Molecular Genetics Neurodegeneration with Brain Iron Accumulation (NBIA) Sequencing

Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration.  The age of onset ranges from infancy to late adulthood, and the rate of progression varies. Cognitive decline occurs in some subtypes, but more often cognition is relatively spared. Cerebellar atrophy is a frequent finding in some subtypes. The diagnosis is usually suspected with findings of brain iron accumulation on MRI.  However, both clinical findings and molecular genetic testing establish the diagnosis of specific subtypes.

Our panel is the most comprehensive clinical NBIA testing panel currently available. It includes genes associated with well-described NBIA disorders, as well as genes associated with new emerging phenotypes and related disorders. The results of each NBIA panel are reviewed by Dr. Susan Hayflick, who discovered many of these genes, and provides consultation on NBIA disorders to physicians around the world.

Reasons for Referral:

• Confirmation of a suspected clinical diagnosis in patients with the hallmark findings of NBIA.
• Further assessment of patients with clinical diagnosis of idiopathic NBIA who have had mutations ruled out in other genes.

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 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.

The 28 NBIA-associated genes are listed below:

Blood:  EDTA or ACD (Solution 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.

• 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)

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information, including ethnicity, clinical history, and family history.

Weekly

6-8 weeks

1. Gregory A. and Hayflick S., 2014. GeneReviews: http://www.ncbi.nlm.nih.gov/books/NBK121988/
2. Maio N., Orbach R., Zaharieva I., Töpf A., Donkervoort S., Munot P., Mueller J., Wilis T., Verma S, Peric S., Krishnakumar D., Sudhakar S., Foley A.R., Silverstein S., Douglas G., Pais L., DiTroia S., Grunseich C., Hu Y., Sewry C., Sarkozy A., Straub V., Muntoni F, Rouault T., Bönnemann C.G. Loss of Function of the Cytoplasmic Fe-S Assembly Protein CIAO1 Causes a Neuromuscular Disorder with Compromise of Nucleocytoplasmic Fe-S Enzymes. MedRxiv. 2020 Dec 20. Loss of Function of the Cytoplasmic Fe-S Assembly Protein CIAO1