Forms

Mitochondrial Complex II Deficiency Sequencing Panel with CNV Detection

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
3457 SDHA 81479, 81479 Add to Order
SDHAF1 81479, 81479
SDHB 81405, 81479
SDHD 81404, 81479
Full Panel Price* $2520.00
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

Targeted Testing

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

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Isolated complex II deficiency is considered a rare form of mitochondrial disease, accounting for approximately 2-23% of all respiratory chain deficiencies (Parfait et al. 2000; Vladutiu and Heffner 2000). Clinical sensitivity for this test is difficult to estimate, however, as no large cohort studies have been reported.

Clinical testing for large deletions and duplications is difficult to predict as no large cohort studies are available.

Only one gross deletion has been described in SDHA to date (Helman et al. 2016).

In a cohort of 24 paraganglioma patients, large deletions in SDHB were reported in 3 individuals (~12%) who previously tested negative for point mutations in paraganglioma-related genes (Cascón et al. 2006).

At the present time, gross deletion/insertion frequencies for the SDHD gene are unknown, although approximately 15 large deletions and one complex rearrangement have been reported in the SDHD gene to date (Human Gene Mutation Database).

See More

See Less

Clinical Features

Isolated mitochondrial complex II (CII) deficiency, a deficit of one of the five oxidative phosphorylation complexes of the mitochondrial respiratory chain, has been associated with a diverse spectrum of clinical disease (Hoekstra and Bayley 2013). In all patients, enzymatic activity of the mitochondrial CII (also referred to as succinate dehydrogenase) is severely reduced in muscle biopsies and cultured fibroblasts.

Leigh syndrome, isolated cardiomyopathy, and infantile leukoencephalopathy are early-onset phenotypes commonly associated with isolated mitochondrial CII deficiency. Patients generally present in infancy or childhood. Leigh syndrome, also known as subacute necrotizing encephalopathy, is characterized by elevated levels of lactate in blood and cerebral spinal fluid; bilateral symmetric necrotic lesions in the basal ganglia, brain stem, thalamus, and/or spinal cord; psychomotor delay or regression; and neurologic manifestations such as hypotonia or ataxia (Wedatilake et al. 2013; Leigh 1951). Alternatively, a small subset of patients present with isolated cardiomyopathy accompanied by mild increases in lactate (Hoekstra and Bayley 2013; Levitas et al. 2010). Finally, defects in mitochondrial CII genes may result in an infantile leukoencephalopathy phenotype, with mild to severe lactic acidosis (Helman et al. 2016; Ghezzi et al. 2009).

Loss of mitochondrial CII activity may also contribute to tumor development, and CII deficiency has been linked to hereditary paraganglioma-pheochromocytoma, gastrointestinal stromal tumor (GIST) development, and renal cell carcinoma (Hoekstra and Bayley 2013; Rutter et al. 2010). For more information regarding hereditary paraganglioma-pheochromocytoma syndrome, see the Hereditary Paraganglioma-Pheochromocytoma Syndrome Sequencing Panel test.

Genetics

Mitochondrial complex II deficiency is the result of defects in the assembly or function of the succinate dehydrogenase (complex II) of the mitochondrial respiratory chain (Hoekstra and Bayley 2013). Complex II (CII) consists of four structural subunits (SDHA, SDHB, SDHC, and SDHD), while at least two accessory factors (SDHAF1 and SDHAF2) play roles in complex II assembly. Two additional accessory factors (SDHAF3 and SDHAF4) may also contribute to complex maturation, although their respective roles in this process have been less well defined (Na et al. 2014; Van Vranken et al. 2014).

This panel covers four nuclear-encoded genes that have been linked to mitochondrial CII deficiency to date (SDHA, SDHB, SDHD, and SDHAF1). SDHC and SDHAF2, which have only been associated with hereditary paraganglioma, are not included in this panel but are available either as single gene tests or via the Hereditary Paraganglioma-Pheochromocytoma Syndrome Sequencing Panel.

While disorders such as Leigh syndrome, cardiomyopathy, and infantile leukoencephalopathy are inherited in an autosomal recessive manner, familial paraganglioma may be inherited in an autosomal dominant manner with variable expressivity and age-related penetrance (Hoekstra and Bayley 2013).

SDHA: The 15-exon SDHA gene encodes for the flavoprotein subunit of the mitochondrial CII. Over 40 pathogenic variants have been reported in the SDHA gene to date (Human Gene Mutation Database). The majority of these variants are missense changes, although nonsense and splicing variants have been described, as well as a number of small deletions or insertions and one large deletion.

SDHB: The eight-exon SDHB gene encodes for the iron-sulfur cluster subunit of the mitochondrial CII. Over 200 pathogenic variants have been described in this gene to date, the majority of which are missense variants or deletions (HGMD).

SDHD: The four-exon SDHD gene encodes for an integral membrane structural subunit of the mitochondrial CII. Over 150 pathogenic variants, primarily missense changes, nonsense variants, and deletions, have been reported in this gene (HGMD).

SDHAF1: The single-exon SDHAF1 gene encodes for an LYR-family assembly factor that mediates maturation of SDHB, the iron-sulfur cluster subunit of mitochondrial CII (Na et al. 2014; Maio et al. 2016). Four pathogenic variants have been reported in the SDHAF1 gene, including three missense changes and one nonsense change (Ghezzi et al. 2009; Ohlenbusch et al. 2012).

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. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.

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 full coverage of all coding exons of the genes listed, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Due to homology issues, the entire coding region of SDHA (exons 1-15), in addition to ~20 bp of adjacent noncoding sequence of each exon, is bi-directionally sequenced using Sanger sequencing technology. Exon 4 of SDHD is also covered using Sanger sequencing technology.

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

Candidates for this test include patients with a deficiency of mitochondrial complex II, or those who present with symptoms consistent with a known disease phenotype.

Genes

Official Gene Symbol OMIM ID
SDHA 600857
SDHAF1 612848
SDHB 185470
SDHD 602690
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Mitochondrial Complex II Deficiency AR 252011

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Hereditary Paraganglioma-Pheochromocytoma Syndrome Sequencing Panel
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHA Gene
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHB Gene
Hereditary Paraganglioma-Pheochromocytoma Syndrome via the SDHD Gene
Infantile Leukoencephalopathy Due to Mitochondrial Complex II Deficiency via the SDHAF1 Gene
Renal Cancer Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Cascón A. et al. 2006. Genes, Chromosomes & Cancer. 45: 213-9. PubMed ID: 16258955
  • Ghezzi D. et al. 2009. Nature Genetics. 41:654-6. PubMed ID: 19465911
  • Helman G. et al. 2016. Annals of Neurology. 79:379-86. PubMed ID: 26642834
  • Hoekstra A.S. and Bayley J.P. 2013. Biochimica et Biophysica Acta. 1827:543-51. PubMed ID: 23174333
  • Human Gene Mutation Database (Bio-base).
  • Leigh D. 1951. Journal of Neurology, Neurosurgery, and Psychiatry. 14:216-21. PubMed ID: 14874135
  • Levitas A. et al. 2010. European Journal of Human Genetics. 18:1160-5. PubMed ID: 20551992
  • Maio N. et al. 2016. Cellular Metabolism. 23:292-302. PubMed ID: 26749241
  • Na U. et al. 2014. Cellular Metabolism. 20:253-66. PubMed ID: 24954417
  • Ohlenbusch A. et al. 2012. Orphanet Journal of Rare Diseases. 7:69. PubMed ID: 22995659
  • Parfait B. et al. 2000. Human Genetics. 106:236-43. PubMed ID: 10746566
  • Rutter J. et al. 2010. Mitochondrion. 10:393-401. PubMed ID: 20226277
  • Van Vranken J.G. et al. 2014. Cellular Metabolism. 20:241-52. PubMed ID: 24954416
  • Vladutiu G.D. and Heffner R.R. 2000. Archives of Pathology & Laboratory Medicine. 124:1755-8. PubMed ID: 11100052
  • Wedatilake Y. et al. 2013. Orphanet Journal of Rare Diseases. 8:96. PubMed ID: 23829769
Order Kits
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). Common benign, likely benign, and low quality variants are filtered from analysis. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.

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

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 close to 100%, 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.  

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

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.

Order Kits

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.
loading Loading... ×