Molecular Genetics Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN), C19orf12, Sequencing

Mitochondrial membrane Protein-Associated Neurodegeneration (MPAN) is characterized by progressive dystonia, spasticity, paraparesis or tetraparesis, optic atrophy, psychiatric changes (ADHD-like behavior, mood swings), and evidence of iron accumulation in both the globus pallidus and substantia nigra on T2-weighted MRI.  Onset generally occurs in childhood to early adulthood with slow progression and survival well into adulthood.

MPAN is observed in ~10% of NBIA patients and mutations in c19orf12 are observed in 95% of patients with a clinical diagnosis of MPAN.  Mutations in MMIN are observed in ~40% of patients with a negative mutation test for other NBIA-associated genes, eg. PANK2, PLA2G6, CP and FTL (Hartig et al. 2011).

Reasons for Referral:

• Confirmation of a suspected diagnosis in patients with the hallmark findings of MPAN.
• Further assessment of patients with clinical diagnosis of idiopathic Neurodegeneration with Brain Iron Accumulation (NBIA) who do not have an eye-of-the-tiger sign and/or have had mutations ruled out in PANK2 or PLA2G6.
• Carrier testing of family members of MPAN patients with known mutations.

Sequencing using either Sanger Sequencing or Next-Generation Sequencing.

Sanger Sequencing:  Sequencing of c19orf12 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 c19orf12 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)

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 (1-2 mL)

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 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

• Keep specimen cold during transit, but do not ship on dry ice. 
• Please contact Client Services at (855) 535-1522 for shipping kits and instructions. 
• Use the cold pack provided in the KDL shipping kit. 
• Ship the specimen via overnight express, using the FedEx priority overnight label provided.

1. Hartig MB, Iuso A, Haack T, et al.  Absence of an orphan mitochondrial protein, c19orf12, causes a distinct clinical subtype of neurodegeneration with brain iron accumulation.  Am J Hum Genet. 2011 Oct 7;89(4):543-50.