Role Of NGS And Molecular Pathology In The Diagnosis Of Liver Disorders Of Unknown Etiology

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1 Role Of NGS And Molecular Pathology In The Diagnosis Of Liver Disorders Of Unknown Etiology

2 Financial: Nothing to disclose Professional: I am not a molecular pathologist!

3 Etiology remains unknown despite work-up Clinical history Laboratory work-up Pathologic analysis

4 NAFLD/NASH Autoimmune hepatitis Chronic cholestatic/biliary disorders A1AT inclusion disease Hepatic vascular disorders Occult alcoholic liver disease

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6 Reticulin stain

7 D-PAS stain

8 1965 HBsAg Blumberg BS JAMA 1969 PFIC Clayton RJ Am J Dis Child 1989 HCV Choo QL Science 2004 Occult HCV Castillo J Infect Dis 2009 WES Choi M. PNAS Ng SB. Nature 2017 Refinement of AIH criteria 1998, 2008 Awareness of NAFLD/NASH <1970 >60% 5-30% 5-10% % pts +ve HCV Abs % pts with CK8 mutations 2007 FMF gene mutations 2008 htert & htr mutations Barnes RE. Am J Gastroenterol. Ku NO. N Engl J Med. Tweezer-Zaks N. Medicine (Baltimore) Alder JK. Proc Natl Acad Sci.

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10 NGS is now readily available, reasonably cheap and is used in clinical application including the work up of liver disorders of unknown etiology Whole genome analysis Whole exome analysis (WES) 1% of whole genome, but 85% of all disease causing variants Selected gene panel

11 15 years old female found to have features suggestive of cirrhosis with esophageal varices, splenomegaly and mild thrombocytopenia Labs: Liver transaminases and alkaline phosphatase were mildly elevated AST 141 u/l, ALT 114 u/l, alkaline phosphatase 596 u/l, GGT 734 u/l, albumin 4.2 gm/dl and conjugated bilirubin 1.67 mg/dl Viral (Hepatitis A, B and C) were negative and autoantibody screen was also negative. Serum immunoglobulin levels were normal Increased 24h urine copper [> 100 mcg] Ceruloplasmin level was normal [38 mg/dl (ref 18 46)}, normal serum free copper [0.6 μg/dl (ref )] Liver biopsy

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16 Diagnosis: Wilson disease with cirrhosis Treatment : Started on zinc therapy, but was switched to Trientine due to GI side effects. After >12 months of treatment her transaminase levels did not improve and urine copper remained elevated. Mutations for Wilson disease (ATP7B)were negative Diagnosis: Cryptogenic cirrhosis

17 Nucleotide change Amino acid change Location Zygosity Reference/Comm ents c984t>g py328 Exon 9 heterozygous Nonsense mutation c3218g>a pc1073y Exon 25 heterozygous Unclassified novel variant

18 Progressive familial intrahepatic cholestasis (PFIC-3)

19 Correct diagnosis was established based on WES Identified novel mutations of a known genetic inherited genetic metabolic disorder Helped institute appropriate therapy

20 5 Y female child from India result of an consanguineous marriage presented with cirrhosis Based on clinical features and laboratory data diagnosed as Wilson disease Mutations for Wilson disease (ATP7B)were negative Underwent OLT at age 9 Explant showed Cirrhosis and HCC (2 nodules, 1.9 and 1.5cm) Post-Tx developed progressive neurodegenerative symptoms and died at 10y

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23 Copper

24 WES of the proband revealed a homozygous missense mutation in MPV17gene (c.148t>c) Both parents were heterozygous carriers for the same mutation MPV17 encodes a mitochondrial inner membrane protein of unknown function Bi-allelic mutations in MPV17 underlie hepatocerebral Mitochonfrial depletion syndrome (MDS): Autosomal recessive To date 35 affected individuals have been described in the literature

25 Mitochondrial depletion disorder leading to neurodegenerative disease Neurologic symptoms (90%) Developmental delay Hypotonia, muscle weakness, seizures, ataxia, peripheral neuropathy Liver involvement (90%) Liver failure or cirrhosis in early childhood

26 Correct diagnosis was established based on WES Identified novel mutations of a known but rare inherited genetic metabolic disorder Expanded our current understanding of the clinicopathological spectrum of a known disorder Earlier diagnosis could have prevented organ wastage

27 33/F with early onset hyperlipidemia diagnosed in childhood with Recurrent pancreatitis leading to pancreatectomy Hypertension and pre-eclampsia at the age of 29 Evaluated by expert pediatric and adult physicians at several U.S. tertiary medical centers Working diagnosis was hyperchylomicronemia syndrome However, deficiency of lipoprotein lipase or apolipoprotein CII could not be demonstrated

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31 Heterozygous missense mutation mutation in PPARG (peroxisome proliferator-activated receptor G) (chr3: , G>T, NM_015869, c.g482t, p.g161v) Heterozygous mutations have been related to autosomal dominant Familial Partial Lipodystrophy Type 3 (FPLD3) None of the parents harbor the mutation, suggesting a de novo mutation

32 PPARG is a key transcription factor in adipocyte differentiation Explains the lack of adipose tissue in the patient Striking accumulation of triglycerides in the bloodstream as well as in other organs, such as the liver. Since leptin is mainly produced by adipocytes and as predicted the patient s leptin levels were very low

33 The patient began leptin replacement therapy During next 13 months there was significant amelioration of dyslipidemia Total cholesterol decreased from 238 to 130 mg/dl Triglyceride levels fell from 3532 to 267 mg/dl HDL cholesterol increased from 8 to 24 mg/dl Normalization of her liver transaminases Insulin requirements decreased by approximately half

34 Clinical Utility of Genomic Analysis in Adults with Idiopathic Liver Disease Hakim A, Zhang X, Deslile A, Oral EA, Dykas A, Drzewiecki K 1, Assis A, SilveiraA, Batisti J, Jain D, Nathanson MH, Bale A, Mistry M, Vilarinho S. Gastroenterology, Submitted for publication. Correct diagnosis was established based on WES Identified de novo mutations of a known but rare inherited genetic metabolic disorder Identification of the specific defect predicted biochemical and laboratory abnormalities Understanding the pathophysiology helped institute appropriate therapy

35 8y boy seen in liver clinic with intermittently elevated transaminases (AST U/L; ALT, U/L), normal GGT levels and preserved liver synthetic functions) Turkish decent, parents are 2 nd cousins Full term baby with mildly delayed developmental milestones Transaminase elevations noted since 8 month of age Normal growth (weight and height between 25 th -50 th and 50 th -75 th percentile, respectively) At 6.5 y developed some neurologic symptoms Liver and spleen not palpable

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38 WES revealed homogygous mutation in ACOX2 gene (premature termination at codon 69) ACOX2 encodes a branched-chain Acyl-CoA oxidase, a peroxisomal enzyme expressed in the liver and kidney

39 Elevated serum levels of 3α,7α-dihydroxy-5β-cholestanoic acid (DHCA) and 3α,7α,12α-dihydroxy-5βcholestanoic acid No ACOX2 Low levels of Cholic acids and its conjugates

40 Figure 1 A ACOX2 c. T207A (p.y69*) B N68 69* F70 ACOX2-deficient Patient WT/Mut WT/Mut N68 Y69/* F70 Father Mut/Mut WT/Mut N68 Y69/* F70 Mother Both parents and brother found to be heterozygous, but clinically normal. Brother N68 Y69/* F70

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42 Patient put on replacement bile acids Normalization of transaminases Long term outcome awaited

43 Identified novel genetic inherited disorder based on WES Identification of the specific defect predicted biochemical and laboratory abnormalities Understanding the pathophysiology helped institute appropriate therapy

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45 Retrospective analysis of pediatric patients with advanced cholestatic liver disease (n=98) 61% showed mutations spanning many genes including ones that have only rarely been reported to cause cholestatic liver disease Most commonly mutated genes ABCB11(25%), ABCB4 (20%), UGT1A1(12%) and TJP2 (12%) Other genetic diagnosis made in this patient cohort included PFIC1, Alagille syndrome, bile acid synthesis defect, tyrosinemia, Wilson disease, ABCC2-related Dubin-Jhnson syndrome, G6PC related glycogen storage disease, SLC10A2-related bile acid malabsorption, MYO5b-related microvillus inclusion disease and VIPAS39-related cholestasis, renal impairment and arthrogryposis syndrome. Shagrani M, Burkholder J, Broering D, Abouelhoda M, Faquih T, El-Kalioby M, et al. Genetic profiling of children with advanced cholestatic liver disease. Clin Genet. 2017;92(1):52-61.

46 Retrospective analysis of cryptogenic cirrhosis (age <40Y)(n=30) from FPPE liver biopsies Satisfactory DNA sample (n=15) Mutations identified (n=5) Patient Abnormality detected Significance 29/M ATP7B comp het:p.n1063s; p.g1075s Wilson Disease 1/M MPV17 ( p.r88x)?mds 35/F APOE (p.e31k, chr19: , G>A) 37/F ABCB4 (p.s320c)?pfic3 19/F ABCB4 ( p.r144q)?pfic3 13/M ABCB4 {p.s733l (D), p.i731v (B) - same allele (one het in ExAC)}?PFIC3

47 19 patients with CLD subjected to WES 5 (26%) patients with monogenic disorders Type 3 Familial Partial Lipodystrophy PFIC3 (2 patients) Mitochondrial disorder due to homozygous mutation in NDUFB3 Familial hypobetalipoproteinemia Hakim A, Zhang X, Deslile A, Oral EA, Dykas A, Drzewiecki K 1, Assis A, SilveiraA, Batisti J, Jain D, Nathanson MH, Bale A, Mistry M, Vilarinho S. Clinical Utility of Genomic Analysis in Adults with Idiopathic Liver Disease. Gastroenterology. Submitted for publication.

48 Genomic analysis is likely to find more application is routine clinical practice in the work up of liver disorders of unknown etiology Whole genome analysis Whole exome analysis Selected gene panel Many new genetic alterations are likely to be identified and help Refine the existing knowledge of known disorders Define new disorders Identification of cryptic infections

49 Etiology of the cirrhosis remains unknown despite work-up Clinical history Laboratory work-up Pathologic analysis Molecular and genetic analysis

50 Dr. Silvia Vilarhino Dr. Xuchen Zhang Dr. Pramod Mistry We thank all the patients and their families whose contribution to this study lead to advancing our understanding of liver disease We thank all the staff of the Yale Center for Genome Analysis for exome sequencing Research supported by: The National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award Number K08DK This work was also supported in part by Yale Liver Center P30DK034989, and AASLD Sheila Sherlock Clinical and Translational Research Award in Liver Disease (to S.V.) Alexion Pharmaceuticals.

51 San Sebastian Thank You