Molecular and Genetics-Based Diagnostics

Introduction

In recent decades technologic advances have expanded our knowledge about the genetic determinants of liver diseases. From a disease point of view, genetic tests for liver diseases can be divided into two distinct groups: (1) diagnostic tests for monogenic diseases and (2) assessment of polygenic (complex) disease predisposition (susceptibility tests). With respect to technology, genetic testing is based on (1) genotyping single variants, (2) genotyping multiple variants, (3) gene sequencing (e.g., resequencing chips), or (4) next-generation sequencing (e.g., exon or whole-genome sequencing). Based on these classifications, genetics-based diagnostics can be classified as illustrated in Table 9-1 .

Monogenic Liver Diseases

Hereditary Hemochromatosis

The first genes of monogenic diseases with predominant liver phenotypes that were mapped and cloned were the Wilson disease gene ATP7B (1993) and the hemochromatosis gene HFE (1996; Fig. 9-1 ). The Wilson disease gene was localized by linkage disequilibrium and haplotype analysis in more than 100 families. Functional studies demonstrated that it encodes a P-type ATPase gene with metal binding regions similar to those found in prokaryotic heavy metal transporters. Building on this discovery, subsequent studies were able to determine previously unknown aspects of copper transport and the pathophysiology of Wilson disease. Using a similar strategy 3 years later, the HFE gene in the extended major histocompatibility complex (MHC) was shown to be mutated in patients with autosomal-recessive hemochromatosis. Furthermore, additional types of non-HFE hereditary hemochromatosis were subsequently described, linked to mutations in the ferroportin gene ( SLC40A1 ), the transferrin receptor 2 gene ( TFR2 ), the hepcidin gene ( HAMP ), and the hemojuvelin gene ( HJV ), respectively. These genetic discoveries jointly paved the way for the characterization of hepatic iron metabolism and its regulators, allowing for dissection of mechanisms responsible for the development of liver disease in hereditary hemochromatosis. Ferroportin disease (non-HFE hemochromatosis type 4) is characterized by predominant reticuloendothelial cell (and not hepatocyte) iron overload. Because in clinical practice non-HFE hemochromatosis types are very rare, genetic testing always starts with HFE mutation detection. Because significant hepatic iron accumulation without environmental factors is only observed in patients who are homozygous carriers of the HFE mutation p.C282Y, it is necessary and sufficient to start genetic testing with this specific mutation, which accounts for approximately 95% of patients with hereditary hemochromatosis in populations of European descent. Here, approximately 1 in 260 individuals is a homozygous carrier of the mutation, and marked variations in p.C282Y risk allele frequencies have been reported, ranging from 12.5% in Ireland to 0% in Southern Europe. This is also reflected in the algorithms (see Fig. 9-1 ) of current guidelines. In contrast, the second common HFE mutation p.H63D and compound heterozygosity (p.C282Y/p.H63D) do not cause a significant disease risk per se . Also, p.C282Y heterozygosity does not confer an increased risk for more advanced chronic liver disease, except for porphyria cutanea tarda .

Wilson Disease

Wilson disease shows a different genetic profile, with extensive allelic heterogeneity with more than 500 mutations described in the ATP7B gene to date. For this reason, there is a need for gene sequencing and variant analysis to obtain genetic support for the diagnosis. Hence, genetic testing represents one part of the circumstantial evidence ( Table 9-2 ) that is needed to establish the diagnosis of Wilson disease.

TABLE 9-2

Diagnostic Scoring System for Wilson Disease

From Nicastro E, et al. Reevaluation of the diagnostic criteria for Wilson disease in children with mild liver disease. Hepatology 2010;52:1948-1956.

Biochemistry	Score
Liver copper content (in the absence of cholestasis)
Normal (<50 µg/g of dry weight)	–1
<5 times ULN (50-250 µg/g of dry weight)	+1
>5 times ULN (>250 µg/g of dry weight)	+2
Rhodamine stain *
Absent	0
Present	1
Serum ceruloplasmin
Normal (>20 mg/dL)	0
10-20 mg/dL	+1
<10 mg/dL	+2
Daily urinary copper excretion
Normal	0
1-2 times ULN	+1
>2 times ULN	+2
Normal but >5 times ULN after PCT	+2
Clinical Symptoms and Signs	Score
Kayser-Fleischer rings
Absent	0
Present	+1
Coomb's negative hemolytic anemia
Absent	0
Present	+1
Neuropsychiatric symptoms suggestive of WD and/or typical brain magnetic resonance imaging
Absent	0
Mild	+1
Severe	+2
ATP7B genetic analysis
No mutation found	0
Mutation on one chromosome	+1
Mutation on both chromosomes	+4

A score of greater than or equal to 4 indicates diagnosis is highly likely, a score of 2 to 3 indicates diagnosis is possible and more testing needed and score of 0 to 1 indicates diagnosis is unlikely.

PCT, Penicillamine challenge test.

* When quantitative copper is not available.

α ₁ -Antitrypsin Deficiency

The recommended screening test for α ₁ -antitrypsin deficiency is the determination of α ₁ -antitrypsin serum concentrations. Indications to screen for α ₁ -antitrypsin deficiency are jaundice in newborns, growth retardation, severe chronic obstructive lung disease and emphysema before the age of 50 years (and in nonsmokers), unexplained chronic liver disease, cirrhosis, or hepatocellular carcinoma. As a next step, patients with decreased serum concentrations are tested for the two most common mutations in the SERPINA1 gene (p.E342K and p.E264V). These mutations are denoted PiZ and PiS (Pi = protease inhibitor) for historical reasons, with the letters referring to the letters of the protein bands: the anodal variants were designated from B to L, and variants cathodal to PiM (medium) were designated from N to Z. The interpretation of genetic tests results is hampered by the fact that there is no clear separation but a gradient from normal to abnormal findings ( Table 9-3 ). In contrast to HFE mutations, the heterozygous state appears to confer an increased risk for chronic liver disease, and the extent of PiZ deposits correlates with the inflammatory activity and the stage of fibrosis. The respiratory disease is highly penetrant, whereas neonatal cholestasis is seen in only 10% to 15% of p.E342K homozygotes; the cause of the reduced penetrance seen in this liver phenotype is yet to be explained.

TABLE 9-3

SERPINA1 Gene Mutations and Corresponding α ₁ -Antitrypsin Serum Concentrations

	SERPINA1 Mutation	Prevalence (%)	Serum α 1 -Antitrypsin Concentration (g/L, Median)	Risk of Lung Emphysema	Risk of Liver Disease
PiMM	p.E264+p.E342	92	1.30	Normal	Normal
PiMS	p.E264V	4.2	1.09	Normal	Normal
PiMZ	p.E342K	1.9	0.81	Normal	Normal
PiSS	p.264V	0.05	0.85	20-50%	↑
PiSZ	p.E264V+p.E342K	0.04	0.56	50-70%	↑
PiZZ	p.342K	0.01	0.32	70-100%	↑
PiNullNull	e.g., p.I192N	rare	0	90-100%	Normal

Cystic Fibrosis–Associated Liver Disease

Since its discovery in 1986, more than 2000 mutations of the CFTR gene encoding an ATP-dependent chloride channel (cystic fibrosis transmembrane conductance regulator) have been described. The most common mutation results in deletion of phenylalanine at amino acid position 508. One copy of the ΔF508 mutation is present in 70% of cystic fibrosis patients and is the predominant mutation in Europe and North America. The most frequent hepatobiliary manifestations encompass biliary cirrhosis, microgallbladder, cholelithiasis, and sclerosing cholangitis. They occur almost exclusively in patients with severe class I–III mutations affecting CFTR synthesis, processing, or regulation. The identification of specific mutations is indicated, because genotyped-guided therapy of lung disease in cystic fibrosis has been established. It remains unclear why only a minority of patients with the same severe CFTR mutations develop liver disease. Several factors have been found to be associated with hepatobiliary manifestations, including age at diagnosis, male gender, history of meconium ileus, and pancreatic insufficiency. Notably, the α ₁ -antitrypsin p.G342K allele (PiZ) is associated with cystic fibrosis-related liver disease (odds ratio [OR] = 4 for the development of portal hypertension).

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here