Cholestasis Genetic Test Program

Hereditary cholestatic liver disease is a heterogenous group of disorders with complex pathophysiology, often presenting with overlapping symptoms such as jaundice, pruritus, failure to thrive, hepatomegaly, and other liver abnormalities (Santos et al. 2010. PubMed ID: 20425482, Karlsen et al. 2015. PubMed ID: 25920091, Sticova et al. 2018. PubMed ID: 30148122). At the molecular level, cholestasis is caused by a reduction of bile flow due to impaired hepatocyte secretion or obstruction of bile ducts as a result of defective hepatocyte transport, disorders of bile duct development, inborn errors of bile acid metabolism, and other metabolic disorders impacting the liver (Santos et al. 2010. PubMed ID: 20425482, Karlsen et al. 2015. PubMed ID: 25920091, Sticova et al. 2018. PubMed ID: 30148122, Sundaram et al. 2008. PubMed ID: 18577977, Grochowski et al. 2016. PubMed ID: 26548814, Fawaz et al. 2017. PubMed ID: 27429428). Hereditary cholestasis often, but not always, presents in the neonatal period, and may present with extrahepatic manifestations as well as systemic disease. The estimated worldwide incidence of cholestasis ranges considerably depending on population. Pediatric cholestasis is estimated to affect about 1:2500 in newborns (Karpen. 2020. PubMed ID: 32685137), while intrahepatic cholestasis of pregnancy is estimated to affect between ~0.1-5.2% of pregnancies worldwide, with up to ~9-25% of pregnancies affected in some South American populations (Lee et al. 2006. PubMed ID: 16761011; Floreani and Gervasi. 2016. PubMed ID: 26593298). The prevalence of cholestasis among patients with inflammatory bowel disease is as high as 7% (Girardin et al. 2018. PubMed ID: 29670889). Some hereditary forms of cholestatic liver disease are known to occur more frequently in ethnic groups where consanguineous marriage is prevalent or in some geographic regions due to founder effects (Gunaydin and Bozkurter Cil. 2018. PubMed ID: 30237746).

Advantages of genetic testing for cholestasis include confirmation of diagnosis, identification of other health risks associated with syndromic disease, allowing for targeted testing of other family members, and assistance with reproductive planning. This test especially aids in a differential diagnosis of similar phenotypes by analyzing multiple genes simultaneously that all include the clinical feature of cholestasis.

Cholestatic liver disease is often multifactorial, resulting from a combination of metabolic, genetic, and environmental factors (Santos et al. 2010. PubMed ID: 20425482). Mendelian forms of cholestasis are most often inherited in an autosomal recessive (AR) manner, but may also be inherited in an autosomal dominant (AD) manner, or arise de novo. This test includes genes identified through literature, OMIM, and HGMD searches that have been reported to be associated with cholestasis.

An example of hereditary cholestatic liver disease due to defective hepatocyte transport includes progressive familial intrahepatic cholestasis (PFIC). Five types of PFIC have been classified in terms of causative genes involved in the hepatocellular transport system. Progressive familial intrahepatic cholestasis-1 (PFIC1) and progressive familial intrahepatic cholestasis-2 (PFIC2) are caused by hepatocyte membrane phospholipid asymmetry due to defects in the ATP8B1 and ABCB11 genes, respectively (Sticova et al. 2018. PubMed ID: 30148122). Progressive familial intrahepatic cholestasis-3 (PFIC3) is caused by reduced biliary phospholipid secretion due to pathogenic variants in the ABCB4 gene. Progressive familial intrahepatic cholestasis-4 (PFIC4) is caused by abnormal tight junctions between adjacent hepatocytes and biliary canaliculi in liver tissue due to TJP2 defects (Sambrotta et al. 2014. PubMed ID: 24614073). Progressive familial intrahepatic cholestasis-5 (PFIC5) is caused by defects in the NR1H4 gene, a key regulator of bile salt metabolism (Sticova et al. 2018. PubMed ID: 30148122). PFIC is estimated to occur in 1/50,000 to 1/100,000 births (Davit-Spraul et al. 2009. PubMed ID: 19133130), and while de novo variants have been reported, the majority of causative variants are inherited (Francalanci et al. 2013. PubMed ID: 23437912).

Alagille syndrome, an autosomal dominant disorder caused by defects in the JAG1 and NOTCH2 genes, is an example of hereditary cholestatic liver disease due to disordered bile duct development. The JAG1 and NOTCH2 genes both encode components of the Notch signaling pathway, which is critical for proper development of the biliary tree (Adams and Jafar-Nejad. 2019. PubMed ID: 31615106). Alagille syndrome is estimated to occur in 1/70,000 births and 1/30,000 individuals overall (Turnpenny and Ellard. 2012. PubMed ID: 21934706; Hartley et al. 2013. PubMed ID: 23540503). Approximately 50-70% of cases have a de novo pathogenic variant (Spinner et al. 1993. PubMed ID: 20301450).

A wide variety of causative variants in genes associated with cholestasis have been reported including missense, nonsense, splicing, small insertions/deletions, large deletions/duplications and complex rearrangements (Human Gene Mutation Database). See individual gene summaries for information about the molecular biology of gene products and spectra of pathogenic variants.

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary

This panel typically provides 99% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Detection rate of pathogenic variants in each gene of this panel in a large cohort of patients with cholestasis of undefined etiology is unknown. In a study of 51 subjects with cholestasis of undefined etiology, Matte et al. found causative variants in genes associated with PFIC in 14 patients (27%) (Matte et al. 2010. PubMed ID: 20683201). In a study of 102 pediatric patients with various forms of cholestasis or idiopathic liver diseases, Chen et al. made a molecular diagnosis in 33 of 102 patients (32.4%) using a 44-gene panel (Chen et al. 2019. PubMed ID: 30366773). Those with progressive intrahepatic cholestasis or syndromic cholestasis in infancy had a diagnostic rate of 62.5% (Chen et al. 2019. PubMed ID: 30366773).

Patient must meet ONE of the criteria below:

• Patient is currently cholestatic, or has a history of cholestasis, without an identified cause
• Unexplained chronic liver disease

AND patient must meet ALL criteria below:

• Extrahepatic disorders are not a consideration (eg, biliary atresia, choledochal cyst, large duct PSC
• TPN cholestasis is not suspected as a primary diagnosis
• Patient lives in the U.S.

Specimen Collection

WHOLE BLOOD

Collect 3 mL - 5 mL of whole blood in EDTA (purple top tube) or ACD (yellow top tube), minimum 1 mL for small infants.

SALIVA

Oragene^TM or GeneFiX^TM Saliva Collection kit used according to manufacturer instructions.

DNA

Send in a screw cap tube with at least 5 μg - 10 μg of purified DNA at a concentration of at least 100 ng/μL, minimum 2 μg for limited specimens. Label the tube with the composition of the solute and DNA concentration along with the patient's name, date of birth, and/or ID number. We only accept genomic DNA for testing; we do not accept products of whole genome amplification reactions or other amplification reactions. DNA must be extracted from a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by CAP and/or CMS.

Shipping and Handling Instructions

Label all specimen containers with the patient's name, date of birth, and/or ID number. At least two identifiers should be listed on specimen containers. Specimen deliveries are accepted Monday-Saturday for all specimen types. Holiday schedules will be posted on our website at least one week prior to major holidays.

WHOLE BLOOD

At room temperature or refrigerated, a blood specimen is stable for up to 8 days. Include a refrigerated gel pack in the shipping container. Fresh blood specimens are preferred. If frozen, a blood specimen is stable for up to 1 month before shipping. Frozen blood specimens should be shipped frozen (preferably on dry ice) overnight.

SALIVA AND DNA

Specimens may be shipped at room temperature.