pain, nausea, vomiting and diarrhea for three days following a meal at a restaurant. He was in severe cardiac failure with markedly disturbed liver function (ALT: 1108 U/L, bilirubin: 68 mmol/L INR: 3.3) and acute kidney injury. He was treated conservatively. Liver enzymes, INR and renal function gradually improved to base line levels over the next two weeks. However, abdominal ultrasound revealed a focal hypoechoic lesion measuring 2.1 1 1.6 cm in the right lobe of the liver. Dynamic computerized tomogram (CT) showed a 2 cm lesion in the right lobe of the liver with intense arterial uptake of contrast followed by corresponding washout in the venous-delayed phases confirming the diagnosis of HCC. No stigmata of hepatic cirrhosis were seen on the CT examination. Alpha feto-protein level was raised while serum ferritin concentration was 3872 mg/L and transferrin saturation was 94%. In view of
severe congestive heart failure, instead of radiofrequency ablation, he underwent arterial chemo-embolization of the tumor. He was then commenced on iron-chelation therapy with deferiprone (75 mg/kg/day orally) and deferoxamine (40 mg/kg/day as intravenous infusion). Deferoxamine was stopped after four weeks and he was discharged home on deferiprone and deferasirox. This combination therapy led to a remarkable improvement in his iron indices and liver enzymes levels as serum ferritin was reduced to 295 mg/L in four months. Iron chelating agents were then stopped and regular phlebotomy was resumed, removing 250 ml blood every two weeks. His latest ferritin level was 71.9 mg/L, serum iron 7.9 mmol/L, and a transferrin saturation of 12%. A repeat CT abdomen, four months after chemoembolization showed stable residual hyper vascular mass in liver. He is currently on the waiting list for a liver and heart transplant. 2.2. Patient 2 A 32-year-old male (IV:1; Fig. 1A), elder brother of case 1 was diagnosed based on family screening. He was asymptomatic. Physical examination was unremarkable. His blood test results are shown in Table 1. Abdominal ultrasound scan revealed normal size of liver with slightly echogenic texture. Spleen size was 11.5 cm. Hepatic fibro-scan confirmed significant fibrosis (stage F3) though his echocardiogram was normal and he had no hypogonadism. He was started on regular phlebotomy and is currently under surveillance for HCC. 3. Molecular genetic analysis Genomic DNA was isolated from leukocytes in whole blood using the Gentra DNA Extraction Kit (Qiagen, Germantown,
Fig. 1. Pedigree and molecular genetic analysis of HJV mutation. (A) Family pedigree. Open symbols unaffected; filled black symbols affected; double line consanguineous event. The genotypes are shown below each individual. (B) Homozygosity mapping of HH in the family showing homozygosity block between two affected individuals (IV:1 and IV:3) on chromosome 1q21.1 harboring HJV gene. (C) Sanger sequence chromatograms of HJV showing the normal control, carrier, and affected individuals. The arrow indicates a single base A > G transition in exon 3 of HJV that causes a substitution of histidine to arginine at codon 166 (p.H166R). Mutation name is based on full length HJV transcript (NM_213653) and encoded protein (NP_998818). (D) Multiple-sequence alignment in HJV from different species revealed that codon 166 (indicated by arrow), where the mutation (p.H166R) occurred, was located within a highly conserved region.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
3
Table 1 Comparison of the phenotype including laboratory parameters of the two siblings.
Presentation Hepatic involvement Hypogonadism Hemoglobin g/L (135e180) Iron mmol/L (6e27) Ferritin mg/L (30e400) Transferrin saturation % (8e43) ALT U/L (10e45) ALP U/L (50e116) FSH IU/L (1.5e12.4) LH IU/L (1.7e8.6) Prolactin mg/L (4e18.4) Testosterone nmol/L (9.9e27.8) TSH mU/L (0.27e4.2) Free T4 pmol/L (12e22) Hemoglobin A1C % (<65) Hepatitis A,B,C antibodies & autoimmune serology
Patient 1 (IV:3; 21 years old)
Patient 2 (IV:1; 32 years old)
Cardiac failure Minimal fibrosis Present 156 53.5 7722 95 94 126 0.3 0.6 13.4 0.35 1.9 13.5 60 Negative
Asymptomatic Advanced fibrosis Absent 153 46 9685 94 166 110 2.1 2.9 10.3 9.7 2.7 13.5 62 Negative
Maryland, USA). Initially, the molecular genetic analysis for the entire HFE gene (exon 1e6), which is most commonly associated with HH was performed but no apparent alteration in the HFE gene was detected. Genome-wide single nucleotide polymorphism genotyping was performed on the genomic DNA from the available individuals of the family (Fig. 1A): affected individuals (IV:1 and IV:3), their unaffected father (III:1) and sibling (IV:4) using Axiom® CEU Human Array (Affymetrix Santa Clara, CA, USA) according to the manufacturer's protocol. Considering the consanguinity of our enrolled family with an autosomal recessive mode of inheritance, analysis for runs of homozygosity (ROH) was performed by AutoSNPa software (Carr et al., 2006) to examine all the genes known to play a role in the development of the different subtypes of HH. The two affected siblings had a shared ROH consistent with the HJV on chromosome chr1q21, which was not present in unaffected members of the family (Fig. 1B) and was thus prioritized for sequencing. Three coding exons and flanking introns of HJV gene (transcript NM_213653.3) were PCR amplified by primer pairs designed by using Primer 3 v.0.4.0 program (http://bioinfo.ut.ee/primer3-0.4.0/ ). PCR reactions were typically performed in a 25 ml reaction volume containing standard reagents and 20 ng of genomic DNA (primer sequences and conditions are available on request). PCR amplicons were then purified and sequenced with ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3730 automated DNA Analyzer (Applied Biosystems, Inc., Foster City, CA, USA). Sequence analysis was performed using Lasergene (DNA Star Inc., Madison, WI, USA) software package, and then compared to the reference gene bank sequences (HJV; NM_213653). The only sequence variant identified in the affected individuals was the homozygous single base pair substitution c.497A > G in exon 3 of the HJV gene resulting in a missense variant at amino acid position 166 (p.H166R) (Fig. 1C). The unaffected father and brother were identified as heterozygous carriers and the unaffected sister was homozygous for the wild type allele (Fig. 1A). Prediction of pathogenicity for the variant identified in the study was performed by web-based interactive soft wares. The identified p.H166R substitution was scored “as probably damaging” by Poly-Phen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), “as deleterious” by Provean (http://provean.jcvi.org/index.php), “damaging” by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/www/ SIFT_enst_submit.html) and “as disease causing” by Mutation Taster (http://www.mutationtaster.org/). Project HOPE (Venselaar et al., 2010) (http://www.cmbi.ru.nl/hope/home); was used to predict the possible structural changes in the mutant p.H166R; HJV
protein (Q6ZVN8) by building the structure using template PDB: 4BQ8. Replacement of smaller neutral residue histidine with the bigger positively charged arginine might lead to bumps and repulsion of ligands or other residues with the same charge in its vicinity. According to the PISA-database (http://www.ebi.ac.uk/ pdbe/pisa/), the mutated residue is involved in a multimer contact and is located very close to a residue that makes a cysteine bond. The mutant 166R residue will affect possible rearrangements of surrounding residues disturbing the multimeric interactions and cysteine bond, thus may result in creating structural instability and loss of external interactions. In addition, the full coding regions of SLC40A1 (NM_014585.5) Njajou, O. T., (Njajou et al., 2001), TFR2 (NM_003227.3) (Camaschella et al., 2000), HAMP (NM_021175.3) (Roetto et al., 2003), and BMP6 (NM_001718.5) (Meynard et al., 2009) genes, which are associated with hereditary iron overload were also directly sequenced and found to be clear of any pathogenic variation. (The identified variant in this study has been submitted to the ClinVar database at NCBI archives (ID: SUB2472478; http://www. ncbi.nlm.nih.gov/clinvar/). 4. Discussion To date, there is no report of JH or HFE related HH from Saudi Arabia where autosomal recessive diseases are fairly prevalent due to high consanguinity rate. A study on 540 DNA samples derived from Saudi newborn subjects, demonstrated an extremely low incidence of < 0.001 for HFE p.C282Y mutation, and 0.177 for HFE p.H63D mutation (Alsmadi et al., 2006). This study does not exclude the presence of other novel HFE as well as non-HFE mutations in this population. Adams et al. (2005) have described highest levels of serum ferritin and transferrin saturation seen in Asians in their study, despite the lowest prevalence of p.C282Y homozygotes. Novel mutations in HJV, HAMP, and SLC40A1 genes were reported in eight Asian families who were studied for the primary iron overload (Lok et al., 2009). This suggests that non HFEhemochromatosis may not be as rare in Asia and the Middle East as was previously thought. Forty seven pathogenic mutations in HJV have been identified in the different populations (Human Gene Mutation Database; http:// www.hgmd.cf.ac.uk). In this case report, using a combination of homozygosity mapping and targeted gene sequencing, we identified a unique homozygous variation in HJV causing JH in two brothers born from a consanguineous union. The HJV p.H166R
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011
4
K. Ramzan et al. / European Journal of Medical Genetics xxx (2017) 1e4
amino acid change is predicted to be pathogenic by in-silico analyses. Protein sequence alignment of HJV orthologs using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) demonstrated that the histidine residue is highly conserved across species (Fig. 1D). In addition, the mutant variant segregated completely with the disease phenotype in the family, further strengthening the correlation of this mutation with the phenotype. The identified variant is not present in the dbSNP database (build 138; http:// www.ncbi.nlm.nih.gov/SNP/) 1000 Genome database (http:// www.1000genomes.org/), or in 200 controls from the same ethnic group. Hypogonadotrophic hypogonadism and cardiomyopathy are frequently the presenting features of JH (De Gobbi et al., 2002). Cardiomyocytes and the cells of the endocrine system have abundant mitochondria and less antioxidant (Lesnefsky et al., 2001). These organs are therefore, more susceptible to iron induced oxidative stress. Excess intracellular iron in HH, is catalyzed to form highly reactive oxygen species (ROS) and free radical hydroxyl ions which damage the cells leading to cell death and fibrosis. However, Lok and colleagues reported a lack of cardiomyopathy in the families with JH in their study (Lok et al., 2009). Our index case presented with severe dilated cardiomyopathy. He was then discovered to have hypogonadism and early hepatic fibrosis prior to developing HCC in the absence of cirrhosis. In contrast, his asymptomatic elder brother with the same HJV mutation had advanced liver fibrosis but to date, he has not developed cardiomyopathy, hypogonadism or HCC. No acquired risk factor for iron overload related organ damage was identified. The discordant phenotype of the two affected brothers suggests that there may be local environmental or other genetic factors, modifying organ specific damage in JH. Recent research is unravelling the role of associated modifying genes in modulating phenotype expression in HH. Exome sequencing of DNA from HFE p.C282Y homozygote males with or without iron overload, revealed a GNPAT gene variant associated with a high-iron phenotype (McLaren et al., 2015). An earlier case report of significant iron overload associated with heterozygous mutation in HJV with wild type HFE, TFR2, HAMP and FPN1, also suggests that other genetic variant might have contributed to the pathogenesis of JH (Pelusi et al., 2014). JH should be suspected in any young patient with unexplained cardiac failure, hypogonadism or cirrhosis. Screening with plasma transferrin-iron saturation and serum ferritin confirms systemic iron overload. In the absence of secondary causes of iron overload, patients under 30 years of age should be first tested for the HJV gene mutation which accounts for more than 90% cases of JH. HCC is a well-known complication of HFE related HH, especially in patients with liver cirrhosis (Harrison and Bacon, 2005). Interestingly, a recent meta-analysis of 36 studies to investigate the association of two common HFE mutations with the risk of cancer, demonstrated increased risk of HCC, breast cancer, colorectal cancer in HH due to HFE p.C282Y mutation while p.H63D mutation was not associated with significantly increased cancer risk (Lv et al., 2016). This study also showed higher risk of cancer in Asian population as compared to the Caucasians. This suggests that in addition to iron induced oxidative damage, other factors such as genegene and gene-environmental interactions might be playing significant role in cancer development by causing activation of oncogene or inactivation of cancer suppressor gene. To the best of our knowledge this is the first case report of HCC developing in a young patient of JH with a novel HJV mutation, who did not have hepatic cirrhosis or exposure to viral hepatitis. The exact etiology of cancer and the molecular mechanism of how certain mutations increase the cancer risk, remain to be elucidated.
Conflict of interest Authors declare no conflict of interest relevant to this manuscript.
Acknowledgements The authors wish to thank the family for the necessary medical data and blood samples. We also thank the Genotyping and Sequencing Core Facilities at the Department of genetics, KFSH&RC, for their technical help. We are also grateful to our colleagues in the departments of cardiology, endocrinology and intervention radiology for their contribution in the management of index case.
References Adams, P.C., Reboussin, D.M., Barton, J.C., McLaren, C.E., Eckfeldt, J.H., McLaren, G.D., Dawkins, F.W., Acton, R.T., Harris, E.L., Gordeuk, V.R., Leiendecker-Foster, C., Speechley, M., Snively, B.M., Holup, J.L., Thomson, E., Sholinsky, P., 2005. Hemochromatosis and iron-overload screening in a racially diverse population. N. Engl. J. Med. 352 (17), 1769e1778. Alsmadi, O.A., Al-Kayal, F., Al-Hamed, M., Meyer, B.F., 2006. Frequency of common HFE variants in the Saudi population: a high throughput molecular beaconbased study. BMC Med. Genet. 7, 43. Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., Gasparini, P., 2000. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat. Genet. 25 (1), 14e15. Carr, I.M., Flintoff, K.J., Taylor, G.R., Markham, A.F., Bonthron, D.T., 2006. Interactive visual analysis of SNP data for rapid autozygosity mapping in consanguineous families. Hum. Mutat. 27 (10), 1041e1046. De Gobbi, M., Roetto, A., Piperno, A., Mariani, R., Alberti, F., Papanikolaou, G., Politou, M., Lockitch, G., Girelli, D., Fargion, S., Cox, T.M., Gasparini, P., Cazzola, M., Camaschella, C., 2002. Natural history of juvenile haemochromatosis. Br. J. Haematol. 117 (4), 973e979. Harrison, S.A., Bacon, B.R., 2005. Relation of hemochromatosis with hepatocellular carcinoma: epidemiology, natural history, pathophysiology, screening, treatment, and prevention. Med. Clin. N. Am. 89 (2), 391e409. Lesnefsky, E.J., Moghaddas, S., Tandler, B., Kerner, J., Hoppel, C.L., 2001. Mitochondrial dysfunction in cardiac disease: ischemiaereperfusion, aging, and heart failure. J. Mol. Cell Cardiol. 33 (6), 1065e1089. Lok, C.Y., Merryweather-Clarke, A.T., Viprakasit, V., Chinthammitr, Y., Srichairatanakool, S., Limwongse, C., Oleesky, D., Robins, A.J., Hudson, J., Wai, P., Premawardhena, A., de Silva, H.J., Dassanayake, A., McKeown, C., Jackson, M., Gama, R., Khan, N., Newman, W., Banait, G., Chilton, A., Wilson-Morkeh, I., Weatherall, D.J., Robson, K.J., 2009. Iron overload in the Asian community. Blood 114 (1), 20e25. Lv, Y.F., Chang, X., Hua, R.X., Yan, G.N., Meng, G., Liao, X.Y., Zhang, X., Guo, Q.N., 2016. The risk of new-onset cancer associated with HFE C282Y and H63D mutations: evidence from 87,028 participants. J. Cell Mol. Med. 20 (7), 1219e1233. McLaren, C.E., Emond, M.J., Subramaniam, V.N., Phatak, P.D., Barton, J.C., Adams, P.C., Goh, J.B., McDonald, C.J., Powell, L.W., Gurrin, L.C., Allen, K.J., Nickerson, D.A., Louie, T., Ramm, G.A., Anderson, G.J., McLaren, G.D., 2015. Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload. Hepatology 62 (2), 429e439. Meynard, D., Kautz, L., Darnaud, V., Canonne-Hergaux, F., Coppin, H., Roth, M.P., 2009. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat. Genet. 41 (4), 478e481. Njajou, O.T., Vaessen, N., Joosse, M., Berghuis, B., van Dongen, J.W., Breuning, M.H., Snijders, P.J., Rutten, W.P., Sandkuijl, L.A., Oostra, B.A., van Duijn, C.M., Heutink, P., 2001. A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis. Nat. Genet. 28 (3), 213e214. Pelusi, S., Rametta, R., Della Corte, C., Congia, R., Dongiovanni, P., Pulixi, E.A., Fargion, S., Fracanzani, A.L., Nobili, V., Valenti, L., 2014. Juvenile hemochromatosis associated with heterozygosity for novel hemojuvelin mutations and with unknown cofactors. Ann. Hepatol. 13 (5), 568e571. Roetto, A., Papanikolaou, G., Politou, M., Alberti, F., Girelli, D., Christakis, J., Loukopoulos, D., Camaschella, C., 2003. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat. Genet. 33 (1), 21e22. Santos, P.C., Dinardo, C.L., Cancado, R.D., Schettert, I.T., Krieger, J.E., Pereira, A.C., 2012. Non-HFE hemochromatosis. Rev. Bras. Hematol. Hemoter. 34 (4), 311e316. Venselaar, H., Te Beek, T.A., Kuipers, R.K., Hekkelman, M.L., Vriend, G., 2010. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinform. 11, 548.
Please cite this article in press as: Ramzan, K., et al., Juvenile hemochromatosis and hepatocellular carcinoma in a patient with a novel mutation in the HJV gene, European Journal of Medical Genetics (2017), http://dx.doi.org/10.1016/j.ejmg.2017.03.011