Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. 
Clinically, no efficient assay protocols have been available. In this study, we 
aimed to develop an oligonucleotide biochip specialized for detection of known 
base substitution mutations in mitochondrial DNA causing LHON and to investigate 
frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs 
of oligonucleotide probes matched with the mutations potentially linked to LHON 
were covalently immobilized. Cy5-lablled targets were amplified from blood DNA 
samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A 
and 14484 T > C from six confirmed LHON patients were interrogated to validate 
this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five 
unrelated healthy individuals were investigated by the LHON biochip, direct 
sequencing and pyrosequencing, respectively. The biochip was found to be able 
efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in 
LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases 
from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > 
C or 3460G <formula>></formula> A, respectively, accounting for 66.7%. The 
mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 
of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in 
one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller 
amount of sampling and reaction volume, easier target preparation, fast and 
high-throughput were the main advantages of the biochip over direct DNA 
sequencing and pyrosequencing. Our findings suggested that primary mutations of 
11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to 
LHON in China. The biochip would easily be implemented in clinical diagnosis.