Biochemical and genetic analysis of Leigh syndrome patients in Korea

1. Introduction

Originally described by Dr. Dennis Leigh in 1951, Leigh syndrome (LS) is a progressive neurodegenerative disorder typically beginning in infancy or childhood and is characterized by symmetrical necrosis in the brain stem, basal ganglia, and thalamus [1]. LS is one of the most common mitochondrial disorders and is caused by heterogeneous genetic and biochemical defects, including deficiencies of pyruvate dehydrogenase complex and mitochondrial respiratory chain enzymes. Because respiratory chain enzyme complexes are encoded in mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), LS can be caused by mutations in either genome [2-4]. Little is known about LS in the Korean population. In this study, we characterized the clinical, biochemical, and molecular genetic features of 16 Korean patients with LS evaluated at a major tertiary-care pediatric hospital in Seoul. This is the first report describing the molecular genetic and biochemical features of LS in Korea.

2. Patients and methods

In 2001-2006, 46 patients were evaluated for possible mitochondrial disorders at Seoul National University Childrens' Hospital. All of the patients were screened for other metabolic disorders by analyses of amino acids and organic acids. Sixteen patients were diagnosed with LS based on the following clinical criteria: encephalopathy, psychomotor regression, and characteristic brain magnetic resonance imaging (MRI) with symmetric lesions indicating bilateral necrosis of the basal ganglia, thalamus, and brain stem. None of the patients had MRI findings of central necrosis in the caudate head with involvement of the putamen that are typical of biotin-responsive basal ganglia disease (BBGD) [5]. Using skeletal muscle biopsy samples from all 16 LS patients, we performed histological studies, biochemical measurement of respiratory chain enzyme activities, and mtDNA genetic analyses. Multiple vitamin cocktails were administered to the patients without overt benefits.

3. Results

4. Discussion

We performed the first systematic biochemical and molecular genetic studies of Korean LS patients; defects were identified in 14 out of 16 patients (Table 1). Biochemical analyses of mitochondrial respiratory chain activities in muscle biopsies of 15 patients revealed deficiency of respiratory chain complex I in 9 individuals, combined defects of complexes I, III, and IV in 4, and normal enzyme activities in 2. Among patients with complex I deficiency, one had a previously reported G13513A mtDNA mutation in ND5 and 3 had a novel pathogenic G10197A in ND3 [7]. The other 5 patients did not have mtDNA mutations in ND genes and therefore are likely to harbor mutations in nDNA. In the 4 LS patients with combined defects of complexes I, III, and IV, sequencing of all mtDNA-encoded tRNAs did not reveal any mutations. These patients also may have a mutation in either the mtDNA-encoded ribosomal RNA genes or more likely, a mutation in a nDNA gene encoding proteins required for mitochondrial translation. One patient had a heteroplasmic T8993G mutation, which is common cause of LS.

Although a limited study based in a single tertiary medical center, our results suggest that isolated complex I defects may be the most frequent cause of LS in Korea and more common than previously reported [8,9]. An increasing number of papers have described mutations in nuclear encoded complex I subunits [10-12]; however, in our series, we identified mutations of mtDNA encoded ND genes in 4 (44%) out of 9 patients with isolated complex I deficiency. This remarkable result indicates that, in contrast to previous reports [13], mtDNA mutations may be responsible for a significant proportion of the infants and children with complex I defects, and targeted mtDNA screening based on biochemical results is an essential step in the diagnostic evaluation of pediatric mitochondrial disorders.

A major cause of mitochondrial disease in infancy and childhood, isolated complex I deficiency manifests a wide clinical spectrum ranging from severe encephalopathy to isolated hypertrophic cardiomyopathy [12,14]. Mutations in 16 mtDNA and nuclear genes have accounted for less than half of all patients with complex I deficiency [15,16], so many other causative genes are likely to be identified.

Although persistently elevated plasma lactate or characteristic histological changes of mitochondria in muscle are typical features of mitochondrial diseases, in our LS patients, half had normal plasma lactate and the majority had normal muscle morphology. Therefore, the usual screening tests for mitochondrial disease are not very sensitive in patients with LS, particularly when due to mtDNA mutations in protein coding genes [17,18]. Brain magnetic resonance spectroscopy (MRS) with voxels on brain lesions can reveal elevated lactate, which can be an important clue to the diagnosis of LS [19].

In this study, biochemical or molecular defects were identified in 14 patients (87.5%). We identified mtDNA mutations in one-third of the patients. In the remaining two-thirds, who are likely to have mutations in nDNA-encoded subunits of respiratory chain enzymes, we could not identify genetic causes. Identifying the genetic etiology of Leigh syndrome in Korean patients remains a persistent challenge for clinicians.