Background:
Rett syndrome (RTT) is a severe progressive neurodevelopmental disorder that presents early in childhood and affects primarily females (OMIM #312750). This X-linked dominant disorder affects 1:10,000-1:15,000 girls. In classic RTT, normal growth and development occurs during approximately the first 6-18 months of life. This is followed by an arrest in development and subsequent progressive loss of acquired motor skills and language and the onset of stereotypical hand movements. Other variants of RTT include congenital Rett syndrome, “forme fruste” and preserved speech variant. RTT has generally been considered to be lethal in males, but recent studies have identified males with RTT with varying degrees of severity. Rett syndrome is caused by mutations in the methyl CpG binding protein 2 (MECP2) located at chromosome Xq28. This comprehensive analysis includes Sequencing and Deletion/ Duplication Analysis.
Reasons for Referral:
- Confirmation of clinical diagnosis in patients with classical Rett syndrome.
- Diagnostic testing in patients with variants of Rett syndrome.
- Testing of family members of Rett syndrome patients or those who have non-syndromic mental retardation or autistic spectrum disorder.
- Prenatal diagnosis.
Methodology:
NGS Sequencing: 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.
80% detection rate in females with classical Rett syndrome.
Deletion/Duplication: A customized CytoSure “exon-centric” array (Oxford Gene Technology) will be used to detect deletions and duplications. The targeted array has enhanced probes targeted to the exonic regions of MECP2. The arrays are run using Agilent SureScan technology.
Specimen Requirements:
Blood: EDTA or ACD (Solution Aor B):
- Adult: 5mL
- Child: 5mL
- Infant: 2-3mL
Saliva: 2 ORAgene Saliva 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 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 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)
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).
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 cool, but not frozen.
- Please use the cold pack provided in the kit.
- 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. Please contact Client Services at (855) 535-1522 for forms and information about prenatal diagnostic testing.