Molecular Genetics Neuroferritinopathy, FTL, Sequencing

NBIA is a group of neurodegenerative diseases caused by iron accumulation in the basal ganglia leading to dystonia, parkinsonism, neurocognitive anomalies, and ophthalmologic disorders.  Neuroferritinopathy is caused by mutations in the FTL gene (Ferritin Light chain), and is inherited in an autosomal dominant manner.  This disease is characterized by progressive adult-onset chorea or dystonia and cognitive deficits, and shows the presence of focal spherical inclusions in the brain and other organs, including liver and muscle.  In addition, mutations in the iron response element (IRE) of the FTL gene cause hereditary hyperferritinemia cataract syndrome (HHCS), a syndrome of early-onset cataract and hyperferritinemia.  The IRE is located in the 5’ untranslated region of the FTL mRNA.

Reasons for Referral:

• Confirmation of a suspected clinical diagnosis in patients with the hallmark findings of neuroferritinopathy.
• Further assessment of patients with clinical diagnosis of idiopathic Neurodegeneration with Brain Iron Accumulation (NBIA) who have had mutations ruled out in other NBIA genes.
• Carrier testing of family members of FTL patients with known mutations.

Sequencing can be performed by either Sanger Sequencing or Next-Generation Sequencing.

Sanger Sequencing: Sequencing of FTL is carried out by amplification of all 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 FTL using
Illumina NextSeq 500/550 technology. Samples are prepared 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.

Blood: EDTA (purple top) or ACD (yellow top) (Solutions A or B):

• 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).

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 CVS or Amniotic Fluid Specimens:       

• Maternal cell rule-out testing will be performed on all prenatal specimens received. Please provide maternal blood 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 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.

Weekly

14-21 days

• 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.

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2. Vidal R et al., (2004) J Neuropathol Exp Neurol. 63:363-380.
3. Chinnery PF et al., (2006) Brain 130:110-119.
4. Maciel P et al., (2005) Neurology 65:603-605.
5. Mancuso M et al., (2005) J Neuropathol Exp Neurol. 64:280-294.