Molecular Genetics Cholestasis Gene Panel

Although jaundice is relatively common in the neonatal period, persistent jaundice is abnormal. When it persists beyond the second week of life, cholestasis or conjugated hyperbilirubinemia may be considered. Cholestasis is defined by impaired bilirubin excretion resulting in excess conjugated bilirubin in the bloodstream and decreased bile salts in the GI tract. This impaired flow of bile can be caused by an intrahepatic or extrahepatic disorder. Infants may present with jaundice, dark urine (containing conjugated bilirubin), hepatosplenomegaly, poor weight gain, and hypopigmented or acholic stools.

Cholestasis has many etiologies. It is estimated that the most common causes of neonatal cholestasis are biliary atresia (25-35%), genetic disorders (25%) and metabolic diseases (20%). Genetic testing may confirm a diagnosis of a genetic disorder, which may affect medical management or treatment of the condition. It is recommended that this testing be accompanied by a complete family history and genetic counseling.

Reasons for Referral:

• Molecular confirmation of a clinical diagnosis
• To aid in decision-making for treatment and management of individuals with inherited cholestatic diseases.
• Testing for individuals with a positive family history for inherited cholestasis (targeted testing is available if the familial mutation is known).

Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs <50 bp, the analytical sensitivity is >87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.

Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmental duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.

This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.

ABCB11, ABCB4, ABCC2, ABCG5, ABCG8, AKR1D1, ALDOB, AMACR, ATP8B1, BAAT, CC2D2A, CFTR, CLDN1, CYP27A1, CYP7A1, CYP7B1, DCDC2, DGUOK, DHCR7, FAH, GPBAR1, HNF1B, HSD17B4, HSD3B7, INVS, JAG1, LIPA, MKS1, MPV17, NOTCH2, NPC1, NPC2, NPHP1, NPHP3, NPHP4, NR1H4, PEX1, PEX10, PEX11B, PEX12, PEX13, PEX14, PEX16, PEX19, PEX2, PEX26, PEX3, PEX5, PEX6, PEX7, PKHD1, POLG, SCP2, SERPINA1, SLC10A1, SLC10A2, SLC25A13, SLC27A5, SMPD1, TJP2, TMEM216, TRMU, UGT1A1, VIPAS39, VPS33B

For a comprehensive list of regions with limited coverage on this panel, please refer to the following table.

Blood:  EDTA or ACD (Solution A or B):

• Adult: 5 mL
• Child: 5 mL
• Infant: 2-3 mL

Saliva: 2 ORAgene™ Saliva Collection Kit(s) (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.

Assisted Saliva: 4 ORAgene™ Assisted Saliva Collection Kits (OGR-575) used according to manufacturer instructions.  Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.

Buccal Cells: 4 CytoSoft™ Cytology Brush (Medical Packaging CYB-1) used according to manufacturer instructions.  Please contact KDL Client Services for a Buccal Collection Kit for patients that cannot provide a blood sample.

Skin Fibroblast: Punch Biopsy (Cell cultures will be prepared at KDL and used for testing), or 2 T-25 confluent flasks

Prenatal:

• Direct Amniotic Fluid (10-20mL)
• Direct CVS
• Cultured Amnio (2-T25 flasks)
• Cultured CVS (2-T25 flasks)
• Cultured Fetal Tissue: Product of Conception (2-T25 flasks)
• Cord Blood (1-2mL)

DNA: 5-10µg at a minimum of 60-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).

Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood (or saliva) in addition to the fetal specimen. Additional charges apply for the maternal cell rule-out test.

For routine testing of blood, saliva and buccal swabs, KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion.  For extraordinary circumstances, where testing must be performed within the above windows, please contact our lab.

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information, including ethnicity, clinical history, and family history.

Weekly

8 weeks

• Package and ship specimen to remain cold, but not frozen. 
• Ship via overnight express, using the FedEx priority overnight label provided. 
• Contact Client Services for shipping kits and instructions at (855) 535-1522.

1. Balistreri WF, Bezerra JA. Whatever happened to “neonatal hepatitis.” Clin Liver Dis. 2006;10(1):27–53.
2. Feldman AG, Sokol RJ. Neonatal Cholestasis. NeoReviews. 2013;14(2):63-73.14-2-e63.