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.

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

Sanger Sequencing: Sequencing of FA2H 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 FA2H 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 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.

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)

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.

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.