Molecular Genetics Fatty Acid Hydroxylase-Associated Neurodegeneration (FAHN), FA2H, Sequencing

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is an autosomal recessive disorder characterized by spastic paraplegia or quadriplegia, pyramidal tract signs, dystonia, ataxia, and brain MRI findings that include T2 hypointensity in the globus pallidus, progressive atrophy of the cerebellum, pons, medulla and spinal cord, thinning of the corpus callosum, and variable T2 hyperintensities in the periventricular and/or subcortical white matter.  Mutations in the fatty acid 2-hydroxylase gene (FA2H; OMIM 611026) are found in patients clinically diagnosed with FAHN (Evardson et al. 2008; Dick et al. 2008; Dick et al. 2010; Kruer et al. 2010).  Onset occurs in the first to second decade and the disorder is progressive.

Reasons for Referral:

• Confirmation of a suspected clinical diagnosis in patients with the hallmark findings of FAHN.
• Further assessment of patients with clinical diagnosis of idiopathic Neurodegeneration with Brain Iron Accumulation (NBIA) who have had mutations ruled out in PANK2 and/or PLA2G6.
• Carrier testing of family members of FAHN patients with known mutations.

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.

Blood: EDTA or ACD (Solution A or B):

• Adult: 5mL
• Child: 5mL
• Infant: 2-3mL

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).

• 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)

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.

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.

1. Kruer, M. et al.  Fatty Acid Hydroxylase-Associated Neurodegeneration. Synonym: FAHN. Includes: Dysmyelinating Leukodystrophy and Spastic Paraparesis with or without Dystonia, Spastic Paraplegia 35.  Gene Reviews. http://www.ncbi.nlm.nih.gov/books/NBK56080/.  Initial Posting: June 28, 2011.
2. Edvardson et al. Am J Hum Genet. 2008 Nov;83(5):643-8.
3. Dick et al. Neurology. 2008 Jul 22;71(4):248-52.
4. Dick et al. Hum Mutat. 2010 Apr;31(4):E1251-60.
5. Kruer et al. Ann Neurol. 2010 Nov;68(5):611-8.