Background:
Multiple endocrine neoplasia type 2 includes subtypes A and B (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC); all are autosomal dominant disorders characterized by mutations in the RET gene. All three of the subtypes comprising MEN2 are associated with a high risk for developing medullary carcinoma of the thyroid (MTC), although at distinct age ranges. MEN2A (OMIM 171400) and MEN2B (OMIM 162300) have an increased risk for pheochromocytoma and MEN2A has an increased risk for parathyroid hyperplasia or adenoma. MEN2B patients may also present with neuromas of the lips and tongue and a Marfanoid habitus; this phenotype may be apparent in infants as may the associated MTC. Approximately 75% of patients with MEN2 have the MEN2A subtype. The diagnosis is based on clinical evaluation and family history.
Reasons for Referral:
- Individuals presenting with clinical features of MEN2A.
- Individuals with MTC and clinical diagnosis of MEN2 or primary C-cell hyperplasia.
- Individuals presenting with adrenal pheochromocytoma.
- Individuals presenting with clinical features suspicious for MEN2B.
- Predictive testing for at risk asymptomatic family members.
- Individuals with Hirschsprung disease.
Methodology:
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.
Specimen Requirements:
Blood: EDTA (purple-top) or ACD (yellow-top Solution A or B) tube
- Adult: 5 mL
- Child: 5 mL
- Infant: 2 - 3 mL
Saliva: 2 ORAgene™ Saliva Collection Kits (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.
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).
Prenatal:
- Direct Amniotic Fluid (10-20mL)
- Direct CVS
- Direct POC
- Cultured Amniocytes (2 T-25 flasks)
- Cultured CVS (2 T-25 flasks)
- Cultured Fetal Tissue: Product of Conception (2 T-25 flasks)
- Cord Blood (1-2mL)
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 and saliva (or DNA extracted from them), 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:
14-21 Days
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:
Additional Info:
Prior to any genetic testing we recommend genetic counseling. To receive forms and information about prenatal diagnostic testing, please contact Client Services at (855) 535-1522.