Leigh Syndrome Associated with Isolated Complex I Deficiency via the NDUFA12 Gene
- Summary and Pricing
- Clinical Features and Genetics
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At this time, due to the limited number of reported cases, the clinical sensitivity of NDUFA12-related Leigh Syndrome is difficult to estimate. Analytical sensitivity should be high as the only reported pathogenic variant is detectable by sequencing.
Leigh Syndrome (LS), also known as subacute necrotizing encephalomyelopathy, is a severe neurodegenerative disorder resulting from defects in the mitochondrial respiratory chain (Ruhoy and Saneto 2014; Zhu et al. 1998; Leigh 1951). The disease incidence for LS is estimated to be around 1:32,000 to 1:40,000 live births (Darin et al. 2001; Rahman et al. 1996).
The hallmark features that characterize this syndrome include elevated levels of lactate in blood and cerebral spinal fluid, in addition to the presence of bilateral symmetric necrotic lesions in the basal ganglia, brain stem, thalamus, and/or spinal cord (Wedatilake et al. 2013; Leigh 1951). Patients also present with isolated or combined mitochondrial complex deficiencies, psychomotor delay or regression, and neurologic manifestations such as hypotonia or ataxia. The term ‘Leigh-Like Syndrome (LLS)’ is used to describe a similar clinical presentation in which one or more of these diagnostic characteristics is atypical.
Symptomatic onset of this disorder usually occurs shortly after birth or within the first three years of life, although cases of adult-onset LS/LLS have been reported (Ronchi et al. 2011). LS/LLS infants often present with feeding difficulties, gastrointestinal distress, hypotonia, and growth delays, while older children (>1 years) may develop additional symptoms including developmental regression (loss of cognitive or motor skills), dysphagia, hypertrichosis, dystonic posturing, nystagmus, and opthalmoplegia (Wedatilake et al. 2013).
LS and LLS have been linked to pathogenic variants in over 60 different genes (Rahman 2015). However, while defects in certain genes are more likely to result in classic LS rather than atypical LLS, genotype-phenotype correlations are still poorly understood. To date, there has been only one report of a patient with NDUFA12-associated LS (Ostergaard et al. 2011). The affected individual presented with isolated complex I deficiency, lactic acidosis, developmental delay, loss of motor abilities, severe muscular atrophy, hypotonia, dystonia, and hypertrichosis.
Leigh and Leigh-Like Syndromes (LS/LLS) are caused by defects in the mitochondrial oxidative phosphorylation (OXPHOS) complexes or associated proteins, such as the OXPHOS assembly factors or components of the pyruvate dehydrogenase (PDH) complex (Rahman 2015). As a result, the LS/LLS phenotypes exhibit significant genetic heterogeneity, and pathogenic variants in over 60 different genes have been reported to be causative for this disorder. Depending on the cellular localization of the affected gene(s), these syndromes may be inherited in an autosomal recessive, maternal, or X-linked recessive manner.
Nuclear genes associated with autosomal recessive inheritance of LS/LLS include: SURF1, BCS1L, C12ORF65, COX10, COX15, FOXRED1, GFM1, LRPPRC, NDUFA2, NDUFA4, NDUFA9, NDUFA10, NDUFA11, NDUFA12, NDUFAF2, NDUFAF5, NDUFAF6, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, PDSS2, PET100, SCO2, SDHA, SDHAF1, SLC19A3, SUCLA2, SUCLG1, TACO1, TTC19, UQCRQ, SERAC1, NDUFV2, MTFMT, HIBCH, TSFM, ECHS1, LIAS, PNPT1, POLG, LIPT1, DLD, TPK1, and ETHE1.
Mitochondrial genes associated with maternal inheritance of LS/LLS include: MT-ATP6, MT-TL1, MT-TK, MT-TW, MT-TV, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-ND6, and MT-CO3 .
X-linked genes associated with X-linked recessive inheritance of LS/LLS include: NDUFA1, AIFM1, PDHA1, PDHB, and PDHX. In this form of inheritance, male patients are more frequently affected, although heterozygous females may present with LS/LLS due to skewed X-inactivation (Patel et al. 2012).
The four-exon NDUFA12 gene encodes for an accessory subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I), although the precise role of this protein is not yet clear (Rak and Rustin 2014). One pathogenic nonsense variant has been reported in NDUFA12 to date (Ostergaard et al. 2011).
Full gene sequencing of NDUFA12 is performed, with bidirectional sequencing of exons 1-4. 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
NDUFA12 sequencing could be considered in patients with symptoms consistent with LS or a family history of childhood encephalopathy, particularly if those individuals also have negative sequencing results for more frequently reported LS genes, such as SURF1. We will also sequence the NDUFA12 gene to determine carrier status.
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- Darin N. et al. 2001. Annals of Neurology. 49: 377-83. PubMed ID: 11261513
- Leigh D. 1951. Journal of Neurology, Neurosurgery, and Psychiatry. 14: 216-21. PubMed ID: 14874135
- Ostergaard E. et al. 2011. Journal of Medical Genetics. 48: 737-40. PubMed ID: 21617257
- Patel K.P. et al. 2012. Molecular Genetics and Metabolism. 105: 34-43. PubMed ID: 22079328
- Rahman S. 2015. Nuclear Gene-Encoded Leigh Syndrome Overview. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 26425749
- Rahman S. et al. 1996. Annals of Neurology. 39: 343-51. PubMed ID: 8602753
- Rak M, Rustin P. 2014. FEBS Letters. 588: 1832-8. PubMed ID: 24717771
- Ronchi D. et al. 2011. Biochemical and Biophysical Research Communications. 412: 245-8. PubMed ID: 21819970
- Ruhoy I.S., Saneto R.P. 2014. The Application of Clinical Genetics. 7: 221-34. PubMed ID: 25419155
- Wedatilake Y. et al. 2013. Orphanet Journal of Rare Diseases. 8: 96. PubMed ID: 23829769
- Zhu Z. et al. 1998. Nature Genetics. 20: 337-43. PubMed ID: 9843204
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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- 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.
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- Only one blood tube is required for multiple tests.
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- 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.
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- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
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(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.
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- 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.