Infantile Leukoencephalopathy Due to Mitochondrial Complex II Deficiency via the SDHAF1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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At this time, due to the limited number of reported cases (<15), the clinical sensitivity of SDHAF1-related mitochondrial complex II deficiency is difficult to estimate. Analytical sensitivity should be high because all pathogenic variants reported are detectable by sequencing.
Isolated mitochondrial complex II deficiency (also known as succinate dehydrogenase deficiency) has been associated with a diverse clinical spectrum of disease, including cardiomyopathy, Leigh syndrome, infantile leukoencephalopathy, familial paraganglioma, renal cell carcinoma, and gastrointestinal stromal tumor development (Hoekstra and Bayley 2013).
Pathogenic variants in at least three genes (SDHA, SDHB, and SDHAF1) have been linked to infantile leukoencephalopathy (Hoekstra and Bayley 2013; Alston et al. 2012; Renkema et al. 2015). In patients with SDHAF1-related infantile leukoencephalopathy, symptoms generally manifest between 6-20 months following birth, and consist of rapid psychomotor regression, lack of speech development, progression of spastic quadriparesis, and partial loss of postural control with dystonia (Ghezzi et al. 2009; Ohlenbusch et al. 2012). Biochemically, this disease is characterized by elevated levels of blood lactate and pyruvate (Ghezzi et al. 2009). Succinate dehydrogenase holoenzyme quantity and activity are severely reduced in patient muscle and fibroblasts, and proton magnetic resonance spectroscopy (MRS) of the brain often reveals a high succinate peak, indicative of mitochondrial complex II deficiency. A distinctive MRI pattern has also been reported, which is delineated by cerebral hemispheric white matter abnormalities with sparing of the U fibers, corpus callosum involvement with sparing of the outer blades, and involvement of the corticospinal tracts, thalami, and spinal cord (Helman et al. 2016).
Mitochondrial complex II deficiency, an autosomal recessive disorder, is the result of defects in the assembly or function of the mitochondrial succinate dehydrogenase (complex II) (Hoekstra and Bayley 2013). Complex II 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).
The single-exon SDHAF1 gene encodes for an LYR-family assembly factor that mediates maturation of SDHB, the iron-sulfur cluster subunit of mitochondrial complex II (Na et al. 2014; Maio et al. 2016). Four pathogenic variants have been reported in the SDHAF1 gene to date, including three missense changes and one nonsense change (Ghezzi et al. 2009; Ohlenbusch et al. 2012).
Full gene sequencing of the SDHAF1 gene is performed, with bidirectional sequencing of exon 1. Testing is accomplished by amplifying the single coding exon and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard Sanger dideoxy sequencing methods and a capillary electrophoresis instrument. We will also sequence any single portion (Test #100) of this exon in family members of patients with a known mutation or to confirm research results.
Indications for Test
SDHAF1 sequencing should be considered in patients with symptoms consistent with infantile leukoencephalopathy or a family history of this disease. We will also sequence the SDHAF1 gene to determine carrier status.
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- Alston C.L. et al. 2012. Journal of Medical Genetics. 49:569-77. PubMed ID: 22972948
- 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
- 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
- Renkema G.H. et al. 2015. European Journal of Human Genetics. 23:202-9. PubMed ID: 24781757
- Van Vranken J.G. et al. 2014. Cellular Metabolism. 20:241-52. PubMed ID: 24954416
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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