Screening for mitochondrial DNA heteroplasmy in children at risk for mitochondrial disease
Introduction
Mitochondrial disorders are present in some patients with a rapidly increasing list of common and uncommon neuromuscular and/or multi-system disorders. A minority of cases show clear maternal, recessive or X-linked inheritance patterns, while in clinical practice, most cases are sporadic and the genome containing the mutation is unknown (Shoffner and Wallace, 1995). In many centers, at risk patients are screened by ‘standard mtDNA analyses’, often consisting of PCR-based methods to detect ∼3–10 known point mutations and Southern blot analysis for large rearrangements. General screening methods such as single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and high performance liquid chromatography (HPLC) employed by research laboratories in at risk patient populations have recently greatly increased the number of suspected pathological mtDNA point mutations to over 100 (MITOMAP database: Kogelnik et al., 1996), suggesting that many additional mtDNA mutations remain to be discovered.
Most known pathogenic mtDNA mutations reported to date co-exist with wild type genomes (‘heteroplasmy’), while benign polymorphisms are generally homoplasmic (Shoffner and Wallace, 1995, Kogelnik et al., 1996). This contrast suggests that a heteroplasmy detection assay could be an effective way to screen for novel mtDNA mutations in at risk children. One such method, temporal temperature gradient gel electrophoresis (TTGE), is relatively sensitive and specific in the detection of mtDNA heteroplasmy (Chen et al., 1997, Chen et al., 1999, Higashimoto et al., 1999). In this study, we applied standard PCR/ASO and Southern analyses, followed by TTGE screening of 70% of the entire mtDNA, including all 22 tRNA genes, for heteroplasmy in 75 children at risk for possible mitochondrial disease and in 95 controls.
Section snippets
Materials and methods
The ‘experimental group’ consisted of 75 unrelated children evaluated by the last author in the Childrens Hospital Los Angeles (CHLA) genetics clinic over a period of 6 years. All qualifying children were retrospectively recruited based upon the presence of neuromuscular and/or multi-system disease, an elevated body fluid lactate concentration (plasma, urine and/or CSF; usually in plasma and often minimally elevated, >2.0 mM or 18 mg/dl) and the absence of another diagnosis despite an extensive
Results
‘Common’ mtDNA mutations were identified in two of the 75 patients using ‘standard methods’: A3243G in an infant with infantile spasms and a near-sudden infant death syndrome (SIDS) event (to be published separately), and the 4.9 kb ‘common deletion’ in a child with adrenal failure and Kearns–Sayre syndrome (Boles et al., 1998). Distinct TTGE band patterns consistent in our experience with the presence of heteroplasmic single nucleotide substitution(s) (‘point heteroplasmy’; Fig. 2) were found
Discussion
The Emory University MITOMAP website (Kogelnik et al., 1996) which lists all reported mtDNA variants is heavily weighed in point mutations towards those in the tRNA genes (65 of a total of 116 established or provisional, pathological point mutations at the time of this writing). As these 22 genes collectively constitute, in area, only about 10% of the mtDNA, and many of the most frequently reported point mutations, including A3243G, occur in tRNA genes, there is an impression that these genes
Acknowledgements
This work was supported by grants from the United Mitochondrial Disease Foundation and the National Institute of Health 1R21NS40462, a Research Career Development Award from the Childrens Hospital Los Angeles Research Institute, and the CHLA Division of Medical Genetics (all to RGB). The authors also thank the numerous clinicians who referred patients and provided samples for DNA analysis.
References (27)
- et al.
The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age
Am. J. Hum. Genet.
(2000) Slowly but surely towards better scanning for mutations
Trends Genet.
(1997)- et al.
Persistent heteroplasmy of a mutation in the human mtDNA control region: hypermutation as an apparent consequence of simple-repeat expansion/contraction
Am. J. Hum. Genet.
(2000) - et al.
Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients
Genomics
(1993) - et al.
A sensitive denaturing gradient-gel electrophoresis assay reveals high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region
Am. J. Hum. Genet.
(2000) - et al.
Identification in Portugal and Brazil of a mtDNA lineage containing a 9-bp triplication of the intergenic COII/tRNALys region
Hum. Hered.
(1999) - et al.
Susceptibility mutations in the mitochondrial small ribosomal RNA gene in aminoglycoside induced deafness
Pharmacogenetics
(1995) - et al.
Mitochondrial Disease and CVS
Digest. Dis. Sci.
(1999) - et al.
Mitochondrial DNA deletion with Kearns Sayre syndrome in a child with Addison disease
Eur. J. Pediatr.
(1998) - et al.
Detection of heteroplasmic mtDNA mutations by temporal temperature gradient gel electrophoresis
Am. J. Hum. Genet.
(1997)
Detection of mitochondrial DNA mutations by temporal temperature gradient gel electrophoresis
Clin. Chem.
Transmission of mitochondrial DNA heteroplasmy in normal pedigrees
Hum. Genet.
Rapid detection of FGFR mutations in syndromic craniosynostosis by temporal temperature gradient gel electrophoresis
Clin. Chem.
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