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.
Known genetic causes of this disorder are mutations in the ret proto-oncogene, RET. Mutations in RET account for 98%, 98% and 95% of individuals with MEN 2A, MEN 2B, and FMTC, respectively. De novo mutations occur in approximately 50% of MEN2B cases (however, the hotspot mutation is the same as with inherited cases, so the tiered strategy presented below remains valid). Recurrent point mutations have been describe, thus a tiered approach to testing based on clinical finding and/or family history will be used and is outlined below.
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:
We sequence exons and exon/intron boundaries of exons 10, 11, 13, 14, 15 and 16 of RET. Based on published studies, this approach is expected to yield approximately 95%-98% of known RET pathogenic variants in MEN2 disorders.
Sequencing using either Sanger Sequencing or Next-Generation Sequencing.
Sanger Sequencing: Sequencing of RET is carried out by amplification of the above mentioned exons and intron/exon boundaries followed by bi-directional Sanger sequencing. The sensitivity of full gene sequencing is estimated to be approximately 99% for single nucleotide substitutions and small insertions/deletions. All nucleotide changes are analyzed within the context of current databases and literature to predict pathogenicity.
NGS: Next generation sequencing will analyze the exons or coding regions of RET using Illumina NextSeq 500 technology. Samples are prepared using hybridization probes to enrich exonic regions. Promoter, intronic, etc. regions are not assessed on our assay, but may contain variants that impact gene function.
Test reporting follows the ACMG Standards & Guidelines for Clinical Genetics Laboratories, Ultra-Rare Disorders Guidelines, and Interpretation of Sequence Variants Guidelines.
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.
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).
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 outside of 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.