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LHON

LHON. Caroline Miller Meseret Hailu. Human Eye Background. Main role: to focus light The optic nerve is a bundle of more than 1 million axons (from ganglion cells) Papillomacular bundle nerve fibers are axons of ganglion cells, enter the temporal portion of the optic disc

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LHON

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  1. LHON Caroline Miller Meseret Hailu

  2. Human Eye Background • Main role: to focus light • The optic nerve is a bundle of more than 1 million axons (from ganglion cells) • Papillomacular bundle nerve fibers are axons of ganglion cells, enter the temporal portion of the optic disc • In standard ophthalmic exam, normal vision is 20/20

  3. Human Eye Anatomy

  4. Mitochondria • Structure of mitochondrion: inner and outer • Site of oxidative phosphorylation • Human mitochondria is maternally inherited • All progeny have mother's mitochondrial genotype and phenotype • Do not follow laws of Mendelian segregation • Mt genome in matrix varies amongst species, between 6 kb to 186 kb • human 16.5, yeast 75 kb

  5. Fig 16.2b: Pedigree showing maternal inheritance of mitochondrial DNA

  6. Genes and regulatory sites in human mitochondrial DNA Human mt DNA is 16.5 kb: Mutations can cause disease phenotypes Fig 16.4: Genes in human mitochondrial DNA

  7. Mitochondrial mutations cause disease Phenotypes

  8. Severity of disease depends on percentage of mutant mitochondriaNote variation in tissues affected – those needing lots of ATP are more sensitive

  9. Mitochondria & Disease • Homoplasmic = one type of organelle genome in cell • Heteroplasmic = mixture of organelle genomes • Severity of disease depends onproportion of normal to diseased mitochondria • Mitochondria are not evenly partitioned at mitosis. • Lethal condition if all mitochondria are mutant • mixture of types allows cell/organism viability

  10. LHON Definition • Leber hereditary optic neuropathy • Named after Theodore Leber, who published a well-known paper about it in 1871 • Maternally inherited disease, about 15% of carriers are heteroplasmic • Leads to acute bilateral blindness due to loss of the optic nerve and papillomacular bundle nerve fibers in young men • Prevalence 1:50,000 • Symptoms begin in young adults, mean age of onset 18-35 • Other symptoms include: cardiac arrhythmias, neurologic abnormalities, postural tremor, peripheral neuropathy, nonspecific myopathy and movement disorder

  11. Current Treatment • No proven treatment • Best technique is prevention, especially minimizing use of potential triggers like tobacco and alcohol • Balanced diet rich in antioxidants (including Vitamins A, C, and E, and Zinc) might help • Diet supplements like Co-enzyme Q10 (Ubiquinone) and have also been considered

  12. Summary of Study • Investigated a series of patients with sub-acute visual failure and who were suspected of having LHON. • Goal: to screen LHON cases for mt-DNA sequence variations by PCR-DNA sequencing • Done in New Delhi, India

  13. Methods • Sample Collection and DNA isolation • from peripheral blood cells (white blood cells) • PCR amplification and sequence analysis of the mitochondrial DNA coding region • sequenced forward and reverse to confirmation of any nucleotide variation • variants compared to the human mitochondrial reference sequence • D-loop was not sequenced. • D-loop is hypervariable region of mitochondrial genome

  14. Methods (continued) • Computational assessment of missense mutations using 2 techniques: • Polyphen: looks to see whether the amino acid change is likely to be deleterious to protein function • SIFT: predicts whether an amino acid substitution in a protein will have a phenotypic effect

  15. Table 1 • Titled “Clinical Phenotypes of Leber Hereditary Optic Neuropathy Patients” • Lists the clinical manifestations of the LHON-positive individuals in the study • All patients are male • Fundus is the back of the eye

  16. Table 2 • Titled “Primers Used for Polymerase Chain Reaction Used for Amplification of Mitochondrial Genome.” • Total of 24 pairs of primers were used • PCR was used to amplify mitochondrial genome • 30 cycles of amplification • Amplified products purified using a gel and PCR DNA fragments extraction kit • Purified PCR products were sent off for sequencing

  17. Table 3 • Titled “Mitochondrial DNA Sequence Changes in Leber Hereditary Optic Neuropathy Patients • * = primary LHON mutation, only mutation observed in patient, different from an mtDNA polymorphism (2 or more alleles at the same locus) • SYN = synonymous codons, does not lead to a change in the amino acid sequence • NS = Non synonymous codons, lead to dysfunction and consequently reduced respiration, leads to a change in the amino acid sequence • NA = not applicable • Transition = a mutation in which a purine/pyrimidine base pair is replaced with a base pair in the same purine/pyrimidine relationship (A:T>G:C or C:G>T:A) • Transversion = a mutation in which a purine/pyrimidine replaces a pyrimidine/purine base pair or vice versa (G:C>T:A or C:G, or A:T>T:A or C:G)

  18. Table 4 • Titled “Mitochondrial DNA Sequence Changes in Controls” • Controls were 20 ethnically/age matched males • without any history of ocular disorders • mean age 23.85 • Base substitutions in control individuals does not alter protein products. • Subunits are in reference to the various components of NADH dehydrogenase

  19. NADH dehydrogenase • Also known as Complex 1 • Enzyme located in the inner mitochondrial membrane • Catalyzes transfer of electrons from NADH to coenzymeQ (CoQ) • Considered the entry enzyme of oxidative phosphorylation in mitochondria Structure of NADH dehydrogenase, Image from <en.wikipedia.org>

  20. Complex I, II, III, IV and V shown in the mitochondria • Complex I is NADH dehydrogenase • Complex II is succinate dehydrogenase • Complex III is cytochrome b • Complex IV cytochrome c oxidase • Complex V is ATP synthase

  21. Table 5 • Titled “Patient and Gene Wise Distribution of Mitochondrial DNA Variations” • Highest ro. of variations (6) were found in: • NADH dehydrogenase subunit 2 • NADH dehydrogenase subunit 5 • Cytochrome B • Least no. of variations (1) were found in: • NADH dehydrogenase subunit 1 • NADH dehydrogenase subunit 4

  22. Results • 30 nucleotide variations in the 10 LHON patients • 30% were nonsynonymous • 29 nucleotide changes in 20 controls • 17.24% were nonsynonymous

  23. Results (continued) • Highest number of changes were present in complex I genes followed by complex 4 then complex 3 then complex 5. • Of the nonsynonymous changes, 4 were pathogenic changes according to the PolyPhen and SIFT • Four patients were positive for at least one of these pathologic mtDNA nucleotide changes, but none of the controls harbored any pathogenic nucleotide change

  24. Conclusions • Mutations in complex 1 genes account for 50-90% of LHON pedigrees in different ethnic pedigrees • In this study 46.66% of variations were reported in complex 1 • 3 novel changes • 3 pathogenic mutations • 5 novel mtDNA variations were identified • one was pathogenic • NonsynonymousmtDNA variations may adversely affect the respiratory chain: • impair the oxidative phosphorylation pathway • Less ATP • more oxidative stress • damages both nuclear and mtDNA

  25. Key Idea: Optic Nerve • LHON preferentially affects the optic nerve • Neurons need lots of energy so they depend on mitochondria • Damage to mitochondrial ETC  free radicals and less ATP production • Oxidative stress (OS)  oxidative damage of cellular macromolecules, like mtDNA • OS can in turn lead to apoptosis in affected tissue

  26. Study Limitations • Small number of LHON patients of North Indian ethnic origin • Needs to be confirmed in other populations • Diagnosis of exclusion • All other causes were ruled out • Relatively thorough, but still leaves room for error

  27. Future Research • Looking at incomplete penetrance • Complete penetrance = when clinical symptoms are present in all individuals who have the disease-causing mutation • Incomplete penetrance = clinical symptoms are not always present in individuals who have the disease-causing mutation • Study mentioned that only 50% of males and 10% of females who have 1 of the 3 primary mutations ever develop LHON • Indicates that other genetic and/or environmental factors induce phenotypic expression of LHON

  28. Sources • Betz, Joan. Mitochondria Presentation. BL 414 Genetics, Regis University. 2010 • Eye Anatomy and Function. <http://faculty.washington.edu>. • Hartl and Jones. Genetics. • Image. http://www.mastereyeassociates.com. • Kumar, Manoj, MukeshTanwar, RohitSaxena, Pradeep Sharma, and RimaDada.Identification of novel mitochondrial mutations in Leber’s hereditary optic neuropathy. Molecular Vision 2010; 16: 782-792. • Leeder, Jim. LHON Treatment. “http://jim.leeder.users.btopenworld.com>. • LHON Treatment. http://jim.leeder.users.btopenworld.com • Orssaud, Christophe. “Leber’s Hereditary Optic Neuropathy.” <http://www.orpha.net>. • NADH Dehydrogenase. <http://en.wikipedia.org/wiki/NADH_dehydrogenase> • Penetrance. http://ghr.nlm.nih.gov/glossary=penetrance • Riordan-Eva, P. and A.E. Harding. Leber's hereditary optic neuropathy: the clinical relevance of different mitochondrial DNA mutations. J Med Genet 1995;32:81-87. • Standard Ophthalmic Exam. Medline Plus. • http://en.wikipedia.org/wiki/File:ETC.PNG

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