Mitochondrial Complex IV Deficiency via the APOPT1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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At this time, due to the limited number of reported cases (<10), the clinical sensitivity of APOPT1-related mitochondrial complex IV deficiency is difficult to estimate, although it is expected to be a rare cause of this disease. Four of the five reported variants in this gene are detectable by sequencing.
Mitochondrial complex IV (CIV) deficiency is characterized by a deficiency of the fourth oxidative phosphorylation (OXPHOS) complex of the mitochondrial respiratory chain (Fassone and Rahman 2012). Primary mitochondrial CIV deficiency is estimated to account for approximately one-fifth of all OXPHOS disorders (Skladal et al. 2003; Scaglia et al. 2004).
The majority of CIV-deficient patients present with a severe, early-onset disease within the first year of life. Similar to other OXPHOS disorders, recurrent lactic acidosis is a prevalent finding in affected individuals. Patients may display significant heterogeneity in additional clinical features, which can include encephalopathy, hypertropic cardiomyopathy, hypotonia, epilepsy, microcephaly, dystonia, psychomotor delay or impairment, nystagmus, respiratory insufficiency, ataxia, muscle weakness, and/or CIV-deficient Leigh or Leigh-like syndrome (Pecina et al. 2004; Darin et al. 2003; Alfadhel et al. 2011). Leigh syndrome (LS) is a severe, progressive encephalopathy characterized by psychomotor delay or regression; isolated or combined mitochondrial complex deficiencies; elevated levels of lactate in the blood and/or cerebral spinal fluid; bilateral symmetrical lesions in the brainstem and basal ganglia; and neurologic manifestations such as hypotonia or ataxia (Rahman and Thorburn 2015; Lake et al. 2015).
APOPT1-related CIV deficiency has been reported in at least five unrelated families to date (Melchionda et al. 2014). In contrast to the majority of CIV-deficient patients, patients with defects in APOPT1 displayed a slightly later age at onset (~2.5-5 years of age); in at least one individual, symptoms were subclinical through early adolescence. All affected individuals underwent a period of acute regression (loss of developmental milestones/speech, episodes with somnolence and seizure, and pyramidal signs that developed into spastic tetraparesis), but stabilized and/or partially recovered after several months or years. In all cases, a distinctive cavitating leukodystrophy involving the posterior cerebral white matter and corpus callosum was identified via MRI during the acute stage of disease. Long-term survival was reported in all patients.
The cytochrome c oxidase enzyme, also referred to as mitochondrial complex IV (CIV) or COX, is the terminal oxidase of the mitochondrial respiratory chain. Over 30 genes are involved in the structure, assembly, or function of this enzyme (Kadenbach and Hüttemann 2015). Primary mitochondrial CIV deficiency has been linked to pathogenic variants in approximately half of these genes to date. Three CIV subunits (MT-CO1, MT-CO2, and MT-CO3), which form the catalytic core of the enzyme, are encoded by the mitochondrial genome. Pathogenic variants in MT-CO1, MT-CO2, and MT-CO3 are maternally inherited (Rak et al. 2016). Defects in the remaining nuclear-encoded genes, including APOPT1, exhibit autosomal recessive inheritance.
The biological function of APOPT1 is still under debate; this protein may have a pro-apoptotic role or may function in mitochondrial anti-ROS defense mechanisms (Yasuda et al. 2006; Melchionda et al. 2014). One missense, one nonsense, and one splicing variant have been identified as causative for APOPT1-related CIV deficiency, in addition to one small and one gross deletion (Melchionda et al. 2014).
Full gene sequencing of APOPT1 is performed, with bidirectional Sanger sequencing of exons 1-5. The full coding region of each exon plus ~20 bp of flanking non-coding DNA on either side are sequenced. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Indications for Test
APOPT1 sequencing should be considered for patients who present with symptoms consistent with mitochondrial complex IV (CIV) deficiency or for individuals with a family history of mitochondrial CIV deficiency. We will also sequence the APOPT1 gene to determine carrier status.
|Official Gene Symbol||OMIM ID|
|Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel|
|Mitochondrial Complex IV Deficiency Sequencing Panel (Nuclear Genes)|
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- Alfadhel M. et al. 2011. American Journal of Medical Genetics A. 155A:840-4. PubMed ID: 21412973
- Darin N. et al. 2003. Neuropediatrics. 34:311-7. PubMed ID: 14681757
- Fassone E., Rahman S. 2012. Journal of Medical Genetics. 49:578-90. PubMed ID: 22972949
- Kadenbach B., Hüttemann M. 2015. Mitochondrion. 24:64-76. PubMed ID: 26190566
- Lake N.J. et al. 2015. Journal of Neuropathology & Experimental Neurology. 74:482-92. PubMed ID: 25978847
- Melchionda L. et al. 2014. American Journal of Human Genetics. 95:315-25. PubMed ID: 25175347
- Pecina P. et al. 2004. Physiological Research. 53 Suppl 1:S213-23. PubMed ID: 15119951
- Rahman S., Thorburn D. 2015. Nuclear Gene-Encoded Leigh Syndrome Overview. In: Pagon RA, Aadam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews (®), Seattle (WA): University of Washington, Seattle. PubMed ID: 26425749
- Rak M. et al. 2016. Clinical Science. 130:393-407. PubMed ID: 26846578
- Scaglia F. et al. 2004. Pediatrics. 114:925-31. PubMed ID: 15466086
- Skladal D. et al. 2003. Brain. 126:1905-12. PubMed ID: 12805096
- Yasuda O. et al. 2006. Journal of Biological Chemistry. 281:23899-907. PubMed ID: 16782708
Bi-Directional Sanger Sequencing
Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org). As required, DNA is extracted from the patient specimen. PCR is used to amplify the indicated exons plus additional flanking non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions. In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.
As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all 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 for example 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 PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
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