Download Genes and regulatory sites in human mitochondrial DNA

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
• In standard ophthalmic exam, normal vision is
20/20
Human Eye Anatomy
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
Fig 16.2b: Pedigree showing maternal
inheritance of mitochondrial DNA
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
Mitochondrial mutations cause disease Phenotypes
Severity of
disease
depends on
percentage of
mutant
mitochondria
Note variation in
tissues affected
– those needing
lots of ATP are
more sensitive
Mitochondria & Disease
• Homoplasmic = one type of organelle genome in cell
• Heteroplasmic = mixture of organelle genomes
• Severity of disease depends on proportion 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
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
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
Summary of Study
• Investigated a series of patients with subacute 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
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
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
Table 1
• Titled “Clinical Phenotypes of Leber Hereditary
Optic Neuropathy Patients”
• Lists the clinical manifestations of the LHONpositive individuals in the study
• All patients are male
• Fundus is the back of the eye
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
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)
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
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>
•
•
•
•
•
•
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
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
Results
• 30 nucleotide variations in the 10 LHON
patients
• 30% were nonsynonymous
• 29 nucleotide changes in 20 controls
• 17.24% were nonsynonymous
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
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
• Nonsynonymous mtDNA variations may adversely affect the respiratory
chain:
•
•
•
impair the oxidative phosphorylation pathway
Less ATP
more oxidative stress
• damages both nuclear and mtDNA
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
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
Future Research
• Looking at incomplete penetrance
• Complete penetrance = when clinical symptoms are
present in all individuals who have the diseasecausing mutation
• Incomplete penetrance = clinical symptoms are not
always present in individuals who have the diseasecausing 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
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, Mukesh Tanwar, Rohit Saxena, Pradeep Sharma, and Rima Dada.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