Leber's Hereditary Optic Neuropathy

Specific mutations at mtDNA positions nt11778 (ND4 gene), nt3460 (ND1 gene), and nt14484 (ND6 gene) are considered primary causes of Leber's Hereditary Optic Neuropathy (LHON) in about 90% of patients worldwide (Mackey et al., 1996; Am. J. Hum. Genet. 59:481-485). In addition, although rare, there is evidence for a primary pathogenic role of the nt15257 (CYB) mutation (Hofmann et al., 1997; Am. J. Hum. Genet. 60:1539-1542). The presence of any one of these mutations in a symptomatic individual can be considered diagnostic for LHON. Only about 50% of index cases have a family history of LHON. Mitochondrial genetics are characterized by maternal transmission only and by mtDNA heteroplasmy (variation in the relative proportions of normal and mutant mtDNA) in tissues. The detection of mutant mitochondrial DNA in peripheral blood does NOT provide a measure of heteroplasmy in other tissues. Hence, it is difficult to provide definitive genetic counseling information to unaffected family members who may harbor a LHON primary mutation. For recent reviews on the genetics of LHON, please refer to Harding et al., 1995 (Am. J. Hum. Genet. 57:77-86) or Riordan-Eva and Harding, 1995 (J. Med. Genet. 32:81-87).

This test involves a PCR assay*. Any individuals that test positive for either the nt3460, nt14484, or nt15257 mutations are additionally evaluated by direct sequencing of the PCR product to confirm the pathogenic LHON mutation rather than a false positive result due to one of the known polymorphisms that occur at these sites (Johns and Neufeld, 1993; (Am. J. Hum. Genet. 53:916-920). Sample positive results are shown below.

DETECTION OF THE nt11778 (ND4) MUTATION

This assay involves two parts as the nt11778 mutation causes both a loss (Sfa NI) and a gain (Mae III) of restriction endonuclease sites. Sfa NI normal gives fragments of 417 and 91 bp while Sfa NI nt11778 gives the full length fragment of 508 bp. Mae III normal gives fragments of 233, 218, and 57 bp while Mae III nt11778 gives fragments of 233, 131, 87, and 57 bp.

Lane 1, DNA size markers; lane 2, full length PCR product; lane 3/6, positive control nt11778 DNA; lane 4/7, query LHON patient; lane 5/8, query LHON patient; lanes 3-5, digestion with Sfa NI; lanes 6-8, digestion with Mae III. Arrowheads indicate either loss of Sfa NI or gain of Mae III sites.

In addition, it is possible to directly sequence the PCR product generated from the mitochondrial DNA of the patient. In the example depicted below, the G > A transistion mutation at mitochondrial nt11778 is clearly seen in an affected LHON patient.

DETECTION OF THE nt3460 (ND1) AND nt14484 (ND6) MUTATIONS

The nt3460 mutation causes loss of a Bsa HI restriction endonuclease site. Bsa HI normal yields fragments of 143 and 102 bp while Bsa HI nt3460 yields the full length fragment of 245 bp.

The nt14484 mutation causes loss of a Sau 3AI restriction endonuclease site. Sau 3AI normal yields fragments of 102 and 22 bp while Sau 3AI nt14484 yields the full length fragment of 124 bp.

Lane 1, DNA size markers; lane 2-5, nt3460 assay; lane 2 full length PCR product; lane 3, control DNA; lane 4, nt 3460 LHON patient; lane 5, query LHON patient; lanes 6-8, nt14484 assay; lane 6, full length PCR product; lane 7, normal DNA; lane 8, query LHON patient. The arrowhead in lane 4 indicates loss of Bsa HI site and heteroplasmy while arrowhead in lane 8 indicates the position of the full-length PCR product in nt14484 assay.

DETECTION OF THE nt15257 (CYB) MUTATION

The nt15257 mutation causes loss of an Acc I restriction endonuclease site. Acc I normal yields fragments of 163 and 97 bp while Acc I nt15257 yields the full length fragment of 250 bp. Lane 1, DNA size markers; lane 2, negative DNA control; lane 3, undigested full length PCR product; lanes 4 - 8, PCR products from query LHON patients digested with Acc I. The arrow indicates the position of the full length undigested product and shows that the patient in lane 6 carries the LHON nt15257 mutation (confirmed by direct DNA sequencing of the PCR product - see below). There is no evidence for heteroplasmy.

In addition, it is possible to directly sequence the PCR product generated from the mitochondrial DNA of the patient. In the example depicted below, the G > A transistion mutation at mitochondrial nt115257 is clearly seen in an affected LHON patient.