BIMM 110 - LECTURES 24-25

MITOCHONDRIAL DISEASES

Textbook:   Strachan and Read, Chapter

Slides 1
Slides 2

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I. INTRODUCTION

• when we speak of genetic diseases we invariably think of heritable diseases
• we think of alterations in genes causing proteins to be defective or missing
• unifactorial diseases like cystic fibrosis
• multifactorial diseases like manic depression
• finally, we have Mendel's laws to interpret pedigrees and classify such genetic diseases as recessive or dominant, autosomal or X-linked, etc.

1. a large heterogeneous group of neurodegenerative diseases and neuro-muscular pathologies with age-dependent onset have come into focus during the past decade
2. a genetic basis for these highly variable symptoms was at first difficult to discern
3. mutations on mtDNA have come sharply into focus, but a whole new set of questions have been raised that constitute a fascinating challenge

1. The mitochondrial genome in humans

the 25 th chromosome

• hundreds to thousands of copies per somatic cell (hundreds of mitochondria)
• ~1/2% of the DNA in a mammalian cell is mitochondrial DNA
• it is a circular genome
• genome size: 16.5 kb; more or less the same size in all vertebrates
• completely sequenced in early 1980's;

2. information content of the mt genome

in vertebrates/mammals it codes for 2 ribosomal RNAs, 22 transfer RNAs, 13 proteins:

• 7 subunits of complex I (NADH dehydrogenase)
• 3 subunits of complex IV (cytochrome oxidase)
• 2 subunits of complex V (ATP synthase)
• 1 subunit of complex III (ubiquinol-cytochrome c oxidoreductase)

- peptides made for complexes I (7), III (1), IV (3) , and V (2) are located on the inner mitochondrial membrane (integral membrane proteins with multiple transmembrane segments)

Protein Synthesis in Mitochondria

- codon usage
- unusual initiation mechanism
- no in vitro system for mitochondrial protein synthesis available

Protein Import and Mitochondrial Biogenesis

- covered in Cell Biology

II. MATERNAL INHERITANCE OF MITOCHONDRIAL DNA

1. RFLPs in mtDNAs are maternally inherited

• formal proof for humans achieved in the early 1980s
• each of us has mtDNA identical to that of our mother

there is no recombination; a formal proof that the enzymatic machinery for recombination is absent in mitochondria is still missing

An example of non-mendelian inheritance

2. Recent Challenges to a strictly maternal inheritance:

• Interspecies vs intra-species crosses of mice
• Oocyte mitochondria vs sperm mitochondria; relative number of mtDNAs in zygote
• Distinction by (surface ?) epitope and targeted destruction of paternal mitochondria

3. Examples of some interesting applications:

a) The analysis of the skeletons of the Russian Tsarist family and comparison with living relatives

b) the search by the "Grandmothers" in Argentina for their grandchildren whose parents were among the "Disappeared" during the military dictatorship 1975-1983 (??)
Marie Claire King from UC Berkeley did the analysis and testified in the courts

3. Applications in Anthropology and Human evolution

• IF there is strictly maternal inheritance: there will also be no genetic recombination, at least not between paternal and maternal mtDNA
• great simplification in the construction of a phylogenetic tree
• general introduction to the construction of a phylogenetic tree

Example 1: Higher primate evolution (Gagneux et al, 1998)

Example 2: Human evolution and migrations in the present distribution of humans on earth

• many interesting applications in the study of human evolution and anthropology make use of distinct mitochondrial DNA sequences found in various population groups
• the time scale of the rate of changes in mtDNA sequences is of the right order of magnitude to be useful in the study of human evolution during the past few 100,000 years, and even useful for studying more recent events such as the population of the North American continent from one or more migrations across the Bering Strait
• the mutation rate is estimated to be 10 times greater than that of the nuclear genome:
  • lack of proof-reading by DNA polymerase?
  • no elaborate mismatch repair system
  • subject to constant attack by reactive oxygen species generated by the electron transport chain

from a practical point of view:

• very little tissue is required: blood, feces, some hairs pulled out (e.g. in nests of gorillas)
• PCR-based amplification of D-loop (control region); ~ 1kb
  no funtional genes encoded, only some very small sequences for DNA replication origins and transcriptional starts, etc.
• high frequency of sequence polymorphisms, even among present day humans

Example 3: Analysis of Neanderthal mtDNA – an example of an application in anthropology/human evolution

3. Sequence polymorphisms and homoplasmy - a dilemma?

• with so much variation in the mt genome one might expect all of us to harbor a whole host of slightly different mtDNA molecules
  this is not the case: we all are generally homoplasmic, i.e. ~ 100% of our mt DNA molecules have the identical sequence
• a very interesting and challenging problem for a population geneticist:
• each one of us can be uniquely identified by our mtDNA:
  • on a time scale of just a few generations there is no significant change (hence applications to forensics, etc)
  • however, on a larger evolutionary time scale there are a lot of changes in the mitochondrial DNA sequence
  • are we truly homoplasmic?
• accumulation of mutations in somatic cells over a life-time (relationship to aging?)

4. Mutations in mtDNA

• mutations vs sequence polymorphisms

• mutants in mtDNA have been known for decades in microorganisms such as yeast ---> respiration-deficient yeast cells
• the first "cytoplasmic" mutations in mammalian cellswere identified in mammalian cells in the 1970s
  chloramphenicol and oligomycin resistant CHO cells in tissue culture:
  shown to be due to mutations in the mitochondrial genome
• respiration deficient mammalian cells in culture (nuclear mutations)
• respiration-deficient people????

5. Assorted myopathies and neuropathies observed clinically in recent years:

• neurologic disorders
• ragged red fibers in muscle biopsies
• weakness exacerbated by exercise
• seizures
• (muscle jumps)
• dementia with early onsetmovement disorders
• stroke-like symptoms
• retinopathy
• hearing loss
• partial blindness
• migraine headaches

general feature:

broad spectrum of symptoms   variable in severity    delayed onset with age

genetic basis?
yes, in many cases, but the pattern of inheritance is distinctly nonmendelian, i.e. there is maternal inheritance, or maternal transmission, but not in all cases

III. SEGREGATION OF MITOCHONDRIAL ALLELES

1. The Problem

• it should be no surprise that there will be mutations in mtDNA
• if there are thousands of copies of the wild type genome, how can a single mutation in one such mtDNA ever have a consequence?
• a single recessive mutation should not cause any change in phenotype
• the problem of ploidy and expression of the phenotype:
• if affected individuals in a pedigree are examined, they are often found to be heteroplasmic:
  there is a mixed population of wild type and mutant mtDNAs
• among offspring from a particular mother (who is normal, but has 10-30% mutant mitochondrial DNA) there are again wide variations in the fraction of mutated mtDNA,
• the more mutated mitochondrial DNA, the more severe the syndromes, BUT,
• the above generalization has to be qualified, because it depends to some extend on the particular function/gene which is mutated
• if a protein is completely inactivated by the mutation, heteroplasmy provides normal genes and a fraction of normal proteins: the person may be sick, but he/she is alive
• if a particular tRNA is altered, one may have close to 100% mutant mtDNA, if the tRNA still can function at lower efficiency

• how do such dramatic fluctuations in mitochondrial DNA populations come about?

2. Mitochondrial DNA and oogenesis

a) the current thought is that there is a "bottleneck"

• only a very small number of mtDNAs are actually involved in the transfer of this genetic information from one generation to the next
• a stochastic (sampling) mechanism operates on mtDNA in the female
• where and how?
• a human oocyte has approximately 100,000 mtDNAs (not a bottleneck!); there once was even a thought that the oocyte has no mtDNA, and that a few mitochondria were introduced by the sperm as a major mechanism of fertilization

b) recent experiments with mice:

• - fuse two zygotes with different mtDNA RFLPs; one zygote is enucleated prior to fusion
• - zygote with heteroplasmic mitochondria is re-implanted into mouse and brought to term
• - female mice with known heteroplasmy
• - analyze their primary and secondary oocytes and mature eggs as well as offspring (early embryos)
• - results: whatever variation has occurred has already occurred in the oocytes
• - a stochastic change in the ratio of mtDNA species appears to occur in the time interval when a population of oogonia goes through a number of rounds of mitotic divisions before they differentiate into oocytes.
• such oogonia are estimated to have only about 200 mtDNAs.

mtDNA replication is relaxed, i.e. a single mtDNA molecule can replicate many times while others do not replicate at all

the above relaxed replication and random partitioning to daughter cells can account for the observed genetic drift

c) another very plausible explanation:

• - a zygote starts with ~ 100,000 mtDNAs
• - there is not mtDNA replication during the first 10-12 zygotic divisions
• - existing mtDNA is diluted to 10 - 100 copies per cell, including future germ cells
• - mtDNA replication start at a later stage in early development

IV. CLINICAL EXAMPLES

A. FAMILIAL MITOCHONDRIAL ENCEPHALOMYOPATHY (MERRF)

Original paper: Shoffner et al. Cell 61:931-937 (1991)

• myoclonic epilepsy with ragged-red muscle fibers
• rare disease of central nervous system and skeletal muscle
• large pedigree discovered, where numerous family members related through the maternal lineage display manifestations of the disease

a. maternal inheritance established

all forms of Mendelian inheritance could be excluded on probabilistic grounds

b. OXPHOS deficiency

• anaerobic threshold: exercise stress testing
• ³¹P-NMR (phosphocreatine/Pi ratios measured during exercise and recovery
• biochemical characterization: deficiency in complexes I and IV, but the basis for the deficiency in complex IV remains unclear, since cytochromes a + a₃ are normal
  

c. Variable OXPHOS deficiency

• the anaerobic thresholds analyzed in 9 family members formed a continuous distribution across the maternal lineage and were directly proportional to the severity of the symptoms
• MERRF is the product of a heteroplasmic mtDNA mutation that undergoes replicative segregation along the maternal lineage

d. Threshold expression

• toxicological studies had indicated that when there is a problem with mitochondrial energy production, individual tissues will be affected differentially: CNS, type I skeletal muscle fibers, heart, kidney, liver (from most to less sensitive)

• detailed clinical evaluation of all patients: CNS electrophysiological aberrations were the most sensitive manifestation of OXPHOS deficiency:
• all members had abnormal VER, EEG
• most had RRF and abnormal mitochondria in type I muscle fibers

• in MERRF the variable symptoms of maternal relatives are the result of variation in the proportion of mutant mtDNA/cell

e. Analysis of mtDNA

no major deletions or RFLPs found, therefore probably a deleterious point mutation

tRNAlys : A-->G in T/C loop

Summary

1. maternal inheritance
2. defects in OXPHOS
3. variable expression of phenotype along maternal lineage
4. different tissues should be affected to varying degrees

B. LEBER'S HEREDITARY OPTIC NEUROPATHY (LHON)

a) Symptoms:

• rapid bilateral loss of central vision caused by neuroretinal degeneration (first described in 1871)
• median age of onset: 20-24 years
• cardiac dysrhythmias also frequent

b) Maternal inheritance

• first described by an Australian clinician, David Wallace, in a large family in Queensland: exposure to an infectious agent in utero???

c) mtDNA sequencing

• in Doug Wallace's lab at Emory
• sequenced 80% (of 16569 bp) of mtDNA from patient from African-American family in Georgia (with several close relatives)
• found 25 mutations by comparison with the human mtDNA sequence first published in 1981 by S. Anderson and colleagues in Cambridge!

d) The 11778 mutation changes an arg to a his at amino acid 340 of subunit 4 (ND4) in complex I. Is this the mutation responsible for LHON?

• from fungi to fruit flies to humans that site codes for arg, - except in Leber's patients
• the mutation can also be recognized by restriction fragment analysis: the normal mtDNA is cut by Sfa NI, mutant mtDNA is not cut; this makes analysis of the Leber's patients much easier: mtDNA from 10 independent normal families did not have the mutation
• the mutation has occurred at least twice in the human population: there is a European pedigree of Leber's patients and a pedigree of a black Georgia family; 7 replacement mutations and 15 synonymous mutations separate the mtDNA of the Georgia patient from those of the European patients;

• use of parsimony computer program PAUP: two pedigrees can be constructed:

e) Recent results

• Independent evidence has been obtained that in all Leber's patients the activity of complex I is reduced

• >50% of LHON patients have mutation at nt 11778 (ND4 gene of complex I)
• two mutations in ND1 gene at nt 3460 and at nt 4160
• one mutation at nt 15257 in cytochrome b
• most recently: two mutations in COI gene of complex IV
• the most severe LHON mutation is a ALA => VAL substitution in the ND6 gene; individuals also suffer from dystonia (movement disorder involving progressive rigidity associated with basal ganglia degeneration or bilateral striatal necrosis)

QUESTIONS:

1. why is the optic nerve specifically affected?
2. why does it take so long for blindness to develop?
3. the most puzzling aspect: EVERY MEMBER OF EACH LHON LINEAGE WAS FOUND TO BE HOMOPLASMIC!
4. only a portion of maternal relatives are affected even if they are homoplasmic for the mutation: is there also a nuclear gene that influences the expression of the phenotype?

C. MELAS

• mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
• seizures, dementia, recurrent headache
• often associated with point mutations in the tRNAleu at positions 3243 or 3771

• similar to MERRF

D. THE KEARNS-SAYRE SYNDROME

• onset before the age 20
• progressive opthalmoplegia
• pigmentary retinopathy
• complete heart block
• cerebellar ataxia

mtDNA has deletions of 2-7 kb

• always heteroplasmic in a given individual: all deleted mtDNAs have the same deletion and represent 45-75% of the total population

• diseases associated with deleted mtDNAs are mostly sporadic; these mitochondrial deletions are thought to arise in early embryogenesis
• no maternal inheritance
• the proportion of deleted mtDNAs varies greatly from tissue to tissue, and may be close to zero in cells such as those found in blood

V. THE STUDY OF MITOCHONDRIAL MUTATIONS IN TISSUE CULTURE

1. Formation of cybrids from fusion of r^o cells (mtDNA less) and cytoplasts

2. Nuclear mutations causing respiration deficiency

a) in tissue culture
b) in cells from human patients with mitochondrial mutations due to nuclear mutations

VI. MITOCHONDRIAL DNA AND THE CLONING OF MAMMALS

- current cloning technology: a somatic cell is fused with an enucleated oocyte
- Where does the majority of the mtDNA come from?
- cloning in the service of species conservation: eg. cloning of gaur (ox-like animal in Asia)
- species compatibility of nuclear and mitochondrial DNA (xenomitochondrial cybrids): which subunits encoded by nuclear genes and by mtDNA interact in the complexes of the mitochondrial electron transport chain?

VII. Accumulation of Mitochondrial Mutations and Aging

role in Alzheimer's disease? Parkinsonism ? Huntington's disease ?

 VIII. The Role of Mitochondria in Apoptosis

- bcl2 and related proteins
- cytochrome c release and caspase activation
- the "apoptosis-inducing factor"

- apoptosis and the mitochodrial permeability transition

    Scheffler, I.E. 1999. MITOCHONDRIA. John Wiley & Sons, Inc., New York, 367 pages.

 1. Shoffner, J. M. and D. C. Wallace. 1990. Oxidative phosphorylation diseases: Disorders of two genomes. Adv. Hum. Genet. 19:267-330.
2. Sokol, R. J. 1996. Expanding spectrum of mitochondrial disorders. J. Pediatr. 128:597-599.
3. Stephenson, J. 1996. A role for mitochondria in age-related disorders? Journal of the American Medical Association 275:1531-1532.
4. Wallace, D. C. 1992. Mitochondrial genetics: A paradigm for aging and degenerative diseases. Science 256:628-632.
5. Wallace, D. C. 1994. Mitochondrial DNA sequence variation in human evolution and disease. Proc. Natl. Acad. Sci. USA 91:8739-8746.