Background:
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).
Methodology:
Next generation sequencing (NGS) will analyze the exons or coding regions of the genes using Illumina NextSeq 500/550 technology and preparing samples using hybridization probes to enrich exonic regions. This assay does not assess regions of insufficient coverage, introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and regions of high GC or polynucleotide repeats, but may contain variants that impact gene function.
Exon-level deletion/duplication analysis is performed by running the NGS data through the Genome Analysis Toolkit (GATK) Germline Copy Number Variation best practices pipeline from GATK, version 4.1.4.1. A Bayesian model was validated clinically in our lab. The model can detect copy changes at a resolution of three (3) or more probe targets (exons) for deletions and duplications in genes that do not have pseudogenes, and is not designed to detect low-level mosaicism or balanced alterations.
The 65 Cholestasis-associated genes are listed below:
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
Specimen Requirements:
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.
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: 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)
Notice Regarding Molecular Genetic Testing on Prenatal Specimens:
- 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.
Test Performed (Days):
Weekly
Turn Around Time:
8 weeks
Shipment Sensitivity Requirements:
- 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.
References:
- Balistreri WF, Bezerra JA. Whatever happened to “neonatal hepatitis.” Clin Liver Dis. 2006;10(1):27–53.
- Feldman AG, Sokol RJ. Neonatal Cholestasis. NeoReviews. 2013;14(2):63-73.14-2-e63.
Additional Info: