Genetic Diagnosis of Liver Diseases

Transcription

1 The Hong Kong Association for the Study of Liver Diseases 27 th Annual Scientific Meeting and International Symposium on Hepatology 16 November 2014 Genetic Diagnosis of Liver Diseases Dr Chloe Mak MD, PhD, FHKCPATH, FHKAM, FRCPA (Scope of Practice in Chemical Pathology & Genetic Pathology) Kowloon West Cluster Laboratory Genetic Service Department of Pathology Princess Margaret Hospital Hong Kong

2 Inherited Liver Diseases *leads to progressive liver disease

3 DR. SAMUEL WILSON ( ) In 1912 Autosomal recessive Neurological deficits Cirrhosis of the liver Progressive Invariably fatal

4 The Local Prevalence of WD in Hong Kong

5 Based on Brewer s method 660 unrelated healthy HK Chinese adults Three p.r778l heterozygotes Carrier rate 1 in 220 All mutant alleles frequencies 1.4% x 6 allele frequency 0.23% Hardy Weinberg Equilibrium WD prevalence in HK = 1 in 5,400

6 Is Wilson Disease so rare 1 in 30,000? Mak et al. (2008) Diagnosis of Wilson's Disease: A Comprehensive Review. Critical Reviews in Clinical Laboratory Sciences

7 Wilson disease Common inheritable liver disorder in Hong Kong Good prognosis if early diagnosis and treatment Challenging diagnosis 1. Neurological deficits 2. Kayser Fleischer rings 3. Serum Ceruloplasmin (Cp) < 0.20 g/l False diagnostic rates up to 50%

8 Diagnostic Challenges KF rings Mak et al. (2008) Diagnosis of Wilson's Disease: A Comprehensive Review. Critical Reviews in Clinical Laboratory Sciences

9 Diagnostic Challenges Ceruloplasmin

10 Ceruloplasmin Problems of Reference Intervals The only clinical use is for the diagnosis of WD Low end is clinically important, however Local Hospital Reference Intervals Method Source A g/l Instrument A Manufacturer insert B g/l Instrument A Manufacturer insert C g/l Instrument A Manufacturer insert D g/l Instrument B Manufacturer insert E g/l Instrument C Literature F g/l Instrument C Manufacturer insert

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13 Serum Ceruloplasmin g/l < 0.20 < 0.14 sensitivity 98.2% 93% specificity 55.9% 100% Comparison of 57 WD patients with 71 family members (49 heterozygotes and 22 wild type homozygotes) (area under the curve, 95% confidence interval ).

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15 Diagnostic Challenges Liver copper Liver biopsy is usually performed in borderline cases in clinical practice which usually give borderline results Mak et al. (2008) Diagnosis of Wilson's Disease: A Comprehensive Review. Critical Reviews in Clinical Laboratory Sciences

16 Diagnostic Challenges Scoring system

17 ATP7B Genomic size bp mrna size 6638 bp 21 exons Up to 500 mutations

18 Screening mutations Extract DNA Amplify ATP7B exons Extractio n (QIAGEN - QIAamp DNA Blood Mini Kit) DNA quantification PCR Sequencing 4 5 Sequencing Reaction (AB - BigDye Terminator v1.1 Cycle Sequencing Kit ) Purification (Amersham - AutoSeq G-50) Sequencing (ABI 3100)

19 ATP7B gene and Population Genetics >500 mutations Founder mutation p.r778l in East Asian p.h1069q in Caucasian Ethnic specific Wilson Disease Mutation Database php

20 Central China Wu et al Shanghai Liu et al R778L 45.6% V1106I 1.7% G943S 0.9% Beijing Gu et al R778L 33.8% P992L 9.5% S974W 2.7% Korea Yoo et al R778L 37.9% N1270S 12.1% A874V 9.4% R778L 37.7% T935M 10% Q1142H 3.1% Hong Kong R778L 16.3% P992L 12.5% I1148T 10.6% Taiwan Tsai et al R778L 28.9% P992L 14.5% G943D 7.9% Japan Shimizu et al delC 30% R778L 25% A874V 7.5%

21 The Best Diagnostic Tool for Wilson Disease Molecular Genetics 98% sensitivity 100% specificity We corrected diagnoses of 12 false positives and 2 false negatives We picked up 10 asymptomatic siblings

22 Mak et al. (2008) Diagnosis of Wilson's Disease: A Comprehensive Review. Critical Reviews in Clinical Laboratory Sciences

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24 Mutational analysis for Wilson s disease Wai Kay Seto*, Chloe Miu Mak*, David But, Ivun Hung, Ching Wan Lam, Sidney Tam, Man Fung Yuen, Ching Lung Lai (Lancet 2009)

25 Cp 0.19 g/l Cp 0.33 g/l Cp 0.16 g/l Cp 0.10 g/l KF ring

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28 Molecular tests diagnostic pitfalls

29 CASE abdominal distension, LL edema at age of 9. significant liver derangement KF ring +ve Serum copper 3.7 umol/l (11 25) ceruloplasmin 0.05 g/l ( ) 24 hours urinary copper excretion 8.9 umol/d.

30 S105X 522_523insA

31 Lam CW and Mak CM, Clin. Chem Mar;52(3):517-20

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35 Summary Sequencing of the remaining exons is usually not proceeded when homozygous mutation is found. False homozygous diagnosis or a false normal in carrier of another unknown mutation Close link of molecular results to clinical data and family history New clinical decision value of ceruloplasmin <0.14 g/l Genetic analysis

36 Hereditary Hemochromatosis Adult onset autosomal diseases Enhanced iron intestinal absorption and iron overload Lead to liver cirrhosis, cardiomyopathy, diabetes, arthritis, skin pigmentation, and hepatocellular carcinoma

37 At least 5 genes with different severities and ages at onset

38 This is not applicable to Chinese?

39 A Chinese patient with non HFElinked iron overload. J Clin Gastroenterol Jul;33(1): Kng C 1, Ng FH, Ng WF, Wong BC, Grosso LE, Brunt EM, Bacon BR. 1 Department of Medicine, Ruttonjee Hospital, Hong Kong, SAR. Abstract The gene for hemochromatosis (HFE) was recently identified and contains two missense mutations: C282Y and H63D. The C282Y mutation is found homozygous in approximately 85% to 90% of patients of Northern European ancestry with hereditary hemochromatosis. There are no previous reports with results of genetic testing in Chinese patients with regard to iron overload. In this case report, we describe a Chinese woman with marked hepatic iron overload that was nonfamilial, with unusual biopsy findings, in whom neither the C282Y nor the H63D mutations in HFE were found.

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41 A recent case from QEH M/43y, incidental finding of high ALT (~120 IU/L) in 2005 Associated with RUQ discomfort and weight loss for months Social drinker, HBsAg ve No history of transfusion/iv use/drug P/E: P J C, no KF rings, no clubings, no stigmata of CLD. Iron 54 umol/l, TIBC <56 umol/l, ferritin 5354 pmol/l MRI liver: increased iron deposition Fibroscan: 7.7 kpa Liver biopsy: grade 2 3 siderosis with fibrosi, steatohepatitis C282Y, H63D of HFE negative With courtesy of Dr Shek Chi Chung and his team in Chemical Pathology Laboratory, QEH

42 Genetic test of SLC40A1 Novel mutation detected in ferroportion Family screening pending (autosomal dominant)

43 Next Generation Sequencing

44 Thank You