Mitochondrial Disorders
Mitochondria – responsible for cellular respiration (the O2-requiring stage of
energy production), which takes place via oxidative phosphorylation.
 Two membranes – outer (MOM) and inner (MIM).
o Between membranes = intermembrane space.
o Inside MIM = matrix.
 Oxidative degradation of foodstuffs results in production of simple energy
carriers, like NADH and FADH2, which are further metabolized to produce the
energy for normal cellular functions.
 ATP is synthesized via oxidative phosphorylation of ADP in the inner
membrane of the mitochondrion.
o OxPhos components (5 multi-protein complexes) are embedded in the
inner membrane (MIM).
o NADH and FADH2 are oxidized by Complexes I and II, then CoQ10
transports electrons (e-) to Complex III.
o Complex III transports e- to cytochrome c, which then transfers them
to Complex IV where they react with O2.
o Proton gradient forms (negative potential inside MIM due to
movement of protons into IMS).
o Complex V uses energy stored in gradient (proton-motive force) to
generate ATP from ADP.
Mitochondrial Genome – circular, double-stranded chromosome (mtDNA) that is
replicated, transcribed and translated within the mitochondrion.
 Unique Characteristics:
o Uses its own genetic code.
o Synthesizes 12S and 16S rRNA.
o Exhibits continuous transcription of multiple genes (some polycistronic mRNAs).
o Complete lack of introns.
o Reminiscent of prokaryotic genomes.
 Mitochondrial genome encodes for:
o 13 polypeptides (in 4 oxphos complexes).
o 2 ribosomal RNAs.
o 22 transfer RNAs.
o **The remainder of proteins that exist in mitochondria (~99% in
humans) are transcribed in the nucleus, translated in the cytoplasm
and transported into mitochondria.
 Ploidy of mitochondrial genome:
o 2-10 copies of mtDNA per mitochondrion.
o Therefore, each cell contains hundreds to thousands of copies of the
mitochondrial genome (useful in forensic and anthropologic
investigations).
Characteristics of mtDNA Affecting Phenotypic Expression
1. mtDNA has a very high mutation rate (7-10 fold higher than nuclear DNA).
a. Thus, the mitochondrial genotype can change within a single
generation through accumulation of somatic mutations.
b. Consequences of mitochondrial mutation rate/accumulation:
i. A study was done in which the proof-reading enzyme for
mtDNA (mtDNA pol ɣ) was mutated in mice via
recombination.
ii. Mice homozygous for the mutation in mtDNA pol ɣ exhibited
progeria-like symptoms (rapid aging, impaired survival).
iii. Phenotype was rescued slightly in mutant mice given
endurance training (treadmill running).
1. Prevented mitochondrial depletion/mutations and
restored mitochondrial morphology, blunting
pathological apoptosis in multiple tissues.
2. Prevented premature mortality and reduced pathology
in multiple organs.
2. Both normal (WT) and mutant mtDNA can co-exist within the same cell:
a. Homoplasmy – one type of mtDNA per cell.
b. Heteroplasmy – multiple types of mtDNA within a cell.
i. In normal individuals, ~99.9% of mtDNA molecules are
identical.
3. Different tissues can contain different proportions of mutant and normal
mtDNA.
a. mtDNA replicates autonomously from nuclear DNA.
b. Replicative Segregation – mitochondria segregate into daughter
cells independently of nuclear DNA.
i. Leads to different proportions of mutant and normal mtDNA in
different tissues.
4. Tissues differ in dependence upon oxidative phosphorylation:
a. Heart, skeletal muscle, brain, and CNS are heavily dependent upon
oxphos.
b. Other tissues that depend heavily on oxphos include the liver, kidney
and insulin-forming islets of the pancreas.
c. **Mitochondrial disorders show their clinical manifestations mainly
in tissues that are heavily dependent on oxphos.
i. Often characterized as myopathies and encephalopathies.
5. Threshold effects in expression of mutant mtDNA --85-90% of
mitochondria in a cell may have to be abnormal to discern an aberrant
phenotype.
 Summary:
o Clinical phenotype in mitochondrial disease is not simply (or directly)
related to mtDNA genotype, rather, it depends on:
 Inherent oxphos capacity, determined by BOTH nuclear and
mitochondrial genes.
 Accumulation of somatic mtDNA mutations.



Degree of heteroplasmy.
Tissue-specific requirements for oxphos.
Age – progressive mtDNA alterations can lead to worsening
conditions with age.
Mitochondrial Diseases
 Maternal Inheritance of mitochondria – non-Mendelian inheritance
pattern exclusively through maternal line.
o Sperm contain few mitochondria, and none survive within fertilized
egg, thus all mitochondria in embryo are maternal in origin (from
oocyte cytoplasm).
o Transmission solely by the mother to BOTH sons and daughters (sex
bias in transmission).
o Equal numbers of males and females are affected (no sex bias in
affected progeny).
o A form of uniparental transmission.
 Diseases caused by:
o Deletions and other structural rearrangements.
 Mitochondrial Myopathy (MM) and Kearns-Sayre (KS)
 Neither size nor position of deletions are well
correlated with either enzyme deficiency or severity of
disease.
 KS is a multi-system disorder characterized by brain
and muscle dysfunction, progressive external
ophthalmoplegia (muscle paralysis), and pigmentary
retinopathy.
o Patients often short of stature, have hearing loss,
mental retardation or dementia, and
endocrinopathy.
 Diabetes and deafness sometimes associated with mtDNA
deletions or duplications in pedigrees showing maternal
inheritance.
o Point mutations in protein-encoding genes and tRNA-encoding
genes.
 Leber’s Hereditary Optic Neuropathy (LHON) is a rare
neurodegenerative disease of young adults that results in
blindness (optic nerve degradation).
 Caused by a point mutation in NADH dehydrogenase
subunit 4.
 Myoclonic Epilepsy with Ragged Red Fiber (MERRF) is
caused by a mutation in tRNALys.
 Mitochondrial Encephalopathy, Lactic Acidosis, and
Stroke-like symptoms (MELAS) is caused by a mutation in
tRNALeu.

Maternal Myopathy and Cardiomyopathy (MMC) is also
caused by a mutation in tRNALeu.
Linkage Analysis
Linkage – two loci on the same chromosome.
 Given two loci that are on the same chromosome in mice (with two alleles for
each locus):
o Determinant of ear color: E – dominant (normal), e- recessive (pale)
o Determinant of eye color: R –dominant (normal), r- recessive (ruby)
 In doubly heterozygous animals (EeRr), in which both dominant alleles are
present on one chromosome and both recessive alleles are on the other, one
might expect only ER and er gametes, but this is not the case.
 Due to crossing over of non-sister chromatids of homologous
chromosomes, all gametes are possible – (ER, Er, eR, er).
o Gametes are classified as either:
 Parental – chromosome with the same genotype as either of
the parental chromosomes (i.e. ER or er).
 Recombinant – chromosome having a different genotype
from either of the parental chromosomes (i.e. eR or Er).
 **There will not always be a crossover event between any two loci on
homologous chromosomes.
 The physical distance between two loci on the same chromosome is roughly
proportional to the probability of a crossover event occurring between two
loci.
o Thus, the proportion of crossover events (and therefore the
proportion of recombinant gametes) is a function of the distance
between these two loci on a chromosome.
o The frequency of observed recombinants is used as a measure of this
distance.
o Distances described in “genetic map units.”
 Map units (m.u.) cannot be directly converted into a physical
distance, but they are roughly equivalent to 1-2 x 106 bp.
 One m.u. is the distance between gene pairs for which 1
product out of every 100 is recombinant.
 A recombinant frequency (RF or �) of 0.01 (i.e. 1%) is
defined as 1 m.u.
 A map unit is also commonly referred to as a centiMorgan
(cM).
Example Backcross –Double heterozygote mouse with a double homozygote
 Recombinant genotypes of offspring arise from crossing over during meiosis
in the EeRr parent.
 Crossing over occurs in eerr parent, but it produces no change in gametes.
Linkage Analysis and Human Disease
 Linkage of a gene and a particular trait begins with studying the segregation
of the disease in large families using polymorphic markers.
 For some diseases, presence of the aberrant phenotype is very closely
associated with the inheritance of particular alleles for a locus or set of loci
(haplotype).
o These loci are said to be in linkage disequilibrium.
o Such associations can be used in populations (instead of families) to
identify candidate genes (genome-wide association studies).
In this pedigree, we
also see that
individual II-3 has a
recombinant
chromosome.
This also indicates
that the disease
gene is NOT
associated with the
A Locus.
The haplotype 2,3,3
for loci B,C,D is
associated with the
disease.
**The polymorphic alleles result from the presence or absence of a BamHI site. 2.0
or 2.7 kb bands are observed after hypridization with a specific DNA probe.
This figure illustrates how crossing over affects the presence of different alleles in
subsequent generations.
Haplotypes and Linkage
 Haplotype – two or more linked loci, considered as a set.
 Recombination fraction (�) = ratio of recombinant to total haplotypes
o � = Recombinant / (Recombinant + Parental)
o Range between 0 (complete linkage, thus no recombination), to 0.5
(unlinked – either on separate chromosomes or far apart on same
chromosome).
o If � = 0.01 = 1 recombinant / 100 gametes = 1 m.u. = 1 cM
Lod Scores
 Linkage analysis is conducted by examination of likelihood (odds) ratios.
o Ratios are studied for � ranging from 0-0.5.
o Geneticists prefer working with smaller numbers, so odds ratios are
transformed by taking their logarithms (base 10).
o Log10 of an odds ratio is called a Lod Score (“log of odds”).
 Lod Scores are calculated as:
o Log10 (likelihood of observed data at specified � / likelihood of
observed data at � =0.5)
o In other words, log10 (likelihood of linkage / likelihood of no
linkage)
 Use of Lod scores:
o Lod scores for a particular trait from different families can be
combined, which is useful in situations in which finding large families
with affected individuals is problematic.
 Maximum likelihood method – the highest Lod score is considered the
best estimate of �.
o Lod > +3.0 = linkage
o Lod < -2.0 = no linkage
It is important to remember that high
Lod scores within families indicate
linkage, but not all families will exhibit
similar linkages.
The graph shows completely different
interactions between loci in different
families.
Heterogeneity between families is
due to random recombination events
that vary from family to family.
When calculating the Lod score for this pedigree, the specified � was 0.05. In each
individual of generation III, the association of a disease phenotype with inheritance
of allele B and a regular phenotype with inheritance of A would exhibit a likelihood
of 1-0.05 = 0.95. At the given � , the Lod score is inconclusive for linkage (<+3.0).
In this example, a larger family exhibiting complete concordance (as in first
example), results in a higher Lod score simply due to a greater amount of data.
Lod Score Example Showing Incomplete Concordance (Presence of
Recombinant Progeny)
In this example, the presence of a recombinant son decreased the Lod score to an
insignificant score.
Genetic Mapping and Quantitative Trait Loci (QTLs)
 A QTL is a chromosomal region containing a gene (or genes) that affect the
phenotypic expression level of a quantitative trait (i.e. blood pressure,
height, intelligence).
 Using genetic mapping, QTLs can be associated (correlated) with specific
phenotypes when they reach a significant Lod threshold:
Genome-Wide Association Studies (GWAS)
 Used to associate loci with disease trait in populations (instead of families).
 Take advantage of linkage disequilibrium.
 Can be used in case-control (i.e. patients with or without disease), or cohort
studies.
 The intention is to identify candidate genes/loci, but to date, rarely identify
causative variant.
 Loci identified usually have modest effects.