Elsevier

Mitochondrion

Volume 1, Issue 3, October 2001, Pages 269-278
Mitochondrion

Screening for mitochondrial DNA heteroplasmy in children at risk for mitochondrial disease

https://doi.org/10.1016/S1567-7249(01)00028-9Get rights and content

Abstract

Temporal temperature gradient gel electrophoresis was used to screen 70% of the mtDNA, including all 22 tRNA genes, for heteroplasmy in 75 children with neuromuscular and/or multi-system dysfunction and elevated lactate levels, and in 95 controls. Standard PCR/ASO (allele specific oligonucleotide) and Southern analyses were also employed. Excluding common length variants, heteroplasmy was found in 22 patients and two controls (P<0.001), with four patients demonstrating heteroplasmy in two locations each. Of the 23 heteroplasmic variants sequenced among the patients, 17 were novel point variants in the control region (CR) and only two involved tRNA genes. Heteroplasmy is highly associated with the disease group, and is predominately found in the CR, an area rarely studied in patient populations. These variants may be pathological mutations or disease markers.

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.

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