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Chronic Progressive External Ophthalmoplegia (CPEO/PEO) Sequencing Panel with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing with CNV

Test Code Test Copy GenesCPT Code Copy CPT Codes
10507 DGUOK 81405,81479 Add to Order
DNA2 81479,81479
MGME1 81479,81479
OPA1 81407,81406
POLG 81406,81479
POLG2 81479,81479
RNASEH1 81479,81479
RRM2B 81405,81479
SLC25A4 81404,81479
SPG7 81406,81405
TK2 81405,81479
TWNK 81404,81479
TYMP 81405,81479
Full Panel Price* $930
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
10507 Genes x (13) $930 81404(x2), 81405(x5), 81406(x3), 81407, 81479(x15) Add to Order
Pricing Comments

We are happy to accommodate requests for testing single genes in this panel or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered via our PGxome Custom Panel tool.

Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Based on a 2014 literature review by Sommerville et al., POLG and TWNK are likely the most common nuclear-encoded genes known to be responsible for chronic progressive external ophthalmoplegia; at the time of publishing, 258 and 143 patients, respectively, had been reported (Sommerville et al. 2014. PubMed ID: 27858775). TYMP, RRM2B, OPA1, and SLC25A4 were found in 30-50 patients each, while SPG7, MGME1, TK2, POLG2, DGUOK, DNA2, and MPV17 were less common causes of disease (<13 individuals each).

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Clinical Features

The accumulation of multiple mtDNA deletions can be a sign of the cell’s inability to maintain mitochondrial genome integrity. Multiple mtDNA deletions are typically seen in the skeletal muscle of patients affected by adult-onset chronic progressive external ophthalmoplegia (weakness of the extraocular muscles, referred to as CPEO or PEO), a disorder that is often accompanied by ptosis, oropharyngeal weakness, and proximal limb weakness (DiMauro and Hirano. 2011. PubMed ID: 20301382). Onset typically ranges between 20-40 years of age (Viscomi and Zeviani. 2017. PubMed ID: 28324239). In contrast to many other mitochondrial diseases, isolated CPEO is a relatively mild phenotype.

When patients display additional, often more severe phenotypes beyond those of the isolated form, this may be referred to as chronic progressive external ophthalmoplegia plus (CPEO+/PEO+). Additional symptoms are often multi-systemic and can include ataxia, psychiatric symptoms, sensory axonal neuropathy, hearing loss and/or optic atrophy, among others (Sommerville et al. 2014. PubMed ID: 27858775; Hanisch et al. 2015. PubMed ID: 25143630). At this time, genotype-phenotype correlation for this spectrum of disease is poorly understood.

Muscle biopsies of patients affected with CPEO are distinguished by the presence of ragged red fibers, a characteristic finding under modified Gomori trichrome staining that is explained by the accumulation of abnormal mitochondria within the subsarcolemmal space (DiMauro and Hirano. 2011. PubMed ID: 20301382; Viscomi and Zeviani. 2017. PubMed ID: 28324239). Patients generally present with decreased activities of the respiratory chain complexes, a consequence of reduced mitochondrial genome integrity, and histochemical staining for cytochrome c oxidase (COX, complex IV) is also either decreased or absent (Viscomi and Zeviani. 2017. PubMed ID: 28324239).

Genetics

Chronic progressive external ophthalmoplegia (CPEO) can be caused by defects in one of a number of genes within the nuclear genome: DGUOK, DNA2, MGME1, OPA1, POLG, POLG2, RNASEH1, RRM2B, SLC25A4, SPG7, TK2, TWNK, or TYMP. Inheritance may be primarily autosomal dominant (POLG2, OPA1, DNA2, SLC25A4) or autosomal recessive (DGUOK, MGME1, RNASEH1, TK2, TYMP), while reports of both recessive and dominant inheritance have been documented for a subset of genes (POLG, RRM2B, TWNK, SPG7).

Please note that CPEO may also present in individuals who harbor de novo or maternally-inherited mtDNA deletions, or pathogenic single nucleotide variants in the mitochondrial genome (DiMauro and Hirano. 2011. PubMed ID: 20301382; Chinnery. 2014. PubMed ID: 20301403). Sequencing of the mitochondrial genome is not offered as part of this test, but could be considered in individuals who test negative for plausible causative variants in the nuclear genes associated with this disorder.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This panel provides 100% coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define coverage as ≥20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome-based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Individuals who present with suspected autosomal dominant or autosomal recessive adult-onset chronic progressive external ophthalmoplegia are good candidates for this test.

Genes

Official Gene Symbol OMIM ID
DGUOK 601465
DNA2 601810
MGME1 615076
OPA1 605290
POLG 174763
POLG2 604983
RNASEH1 604123
RRM2B 604712
SLC25A4 103220
SPG7 602783
TK2 188250
TWNK 606075
TYMP 131222
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
?Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal recessive 3 AR 617069
?Seckel syndrome 8 AR 615807
Behr Syndrome AR 210000
Dominant Hereditary Optic Atrophy AD 165500
Glaucoma, Normal Tension, Susceptibility To AD 606657
Mitochondrial DNA depletion syndrome 11 AR 615084
Mitochondrial DNA Depletion Syndrome 12 (Cardiomyopathic Type) AR 615418
Mitochondrial DNA Depletion Syndrome 12A (Cardiomyopathic Type) AD AD 617184
Mitochondrial DNA Depletion Syndrome 14 (Encephalocardiomyopathic Type) AR 616896
Mitochondrial DNA Depletion Syndrome 2 (Myopathic Type) AR 609560
Mitochondrial DNA Depletion Syndrome 4B, Mngie Type AR 613662
Mitochondrial DNA Depletion Syndrome 7 AR 271245
Mitochondrial DNA Depletion Syndrome, Encephalomyopathic Form, With Renal Tubulopathy AR 612075
Mitochondrial DNA-Depletion Syndrome 3, Hepatocerebral AR 251880
Mitochondrial Neurogastrointestinal Encephalomyopathy Syndrome AR 603041
Optic Atrophy Type 1 AD 125250
Perrault Syndrome 5 AR 616138
Portal hypertension, noncirrhotic AR 617068
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions 1 AD 157640
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions, Autosomal Dominant, 2 AD 609283
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions, Autosomal Dominant, 3 AD 609286
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions, Autosomal Dominant, 4 AD 610131
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions, Autosomal Dominant, 5 AD 613077
Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant, 6 AD 615156
Progressive External Ophthalmoplegia With Mitochondrial DNA Deletions, Autosomal Recessive AR 258450
Progressive External Ophthalmoplegia with Mitochondrial DNA Deletions, Autosomal Recessive 2 AR 616479
Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal recessive 4 AR 617070
Progressive Sclerosing Poliodystrophy AR 203700
Sensory Ataxic Neuropathy, Dysarthria, And Ophthalmoparesis AR 607459
Spastic Paraplegia 7 AD 607259

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Chinnery. 2014. PubMed ID: 20301403
  • DiMauro and Hirano. 2011. PubMed ID: 20301382
  • Hanisch et al. 2015. PubMed ID: 25143630
  • Sommerville et al. 2014. PubMed ID: 27858775
  • Viscomi and Zeviani. 2017. PubMed ID: 28324239
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

NextGen Sequencing: As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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