What is A1AT?

A1AT is a serine protease inhibitor secreted primarily in liver parenchymal cells and to a lesser extent in macrophages.

It protects tissues from enzymes of inflammatory cells by inactivating especially neutrophil elastase (and also a variety of other proteases like collagenase, trypsin and chymotrypsin)

It has a reference range in blood of 1.5 – 3.5 gram/liter but the concentration can rise many fold upon acute inflammation.

In its absence, neutrophil elastase is free to break down elastin, which contributes to the elasticity of the lungs, resulting in respiratory complications such as emphysema, in adults and cirrhosis in adults or children. However the exact mechanism of liver injury is less precise compared to lung injury.

Discuss the phenotypes of A1ATD?

The gene coding for A1AT is located on chromosome 14 and there are more than 90 genetic variants producing different types of A1AT.

Serum AAT when allowed to migrate in a pH gradient gel (IEF or isoelectric focus gel)  travels at different speed according to the protein molecule property- the fastest are termed A and slowest Z.

As every person has two copies of the A1AT gene, a heterozygote with two different copies of the gene may have two different bands showing on electrofocusing. The blood test results (i.e. IEF results) are notated as in PiMM, where Pi stands for protease inhibitor and “MM” is the banding pattern of that patient.

It has a reference range in blood of 1.5 – 3.5 gram/liter but the concentration can rise many fold upon acute inflammation.

The serum levels of some of the common genotypes are:

• PiMM: 100% (normal)
• PiMS: 80% of normal serum level of A1AT
• PiSS: 60% of normal serum level of A1AT
• PiMZ: 60% of normal serum level of A1AT
• PiSZ: 40% of normal serum level of A1AT
• PiZZ: 10-15% (severe alpha 1-antitrypsin deficiency)

M is the most common allele, accounting for 95% of those found in Caucasian people whereas S and Z are the most common mutant alleles.

How common is the condition?

It is the most common metabolic disease affecting the liver and the commonest cause of paediatric liver transplantation.

Prevalence is highest in northern European white population (incidence of homozygous deficiency is 1in 1500). Roughly one in ten in Northern Europe carry a deficiency gene. In USA the incidence is 1 in 1800-2000.

What does liver histology shows?

Periodic acid-Schiff (PAS) positive, diastase resistance globules are seen in periportal hepatocytes. These can be shown to be A1AT using specific antiserum. Fibrosis and cirrhosis can be present in some cases.

How many of the patient with abnormal allele will actually develop the liver disease according to the phenotype?

More than 90 phenotypic variants of alpha1-antitrypsin deficiency have been identified, but one phenotype, PiZZ, is responsible for nearly all cases of AAT deficiency emphysema and liver disease. PiZZ phenotype serum levels range from 3.4-7 µmol/L, about 10-20% of the reference range levels. Other phenotypes associated with alpha1-antitrypsin emphysema and liver disease include PiSZ and PiZ/Null. PiNull/Null is not associated with liver disease but is associated with alpha1-antitrypsin deficiency emphysema.

PiZZ phenotype is associated with development of liver disease in about 15-30% and affects both children and adults.

Prognosis of patients with liver disease presenting in infancy secondary to AAT deficiency (PiZZ) is variable- not all patients progress to end stage liver disease.

Approx 10-15% of the adult patients will develop cirrhosis, usually over 50yrs of age and 75% will have respiratory problems

Heterozygotes (PiSZ or PiMZ) may develop liver disease but the risk is small.

Discuss the clinical features of the condition?

Children and infant normally present with neonatal jaundice, unexplained hepatomegaly, elevated transaminase, failure to thrive.

Adults can present with abnormal LFT, Chronic hepatitis, cirrhosis and its complications or HCC.

Discuss the diagnosis of A1AT deficiency?

1. Low serum AAT level
2. Serum A1 phenotype determination (Pi typing)
3. Liver biopsy: to confirm, exclude other causes and determine prognosis

Discuss the mechanism of damage to liver and lungs in A1AT deficiency?

The most common pathologic form, which causes both lung and liver disease is the PiZZ variant which produces A1ATZ protein.

A1AT is produced in hepatocytes in rough endoplasmic reticulum and the secreted in the circulation via Golgi apparatus. Structural misfolding and polymerization of A1ATZ cause this protein to be retained in the ER but how that subsequently produces liver injury is not clear. However the lung injury is relatively straight forward by uninhibited damage by alveolar elastin fibres by neutrophil elastase present in the alveolar fluid.

Discuss the treatment of the condition?

1. In absence of liver damage- patients are encouraged to avoid smoking (including passive smoking) and alcohol, maintain adequate nutritional intake and take oral fat soluble vitamins if needed.
2. Managing the complications of liver and lung disease once developed.
3. Patients with hepatic decompensation should be considered for liver transplant- donor liver synthesizes normal protein- so condition does not recur in the graft.
4. Experimental treatment for both pulmonary and hepatic disease are infusion/inhalation of purified AAT and gene therapy
5. Offering genetic counselling and phenotype identification of the relatives to determine those with high risk.

It is an autosomal recessive disease characterised by hepatic copper accumulation and injury. The gene ATP7B (on chromosome 13) encodes a transmembrane transporter of copper in hepatocytes. Absent or reduced function of ATP7B protein leads to decreased hepatic excretion of copper into bile. This results in hepatic copper accumulation and injury. Eventually copper is released into the bloodstream and deposited in various other organs, notably the brain, kidneys, and cornea.

Failure to incorporate copper into ceruloplasmin is an additional consequence of the loss of functional ATP7B protein. The hepatic production and secretion of the caeruloplasmin protein without copper (apocaeruloplasmin) results in the decreased blood levels of caeruloplasmin due to the reduced half-life of the apoprotein.

Discuss the copper cycle?

The average diet provides 2-5 mg/day (almost 3 times the recommended intake).
Most dietary copper ends up being excreted. Copper is absorbed mainly in the duodenum and proximal small intestine and transported to the liver, where it is avidly removed from the circulation. The liver utilizes some copper for metabolic needs and excretes excess copper into bile. Processes that impair biliary copper excretion can lead to increases in hepatic copper content

Discuss the clinical presentation of WD?

It can present clinically as liver disease, as a progressive neurologic disorder (hepatic dysfunction being less apparent or occasionally absent), or as psychiatric illness.
The type of the liver disease can be highly variable. It can be asymptomatic with only biochemical abnormalities or present with acute or chronic hepatitis or chronic liver disease with portal hypertension or with acute liver failure.

Discuss the eye manifestations of WD?

Kayser-Fleischer (KF) rings represent deposition of copper in Descemet’s membrane of the cornea. They appear as a band of golden-brownish pigment near the limbus. A slit-lamp examination is required to identify KF rings in most patients. They may also be found in patients with chronic cholestatic diseases. KF ring is present in up to 62% of patients with hepatic disease at the time of diagnosis. KF rings are almost invariably present in patients with a neurological presentation.
Sunflower cataracts, also found by slit-lamp examination, represent deposits of copper in the lens. These typically do not obstruct vision. Both KF rings and sunflower cataracts will gradually disappear with effective medical treatment or following liver transplant.

Discuss the diagnosis of WD?

WD should be considered in any individual between the ages of 3 and 55 years with liver abnormalities of uncertain cause. Age alone should not be the basis for eliminating a diagnosis of WD. Diagnosis of WD is confirmed, if 2 of the following 3 are present:

• Serum caeruloplasmin level <20 mg/dL
• Presence of KF ring
• Hepatic copper content > or =250 µg/g dry weight. (Normal < 50 µg/g).

A basal 24-hour urinary copper of more than 40 µg (>0.6 µmoles or >600 nmoles) suggests WD.

The serum noncaeruloplasmin-bound copper concentration is elevated above 25 µg/dL in most untreated patients (normal <15 µg/dL). Total serum copper (which includes copper incorporated in caeruloplasmin) in WD is usually decreased in proportion to the decreased caeruloplasmin in the circulation.

Mutation analysis by whole-gene sequencing is possible and should be performed on individuals in whom the diagnosis is difficult to establish by clinical and biochemical testing.

When should a diagnosis of WD be considered?

• Adult patients with atypical autoimmune hepatitis or who respond poorly to standard corticosteroid therapy.
• WD should be considered in the differential diagnosis of patients presenting with nonalcoholic fatty liver or who have pathologic findings of NASH.
• WD should be suspected in any patient presenting with fulminant hepatic failure with Coombs-negative intravascular hemolysis, modest elevations in serum amino transferases, low serum alkaline phosphatase, and ratio of alkaline phosphatase to bilirubin of less than 2.
• First-degree relatives of any patient newly diagnosed with WD must be screened for WD.

Discuss the therapeutic options in WD?

D-penicillamine – is the drug of choice.  It is a general chelator of metals and promotes urinary excretion of copper. Recovery from symptomatic liver disease occurs typically during the first 2-6 months of treatment. Penicillamine use is associated with numerous side effects i.e. sensitivity reactions, nephrotoxicity, lupus like syndrome, bone marrow toxicity, hepatotoxicity etc. Dose- 1000-1500 mg/day in 2-4 divided doses. Maintenance dose is usually 750-1000 mg/day.

Trientine – It is another chelator. Like penicillamine, trientine promotes copper excretion by the kidneys.   Trientine is an effective treatment for WD and is indicated especially in patients who are intolerant of penicillamine.  Dose- 750-1500 mg/day in 2-3 divided doses, with 750-1000 mg used for maintenance therapy.   As for penicillamine, trientine should be administered one hour before or two hours after meals.

Zinc – Zinc acts by interfering with the uptake of copper from the GI tract. Zinc induces enterocyte metallothionein, which binds copper present in the enterocyte and inhibits its entry into the portal circulation. Once bound, the copper is not absorbed but is lost into the focal contents as enterocytes are shed in normal turnover.  Zinc is currently reserved for maintenance treatment. Dose- 150 mg of elemental zinc per day in 2 divided doses.

How do you monitor adequacy of treatment?

Adequacy of treatment can be monitored by clinical and biochemical improvement and by measuring 24-hour urinary copper excretion while on treatment. This should run in the vicinity of 200 to 500 µg (3 to 8 µmoles) per day on treatment with chelators (less than 75 µg wit copper). Additionally, estimate of noncaeruloplasmin-bound copper may show normalization with effective treatment.  Urinary excretion of zinc may be measured from time to time to check compliance

Discuss the management of WD?

• Treatment is lifelong and should not be discontinued, unless a liver transplant has been performed.
• Foods with very high concentrations of copper (shellfish, nuts, chocolate, mushrooms, and organ meats) generally should be avoided, at least in the first year of treatment.
• Antioxidants, mainly vitamin E, may have a role as adjunctive treatment.
• Treatment should be initiated with a chelating agent in both symptomatic and asymptomatic or presymptomatic patients identified through family screening. Zinc may be used as the first agent in presymptomatic patients.
• Maintenance therapy – After adequate treatment with a chelator (usually for 1-5 years), stable patients may be transitioned to treatment with zinc. They will be clinically well, with normal serum aminotransferases and hepatic synthetic function, nonceruloplasmin-bound copper concentration in normal range, and 24-hour urinary copper repeatedly in the range of 200 to 500 µg (3 to 8 µmoles) per day on treatment. The advantages of long-term treatment with zinc include that it is more selective for removing copper than penicillamine or trientine and is associated with few side effects. Adequate studies regarding the timing of this change-over in treatment are not available. No matter how well a patient appears, treatment should never be terminated indefinitely. Patients who discontinue treatment altogether risk development of intractable hepatic decompensation.
• In pregnant women, treatment must be maintained throughout the course of pregnancy for all patients with WD. Interruption of treatment during pregnancy have resulted in fulminant hepatic failure.
• Liver transplantation is indicated for all WD patients with decompensated liver disease unresponsive to medical therapy, and it is the only effective option for those who present with fulminant hepatic failure.

Discuss the prognosis?

Long-term outcome is dependent on adherence to lifelong treatment. Patients with WD who commence treatment before onset of symptomatic hepatic or neurologic disease have an excellent long-term prognosis and rarely develop symptoms. Symptomatic patients may expect to stabilize or improve on treatment. A minority of patients with neurologic disease experience worsening with initiation of therapy: some stabilize but some worsen despite treatment. The prognosis for patients with liver disease who adhere to effective treatment is excellent, even if cirrhosis was present at the time of diagnosis.
Development of hepatocellular carcinoma is a rarely reported complication of WD. Screening for hepatocellular carcinoma has not been recommended for WD patients.

Ref

1. AASLD Practice Guidelines: Diagnosis and Treatment of Wilson Disease: An Update

HFE-HC is an autosomal recessive iron-overload disorder caused by pathophysiological predisposition to increased and inappropriate absorption of dietary iron. It is associated with mutation of the HFE gene, located on chromosome 6. In most cases the mutation is a single-base change that results in the substitution of tyrosine for cysteine at position 282 of the HFE protein (C282Y). Other mutations in HFE, less common than C282Y, have also been described. The clinical effects of a mutation in which aspartic acid replaces histidine at position 63 (H63D), for example, appear to be limited, although 1-2% of persons with compound heterozygosity for C282Y and H63D seem predisposed to disease expression. In most studies C282Y/C282Y homozygosity has been found in 80% of patients with HFE?HC, while compound heterozygosity (C282Y/H63D) accounts for 3% to 5% of such cases in published series. Rarer forms of genetic iron overload recently attributed to pathogenic mutations of transferrin receptor 2, (TFR2), hepcidin (HAMP), hemojuvelin (HJV), or to a sub-type of ferroportin (FPN) mutations may account for the rest of the cases.

The penetrance and progression of C282Y mutation is variable. Available data suggest that up to 38% to 50% of C282Y homozygotes may develop iron overload, with 10% to 33% eventually developing haemochromatosis-associated morbidity.

Discuss the clinical manifestations of HFE-HC ?

The iron deposition in HFE-HC primarily occurs in parenchymal cells, with reticuloendothelial (RE) cell accumulation occurring very late in the disease. This is in contrast to transfusional iron overload in which iron deposition occurs first in the RE cells and then in parenchymal cells. The clinical condition of HFE-HC evolves in a series of stages beginning with clinically insignificant iron accumulation (0-20 years of age, 0-5g parenchymal iron storage). This evolves to a stage of iron overload without disease (approximately 20-40 years of age, 10-20g parenchymal iron storage), which if left untreated, may progress to a stage of iron overload with organ damage (usually more than 40 years of age and more than 20g parenchymal iron storage).

Fatigue (75%) and arthralgia (45%) are the most common presenting symptoms.

Skin — A combination of primarily melanin and iron deposition causes skin bronzing, slate grey appearance or hyperpigmentation, Porphyria cutanea tarda may be associated with HFE-HC.

Liver disease — Hepatomegaly, elevated liver enzymes, and cirrhosis. HCC is associated with cirrhosis and the amount of mobilisable iron.

Diabetes mellitus — This complication is due to progressive iron accumulation in the pancreas.

Arthropathy — Second and third MCP joints are characteristically affected. HFE-HC can be associated with an arthropathy that displays the full spectrum of calcium pyrophosphate crystal deposition disease (i.e. pseudogout, chondrocalcinosis, and chronic arthropathy).

Heart disease — Dilated cardiomyopathy and conduction disturbances such as the sick sinus syndrome, due to excess deposition of iron within the myocardium. Treatment with phlebotomy or chelation therapy has been associated with reversal of the left ventricular dysfunction.

Hypogonadism — Due to excess iron deposition in pituitary cells, leading to reduced serum levels of a number of trophic hormones. Secondary hypogonadism causes decreased libido and impotence in men. Other pituitary deficiencies may occur, but are much less frequent. Primary hypogonadism, presumably due to testicular iron deposition, also can occur, but is much less common. Repeated phlebotomy to remove excess tissue iron deposits may reverse the hypogonadism. Amenorrhea can occur in women but appears to be much less common than hypogonadism in men. Hypothyroidism occurs in approximately 10% of males.

Osteoporosis — Patients are at risk of osteoporosis, and should undergo a DEXA scan.

Susceptibility to specific infections — Iron overload states appear to be risk factors for infection with Listeria and Yersinia enterocolitica. Iron overload of macrophages can diminish phagocytosis, while high serum iron levels may increase bacterial virulence. Septicemia from Vibrio vulnificus, another iron-requiring bacterium, is also common in patients with HFE?HC who ingest uncooked seafoods. Accordingly, it has been recommended that patients with HFE?HC avoid consumption of uncooked seafood.

Discuss the diagnosis of HFE-HC ?

Hyperferritinaemia

A search for common causes of hyperferritinaemia should be made initially. Ferritin has low specifity as elevated levels are found in a range conditions including; inflammation (check CRP), neoplastic conditions (ESR, CT scan), chronic alcohol consumption, and hepatocellular or other cell necrosis (check AST/ALT/CK). Of note one third of patients with NAFLD and the metabolic syndrome (check BP, cholesterol, serum glucose, triglycerides), have what is called the dysmetabolic iron overload syndrome.

Transferrin saturation (TS: 100X SI/TIBC) and serum ferritin are used as initial tests. If TS <45% and serum ferritin is normal, a diagnosis of HFE-HC is excluded. A TS >45% in females and >50% in males with a raised serum ferritin will need genetic testing to confirm the diagnosis. If the genotype is homozygous C282Y/C282Y, a diagnosis of HFE-HC is confirmed. Of note, compound heterozygotes for C282Y/H63D usually present with mild iron overload, which is associated with comorbid factors such as obesity, NAFLD, chronic alcohol consumption, and end-stage cirrhosis.

For all other genotypes, then confounding cofactors, non-HFE iron overload disease (ferroportin disease, aceruloplasminemia), hepatic or other haematological diseases will need to be excluded and this may need a liver biopsy/MRI imaging. L-ferritin gene mutations cause hyperferritinaemia-cataract syndrome with an elevated serum ferritin, TS <45% and normal liver iron content.

Discuss the role of genetic testing?

• HFE testing should be considered in patients with unexplained chronic liver disease pre-selected for increased TS.
• HFE testing could be considered in patients with:
  • Porphyria cutanea tarda
  • Well-defined chondrocalcinosis
  • Hepatocellular carcinoma
  • Type 1 Diabetes

Discuss family screening

• Siblings of patients with HFE-related HC must undergo screening, since they have a 25% chance of being susceptible.
• HFE genotyping of the unaffected spouse is valuable to establish the need for genetic testing in children later on in life, once above the age of consent.

Discuss the role of liver biopsy?

• Liver biopsy and hepatic iron evaluation are also recommended in compound heterozygotes (C282Y/H63D), C282Y heterozygotes, or non-HFE mutated individuals who have indirect markers of iron overload, particularly if they also have abnormal liver enzymes or clinical evidence of liver disease.
• Liver biopsy could be offered to document the degree of fibrosis in C282Y homozygotes that are over the age of 40 years, or have an elevated AST level, hepatomegaly, or have a serum ferritin >1,000µg/L
• Patients less than 40 years of age who have no clinical evidence of liver disease (raised AST, hepatomegaly, etc.) and whose serum ferritin <1,000µg/L are unlikely to have significant hepatic injury and thus may be offered therapeutic phlebotomy without the necessity for a liver biopsy.
• Serum ferritin is an extremely useful diagnostic threshold test. If the ferritin is <1000µg/L, the patient has essentially no risk of having cirrhosis and thus liver biopsy is optional. By contrast, if the ferritin is >1000µg/L, there is a fairly high risk that the patient will have cirrhosis and thus liver biopsy is indicated. Documentation of extensive bridging fibrosis or cirrhosis by liver biopsy has a profound impact on the prognosis in HFE-HC patients. HFE-HC patients have a normal life span in the absence of cirrhosis and diabetes.  .
• A hepatic iron index or HII (hepatic iron conc. or HIC/age in years) of >1.9 µmol/g/y is diagnostic of HFE-HC.

Future tests for assessing fibrosis

• Serum hyaluronic acid is reported to correlate with the degree of hepatic fibrosis in HFE-HC, and may provide an alternative approach to liver biopsy for the diagnosis of advanced fibrosis .
• Transient elastography can also be helpful for determination of advanced fibrosis and cirrhosis.

Discuss the management of HFE-HC (C282Y homozygosity)?

• If ferritin is normal then once yearly follow up is advised.
• If ferritin is elevated then initial evaluation should include AST, ALT, fasting serum glucose with further investigations according to clinical features (ECG/ECHO/gonadotropic hormones/liver scanning).
• Liver biopsy should be considered as discussed above.
• Those with cirrhosis should have 6-monthly HCC screening with (Alpha)&-FP and US liver as they have a 100-fold greater chance of developing HCC compared with the normal population.

Therapeutic phlebotomy

Is the most effective way to remove iron. There is no data from which to base the optimal treatment regimen and target serum iron indices. Generally, the indication for initiating phlebotomy is ferritin >200µg/L in females and >300µg/L in males.

• One phlebotomy (removal of 500ml of blood) weekly or biweekly.
• Check haematocrit prior to each phlebotomy; allow haematocrit to fall by no more than 20% of prior level.
• Check ferritin level after 10-12 phlebotomies.
• Stop frequent phlebotomy when ferritin falls below 50µg/L (usually takes 3 to 6 months)
• Continue phlebotomy at intervals to keep serum ferritin between 50-100µg/L. The frequency of maintenance phlebotomy varies from monthly to 3-4 times a year.

The blood withdrawn from a patient with HFE-HC can be used for direct transfusion. Each 500ml of whole blood removed contains 200 to 250mg of iron. The marrow, in providing replacement for the lost Hb, mobilises iron from tissue stores, thereby reducing the degree of iron overload.

Diet

A normal balance diet is sufficient. However, dietary supplements containing iron, iron fortified cereals should be avoided and limit vitamin C intake to 500mg/day.

Discuss quantitative phlebotomy?

This is a way of confirming iron overload when iron overload is suspected and a liver biopsy cannot be performed. Iron overload can be confirmed or refuted by determining the number of weekly 500 mL phlebotomies which is required to produce iron deficient erythropoiesis. Normal men have approximately 1 g of iron stores. As a result, 4-5 phlebotomies will produce an iron deficient state (microcytosis or hypochromia with anaemia). In contrast, patients with significant iron loading usually have at least 5-20g of iron stores, requiring at least 20 units of phlebotomy to induce iron deficiency.

Discuss the prognosis??

The major causes of death are decompensated cirrhosis, hepatocellular carcinoma (HCC), diabetes mellitus, and cardiomyopathy. HCC accounts for about 30% of all deaths in HFE-HC, whereas other complications of cirrhosis account for an additional 20%. HCC is exceedingly rare in non?cirrhotic HFE-HC. There is overwhelming evidence that institution of phlebotomy therapy before cirrhosis and/or diabetes develop will significantly reduce the morbidity and mortality of HFE-HC. Certain clinical features may be ameliorated by phlebotomy (malaise, fatigue, skin pigmentation, insulin requirements in diabetes, abdominal pain), whereas other features are either less responsive to iron removal or do not respond at all (arthropathy, hypogonadism, cirrhosis). Cirrhosis does not reverse with iron removal and the development of decompensated liver disease is an indication to consider OLT.

Discuss secondary causes of iron overload??

Secondary iron overload occurs in ineffective erythopoiesis like thalassaemia major, sideroblastic anaemia, chronic haemolytic anaemia’s etc and chronic liver diseases like hepatitis C and B, alcohol-induced liver disease, porphyria cutanea tarda, fatty liver disease etc. Phlebotomy is useful only in certain forms of secondary iron overload. It has been used in African iron overload and porphyria cutanea tarda with reduction in morbidity and mortality.? In secondary iron overload associated with ineffective erythropoiesis, iron chelation therapy with parenteral deferoxamine is the treatment of choice.

References

1. AASLD Practice Guidelines. Diagnosis and Management of Hemochromatosis.
2. EASL Clinical Practice Guidelines for HFE Hemochromatosis. J Hepatol (2010).
3. Iron in fatty liver and in the metabolic syndrome: A promising therapeutic target. Dongiovanni P, Fracanzani AL, Fargion S, Valent L. J Hepatol. 2011 Oct;55(4):920–932.