1. HEMOCHROMATOSIS: BEING AN “IRON MAN” CARRIES RISK! Gina C. Guzman, MD, DBIM, FALU, FLMI 2nd VP & Medical Director

2. Hereditary Hemochromatosis (HH) 2 Genetic Disorder of Iron Metabolism Autosomal Recessive Excessive Iron Overload Symptoms Due to Iron Accumulation in Various Tissues Cause of Significant Morbidity and Mortality Nov 17, 2015 NHOLUA

3. Total Body Iron ~3.5 grams Nov 17, 2015 NHOLUA 3

4. Normal Iron Absorption and Metabolism Nov 17, 2015 NHOLUA 4 2.5 gms 1 gm Total Body Iron ~3.5 grams Women – 2mg/day Menses Pregnancy Women – 2mg/day Menses Pregnancy 3-7mg

5. Pathophysiology of HH Nov 17, 2015 NHOLUA 5 Persons with Hereditary Hemochromatosis continue to absorb iron even when their body already has enough Excessive Iron Deposition in Tissues (especially the liver, heart, pancreas, and pituitary) except BLOOD LOSS NORMAL PERSON

6. “Classic”19 th Century Presentation Middle-aged man Diabetes Mellitus Bronzed skin Hepatomegaly +/- cirrhosis Bronze diabetic with cirrhosis Advanced end- organ damage with poor mortality Considered “rare” 6 Nov 17, 2015 NHOLUA

7. 21 st Century Presentation Younger person Asymptomatic Elevated liver function tests No end organ damage with good prognosis Common disease 7 Nov 17, 2015 NHOLUA

8. Why the switch? Increased routine iron testing during routine physical exams Increased awareness of the diseaseIncreased diagnosis before symptoms developDevelopment of a genetic test in 1996 Nov 17, 2015 NHOLUA 8

9. Prevalence of HH 1 in 250 persons Most common single-gene genetic disorder in the US  White Northern European descent (1 in 227)  Hispanic (1 in 3700)  Black (1 in 7000)  Asians (<0.001 per 1000) Caucasian population (USA, Western Europe)  10% carrier for the mutation  0.5% homozygous state 9 N Engl J Med 2005;352:1769-78. Nov 17, 2015 NHOLUA

10. Iron Stores & Clinical Manifestations 10 Adapted from Riely CA, Vera SR. Inherited liver disease. American Gastroenterological Association, 1993. Nov 17, 2015 NHOLUA ^ Diagnosis before Sx Dx here RARE

11. Early Symptoms No symptoms  A common early sign is asymptomatic elevation of LFT’s (75%) Early, non-specific  Fatigue/Weakness (74%)  Joint pain (44%)  Weight loss  Abdominal pain  Palpitations 11 Hepatology 1997;25:162-6. Nov 17, 2015 NHOLUA

12. Late Symptoms – Where is Iron Deposited? Nov 17, 2015 NHOLUA 12 Hepatomegaly ^LFTs Cirrhosis Hepatomegaly ^LFTs Cirrhosis Cardiomyopathy Arrhythmia Cardiomyopathy Arrhythmia Bronze Skin Diabetes Impotence Early menopause Impotence Early menopause Hypothyroidism Arthritis

13. CASE FILE REVIEW Nov 17, 2015 NHOLUA 13

14. Case File Review 14  43 y/o NS male  $5,000,000 term life  No admitted medical history  “Normal” physical last year  AST 47 and ALT 72  Hep B & C - negative Nov 17, 2015 NHOLUA

15. Differential Diagnosis of ^LFTs 15 Drugs and Alcohol Nov 17, 2015 NHOLUA

16. Medications and Supplements 16 Antibiotics Lipid Lowering Drugs (Statins) Seizure drugs Herbal therapy Recreational drugs Nov 17, 2015 NHOLUA

17. Liver Disease 17 Viral Hepatitis ( B and C) NAFLD Fatty liver/NASH Hemochromatosis Wilson’s disease Autoimmune Hepatitis Alpha-1-Antitrypsin deficiency Nov 17, 2015 NHOLUA

18. Other Possibilities…  Muscle Disorders  Thyroid Disorders  Celiac Disease  Adrenal insufficiency Nov 17, 2015 NHOLUA 18

19. Sample Evaluation of Mild ^LFTs HepBsAg Hep C Ab Iron studies Abdominal ultrasound of the liver Serum ceruloplasmin - (decreased in Wilson’s) ANA, anti-smooth muscle Ab (ASMA) (+ in autoimmune hepatitis) Serum alpha-1- antitrypsin – (decreased in deficiency) 19 Nov 17, 2015 NHOLUA

20. Iron Studies Serum Fe Total Iron Binding Capacity (TIBC) Ferritin Transferrin Saturation Nov 17, 2015 NHOLUA 20

21. Transferrin Saturation (TS) How much iron is bound to the carrier protein Earliest lab abnormality in iron overload BEST TEST for detecting iron overload Serum Fe/TIBC Abnormal >50% in women >60% in men Should be done fasting 21 Nov 17, 2015 NHOLUA 95% accuracy in identifying iron overload

22. Our case had iron studies… Total Serum Fe = 221 (49-181 ug/dL) TIBC = 261 (250-450 ug/dL) Ferritin = 1277 (20-300 ng/mL) Transferrin Sat = Serum iron/TIBC 221/261 = 85% ABNORMAL (>50% in women, 60% in men) 22 Nov 17, 2015 NHOLUA

23. Disorders Associated with Iron Overload Hereditary hemochromatosis Related to HFE gene Not related to HFE gene  African (Bantu) hemochromatosis  Juvenile hemochromatosis  Neonatal hemochromatosis Chronic anemias Thalassemia major Sideroblastic anemia Congenital dyserythropoietic anemia Congenital atransferrinemia 23 Exogenous iron overload  Chronic iron supplementation (in absence of blood loss)  Transfusion  Iron dextran injection  Oral supplements (rare) Chronic liver diseases  Viral hepatitis  Alcoholic liver disease  Nonalcoholic steatohepatitis  Porphyria cutanea tarda  Portacaval shunt Nov 17, 2015 NHOLUA

24. Work-up for HH Fasting Transferrin Saturation >50% in women >60% in men Ferritin CBC LFT’s Genetic TestingLiver Biopsy 24 J Hepatol 2000;33:485-504. Nov 17, 2015 NHOLUA

25. Ferritin Iron storage protein – reflects the body’s stores of iron Normal levels  20-300 ng/mL (male), 20-150 ng/mL (female) Acute Phase Reactant  Elevated in inflammation, infection, recent trauma, surgery, cancer  Less specific than the iron saturation (PPV 20% vs. 80%) for HH  Should NOT be used as a screening test Ferritin > 600 needs work-up Ferritin > 1000 despite normal TS, may need liver biopsy 25 Clin Chim Acta 1996;245:139-200. Nov 17, 2015 NHOLUA

26. GENETICS Nov 17, 2015 NHOLUA 26

27. Basic Genetics Human DNA 46 Chromosomes (structures that hold our genes) - organized in 23 pairs (one copy from each parent) - 22 pairs of autosomes - 1 pair of sex chromosomes (XX, XY) ~30,000 pairs of Genes Locus – point on the chromosome where the gene is located Alleles – 2 per locus 27 Nov 17, 2015 NHOLUA

28. Alleles 28 Two copies of the sameTwo different HOMOZYGOTE HETEROZYGOTE Nov 17, 2015 NHOLUA

29. More Genetics Definitions Genotype – the genetic makeup of an individual (AA, Aa, aa) Phenotype – the observable appearance of an individual (Black, Brown, Blonde) Autosomal Dominant – only a single abnormal gene from either parent can cause disease Autosomal Recessive – two copies or an abnormal gene must be present for disease Nov 17, 2015 NHOLUA 29

30. Genetic Testing for HH Mutation in the HFE gene – regulates Fe absorptionDiscovered in 1996Short arm of chromosome 6 (6p21.3) 40 allelic variants of the HFE gene have been described to date Only two are significantly correlated with HH 30 Nov 17, 2015 NHOLUA

31. HFE gene defect Two mutations have been described in the majority of patients with HH C282Y Cysteine to Tyrosine at position 282 (C282Y) Strong association with the phenotypic changes of iron overload H63D Histidine to Aspartate at position 63 (H63D) Limited clinical effect 31 Nov 17, 2015 NHOLUA

32. HFE Mutation Combinations Review: Chromosome 6 = 2 alleles 3 possibilities for the HFE gene (C282Y, H63D, Normal version) 6 possible combinations: 1.C282Y/C282Y 2.C282Y/H63D 3.H63D/H63D 4.C282Y/normal 5.H63D/normal 6.Normal/normal Nov 17, 2015 NHOLUA 32

33. Transferrin Sat % and HFE Genotype 33 (N Engl J Med 2005;352:1769-78) Nov 17, 2015 NHOLUA

34. C282Y/C282Y (Homozygote) 69-100% of clinically diagnosed HH (average 83%) Very high prevalence in Caucasians in North America (1:227) More severe than H63D 72-99% likelihood of being free of signs/symptoms of HH at diagnosis Variable penetrance (the proportion of individuals of a particular genotype that express its phenotypic effect)  Up to 50% may never develop clinically significant iron overload (population studies)  Females have lower penetrance over males 34 Amer J of Epidemiology. Vol. 154, No 3, 2001 Lancet 2002;359:211-8. N Engl J Med 2005;352:1769-78 Nov 17, 2015 NHOLUA

35. C282Y/C282Y with NL TS Uncertain at the present time what percentage of these may eventually develop iron overload Cost of follow-up is significant Should be followed yearly (CPE, yearly iron studies) Copenhagen Heart Study monitored 23 homozygotes over 25 years who never developed overt iron overload 35 Blood 2004;103:2914-9. Nov 17, 2015 NHOLUA

36. C282Y/H63D (Compound Heterozygote) 3-5% of clinically diagnosed HH Comorbid factors (Steatosis, DM, Excess Alcohol) ^ risk of developing progressive clinical disease High prevalence of increased iron indices with reduced penetrance of <2%  Only 0.5 – 2 % will develop HH Must have clinical correlation in order to make a diagnosis of HH C282Y/H63D, normal iron studies, no comorbid factors => consider as only a carrier of HH gene with no additional mortality risk 36 Blood Cells Mol Dis. 1997 Aug;23(2):314-20. Clin Gastroenterol Hepatol. 2006 Nov;4(11):1403-10. Nov 17, 2015 NHOLUA

37. H63D/H63D (Homozygote) 1% of diagnosed HH Typically a mild course of disease with less iron accumulation < 1% risk of developing HH Most will NEVER develop any symptoms 4-fold risk of amyotrophic lateral sclerosis (Hepatology. 2007 Sep 7:46(4):1071-1080.) 2-3 fold risk of ischemic stroke (Neurology. 2007 Mar 27;68(13):1025-31.) 37 Nov 17, 2015 NHOLUA

38. C282Y/normal or H63D/normal (Heterozygotes) 3-10% of clinical HH (presumed due to additional unknown mutations) Many have mildly increased iron levels, but no clinical symptoms Majority of these will be HEALTHY CARRIERS with NL iron levels (and no increased mortality risk) 1 in 10 Caucasians in the USA 38 Ann Intern Med. 1999 Jun 15;130(12):953-62. Nov 17, 2015 NHOLUA

39. Caveat about HFE testing HFE gene testing is only 85% accurate Non-HFE related HH (false negatives)  7-10% HH have mutations not commonly tested or not yet discovered  Familial cases of HH without detectable HFE mutation  N Engl J Med 2004;350:2383-97.  Semin Liver Dis 2005;25:450-60.  Amer J of Epidemiology. Vol. 154, No 3, 2001 Variable penetrance (false positives) 39 Nov 17, 2015 NHOLUA

40. Newly, Identified Iron-Related Genes Ferroportin Hemojuvelin Hepcidin Ceruloplasmin Transferrin Receptor 2 Unrelated to HFE Rare cases of iron overload Complex genetic testing Further research ongoing 40 Nov 17, 2015 NHOLUA

41. SCREENING FOR HH Nov 17, 2015 NHOLUA 41

42. Screening for HH American College of Physicians/ NIH/ CDC  (+) Family History and/or (+) symptoms The College of American Pathologists  All adults over 18  (+) Family History - Every 5 years The American Hemochromatosis Society  Age 4 – routine iron testing  (+) Family History – Every 5 years REMEMBER: BEST test for screening Transferrin Sat = Serum Fe / TIBC 42 Nov 17, 2015 NHOLUA

43. U.S. Preventive Services Task Force Against routine genetic testing for HH in the asymptomatic general population Genetic testing should NOT be used as a screening tool 43 Ann Intern Med 2006 Aug 1;145(3):204-8 Nov 17, 2015 NHOLUA

44. USPSTF Grade D 44 Nov 17, 2015 NHOLUA

45. CASE FILE REVIEW Nov 17, 2015 NHOLUA 45

46. Case summary Elevated LFT’s in an asymptomatic male Elevated iron and ferritin Elevated transferrin saturation Genetic Testing reveals: Heterozygote C282Y/H63D mutation Do we have enough now to make the diagnosis of hereditary hemochromatosis? 46 Nov 17, 2015 NHOLUA

47. Minimum Criteria for Diagnosis Increased iron stores  Elevated transferrin saturation  Serum iron/TIBC (+) HFE Gene mutation  C282Y/C282Y or C282Y/H63D 47 Nov 17, 2015 NHOLUA

48. What about liver biopsy? Liver biopsy is no longer essential for the diagnosis Who needs one? What information can be gained? Liver is the major organ affected with HH Easily accessible tissue Prognostic rather than diagnostic 48 Nov 17, 2015 NHOLUA

49. Who should get a liver biopsy? Age over 40 years old Elevated LFT’s Clinical evidence of liver disease History of alcohol abuse Coexisting diabetes, impotence Ferritin level > 1000 ng/mL  ^ likelihood of fibrosis or cirrhosis 49 Gastroenterology 1998;115:929-36. Hepatology 2002;36:673-8. Ann Int Med 2003;138:627-33 Nov 17, 2015 NHOLUA

50. Liver Biopsy 50 Brown pigment = iron Trichrome stained tissue (blue) = fibrosis Nov 17, 2015 NHOLUA

51. Prognostic value of liver biopsy Document the degree of fibrosis, if any  Liver cirrhosis/fibrosis => ^ risk HCC  Normal biopsy => normal life expectancy with treatment Confirm the diagnosis with Hepatic Iron Index  Negative genetic testing  Compound Heterozygote mutation 51 Nov 17, 2015 NHOLUA

52. Hepatic Iron Index (HI) Measurement of the iron concentration HI = Hepatic Iron Concentration (HIC)/patient’s age  Normal HI < 1.0  HI > 1.9 is a strong marker for HH CAVEAT – up to 15 % of homozygous HH will have a normal hepatic iron index despite clinical evidence of disease 52 Nov 17, 2015 NHOLUA

53. Who may NOT get a liver biopsy? Age < 40 years old No clinical evidence of liver disease (e.g. normal LFT’s, no hepatomegaly) Fasting serum ferritin level < 1000 ng/mL These people are unlikely to have significant hepatic injury Clinician may not recommend liver biopsy and proceed directly to treatment A LIVER BIOPSY IS NOT NECESSARY FOR THOSE < 40 Y/O WITH GENOTYPICALLY DEFINED HH (C282Y HOMOZYGOUS) WITH NL LFTS 53 Nov 17, 2015 NHOLUA

54. Back to Our Case One Last Time Applicant has iron overload and probably has hemochromatosis Liver biopsy would be very helpful  Age>40, elevated LFTs, ferritin>1000  Can help confirm diagnosis in a heterozygote Treatment is indicated 54 Nov 17, 2015 NHOLUA

55. Treatment = Phlebotomy 55 Nov 17, 2015 NHOLUA

56. Treatment Goals Initial treatment = “de-ironing”  Removal of 1-2 units of whole blood/week (several months)  1 unit of whole blood = 1 pint = ~ 500 cc = 200-250 mg of iron  Goal Ferritin < 50 ng/mL, TS < 50%  Hgb < 12 for men, Hgb < 11 for women Maintenance  Every 2 to 4 months for life  Maintain Ferritin < 100 ng/mL with normal hemoglobin 56 Nov 17, 2015 NHOLUA

57. Iron Chelation  Binds to iron allowing iron to be excreted in either urine or bile thereby reducing the body burden of iron  Used in the rare case when phlebotomy contraindicated (e.g., HH with severe cardiac involvement – unstable hemodynamic status)  Deferoxamine  Deferiprone  Deferasirox  Almost never necessary due to the ease/efficacy of phlebotomy Nov 17, 2015 NHOLUA 57

58. Dietary Management Avoid iron supplements Avoid excess Vitamin C, which promotes iron absorption  Increased risk of cardiac arrhythmia due to acceleration of iron metabolism Avoid uncooked seafood / raw oysters  Increases the risk of Vibrio vulnificus and Salmonella enteritidis infections  These bacteria grow well in an iron-rich environment  ^ iron may also impair WBC’s fighting capability Limit EtOH consumption  Mild/moderate EtOH consumption ^ prevalence of iron overload  EtOH ^ severity of disease => > 2 drinks/day ^ risk of cirrhosis 58 Nov 17, 2015 NHOLUA

59. Role of Alcohol in HH 59 Scotet V. Am J Epidemiol. 2003 Jul 15;158(2):129-34. Nov 17, 2015 NHOLUA

60. HH and Mortality 60 % of all deaths are related to complications of iron overload #1 LIVER (75% of above)  Cirrhosis  HCC #2 DIABETES #3 CARDIOMYOPATHY Survival is NORMAL in HH patients in whom treatment was initiated before the development of cirrhosis or diabetes 60 Gastroenterology 1996;110:1107-19. Can J Gastroenterol 1993;7:37-41. Nov 17, 2015 NHOLUA

61. Prognosis Early diagnosis => early effective treatment => GOOD prognosis with normal life expectancy  BEST CASE  Compliant with treatment, good follow-up  Ferritin < 100 ng/mL, TS < 50%  No end organ damage  No symptoms  Non-specific symptoms (weakness, fatigue) can resolve  Liver function can return to normal  Endocrine changes may improve (impotence can resolve, insulin requirements can decrease)  Joint pain can resolve 61 Ann Intern Med 1998;129:932-9. Gastroenterology 1996;110:1107-19. Nov 17, 2015 NHOLUA

62. Prognosis Evidence of end organ damage e.g. cirrhosis or DM => WORSE/BAD prognosis  Significant fibrosis in any organ is irreversible  Cirrhosis increases risk for HCC  75% of HH-related deaths  Screen with routine AFP testing  Without treatment, eventual fatal iron overload 62 Gastroenterology 1996;110:1107-19. Hepatology 1992;15:655-9. Nov 17, 2015 NHOLUA

63. Conclusions 63 Nov 17, 2015 NHOLUA Most people with HH will have a normal life expectancyEarly diagnosis and effective treatment is the key Phlebotomy therapy, if initiated early, can prevent cirrhosis, cardiac complications, and diabetes Patients with evidence of iron overload, positive family history, or other risk factors should be screened The best screening test for iron overload is the TRANSFERRIN SATURATION (Serum Fe/TIBC)

64. Thank you! 64