Primordial germ cell Primary oocytes Mature oocytes

Abnormal mitochondrion

Normal mitochondrion

Severe disease

Abnormal function

Normal function

Egg ZygoteSperm

MELAS

LHONMERRF

MIT

OCH

ON

DRIA

L DISEASES

MUTATIONS IN MITOCHONDRIAL DNA

Mitochondria play a crucial role in cellular functions. Each eukaryotic cell

holds numerous energy-producing mitochondria that contain their own

mitochondrial genome. The mitochondrial genome contains 37 protein-encoding

genes that affect a host of cellular processes such as apoptosis, adenosine triphosphate (ATP) creation, and insulin secretion. Mutations in mtDNA affect these important processes and cause

multiple genetic diseases.

Mutations of mitochondrial genes disrupt cellular processes and

reduce energy production capabilities.

Mutations of the MT-TK gene are linked to myoclonic epilepsy

with ragged red fibers (MERRF) syndrome, Leigh syndrome, and

Parkinson's disease. Previous names of this gene include

MERRF and MTTK.

Mutations of the MT-ND1, ND2, ND4, ND4L, and ND6 genes

can result in Leber’s hereditary optic neuropathy (LHON), diabetes,

myopathy, and dystonia.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like

episodes (MELAS) is a rare genetic disorder that causes

strokes and dementia. Mutations in several

mitochondrial genes can cause MELAS — MT-ND1,

MT-ND5, MT-TH, MT-TL1, and MT-TV.

How do mtDNA mutations cause disease?

Each aerobic cell contains numerous mitochondria; those cells requiring more energy have more mitochondria. Additionally, the number of mtDNA copies can vary from several to hundreds within each mitochondrion. In contrast to the uniformity found in somatic nDNA, the occurrence of more than one version of a sequence variant within a single cell results in a unique mitochondrial attribute called heteroplasmy, a critical concept in understanding mitochondrial diseases.

When a cell has only a few mitochondria with mutant mtDNA, the cell can function normally. However, if most of the mitochondria contain mutant DNA, then cell function is affected, and abnormal cellular function or severe disease is observed.

The amount of mutant mitochondria can vary throughout a person’s body such that some organs are affected while other organs in the body function normally. All of these factors make diagnosing and treating diseases caused by mitochondrial mutations very difficult.

From conception to detectionUnderstanding mitochondrial DNA — it’s more than just DNA in a circle

Normal Function

Low percentage of abnormal mitochondria

Abnormal Function

Abnormal mitochondriaat a threshold to affect

cell function

Severe Disease

High percentage ofabnormal mitochondria

Abnormal mitochondrionNormal mitochondrion

How are mtDNA mutations inherited?

The egg cell, or ovum, contributes mitochondria during the formation of the zygote — this means that offspring only inherit mtDNA from their mother. While sperm contain mitochondria in their tails, the paternal mitochondria are actively destroyed during embryogenesis.

Affected individual

Normal mitochondrion

Abnormal mitochondrion

How do mitochondrial diseases affect the body?

How are mtDNA mutations identified?

Mitochondrial diseases have diverse and consequential impacts on the human body. Disease may appear in childhood or as an adult, and determining mitochondrial DNA mutations as the cause of disease may be challenging. Understanding disease-related mutations of the mitochondrial genome will hopefully lead to therapeutic remedies that restore mitochondrial function.

Mitochondrial diseases are often hard to diagnose because they can affect each individual very differently. Genome analysis methods, like next-generation sequencing and Sanger sequencing, are often used for mitochondrial disease research, but these methods are often time consuming and costly. Focusing on the mutations associated with MERRF, MELAS, and LHON, Canon BioMedical developed four assays that identify the underlying mutations that cause these diseases.

LIVERLiver disease

ATP

STOMACHLoss of appetite

BLOODLactic acidosis

BRAINSeizures

MyocolonusAtaxiaStroke

DementiaMigraine

HEARTConduction defects

Cardiomyopathy

INNER EARSensorineural

hearing loss

Optic atrophyOptic neuropathy

Vision loss

EYE SKELETAL

MUSCLEWeakness

FatigueMyopathy

Neuropathy

Renal dysfunction

KIDNEY

INTESTINE

Loss ofbowel control

LHONLHON causes the degeneration of retinal ganglion cells and their axons, which leads to an acute or subacute loss of central vision. The disease predominantly affects young, adult males.

MELASMELAS is a progressive, neurodegenerative disorder primarily affecting the nervous and muscular systems. Symptoms include seizures, recurrent migraines, and loss of appetite and bowel control. Stroke-like episodes with temporary muscle weakness on one side of the body may also occur, leading to intellectual disability and loss of sensorineural hearing, vision, and motor skills.

MERRFMERRF is a multisystem disorder characterized by myoclonus, the involuntary jerking of muscles caused by sudden muscle contractions, followed by generalized epilepsy, ataxia, weakness, and dementia. The disorder can also cause hearing loss, optic atrophy, and cardiomyopathy. Symptoms typically appear during childhood or adolescence.

LHON

LHON

MELAS

MERRF

FV 12s

L1

16S

ND1

Q

EI

TP

M

S2L2

H

ND2

WANCYCOXI

COXII COXIII

S1D

KA8

A6G

RND3ND4L

ND4

ND5

ND6

Cyt b

We are here to assist.Please call or email to talk with our technical support staff.

contactus@canon-biomedical.com | www.canon-biomedical.com | 844-CANONBIO

Target Assay catalog number Control set catalog number

MERRF m.8344A>G 40338 40685

MELAS m.3243A>G 40219 40577

LHON m.3460G>A   40339 40686

LHON m.11778G>A 40225 40583

The Novallele™ genotyping assays provide a sensitive, fast, and cost-effective method to genotype mtDNA mutations. Using polymerase chain reaction (PCR) followed by high-resolution melting (HRM) analysis, the Novallele genotyping assays only require an HRM-enabled thermocycler in a very easy procedure. All Novallele genotyping assays are functionally verified.

How does HRM work?

Mitochondrial mutations can be tricky to detect. Save time and get accurate results with the Novallele genotyping assays.

HRM analysis distinguishes DNA variations by analyzing sequence-specific melt curve patterns. Data analysis using melt profiles simplifies experimental design by not requiring a minimum number of samples to distinguish genotypes. With HRM, you can genotype a single sample when using the applicable controls.

The mutant mtDNA detection limit for HRM is just as sensitive as Sanger sequencing. If you want to reduce the costs of your mtDNA research, then the Novallele genotyping assays are the solution.

1200

800

065

Temperature (°C)

-d(R

FU)/

dT

400

200

600

1000

70 75 80

MERRF m.8344A>G Novallele Genotyping Assay

Wild type

Homozygous variant Heterozygous variant

1400

AccurateThoroughly bench

tested for consistent results

Cost effectiveHRM is cheaper per

sample when compared to other methods

FastAnalysis in minutes

Minimal reagents No need for HPLC

solvents or denaturing gradient gel

electrophoresis

SimpleOnly an HRM-enabled

thermocycler is needed

VersatileUseful for a variety of

applications

HRM is quick because there is no need to process or separate PCR products. Once PCR is finished, a fast HRM step is performed, and

analysis is completed using the free, web-based Novallele HRM Analyzer. Using HRM, you can get your genotyping answers with the

following additional benefits:

The MERRF m.8344A>G Novallele Genotyping Assay identifies a single-nucleotide polymorphism

of m.8344A>G. The graph displays the homozygous wild type in black, heterozygous variant in

blue, and homozygous variant in red. The data were generated using the companion Novallele

Control Set.

If testing samples in your own lab seems overwhelming, our scientifically trained service and support team at Canon BioMedical is ready to assist you with the transition. We can help with such endeavors as designing your experiments, setting up your testing equipment, or understanding your data analysis.

For research use only. Not for use in diagnostic procedures.All referenced product names, and other marks, are trademarks of their respective owners. © 2018 Canon BioMedical, Inc. All rights reserved.

Mitochondrial vs Nuclear DNANuclear DNA (nDNA) is found within the cell nucleus and inherited from both parents. In contrast, mitochondrial DNA (mtDNA) is found in cell mitochondria and inherited only from the mother. While both nDNA and mtDNA are double-stranded, mtDNA is organized in a circular, closed structure. A cell usually contains only two copies of nDNA but hundreds of mtDNA copies within numerous mitochondria.

Nucleus

Cell

Mitochondrion

Chromosome Nuclear DNA

Mitochondrial DNA

Genotyping Workflow for Mitochondrial DNA

Prepare Reactions Amplify and Melt DNA Analyze ResultsExtract Mitochondrial DNA

Novallele genotyping assay

Novallele Genotyping Mastermix

Novallele Control Set

HRM-enabled thermocycler Novallele HRM Analyzer

Unlike nDNA, the inheritance of

mitochondrial mutations is not a simple transfer

of one gene. During ovum creation, mitochondria

are randomly sorted such that offspring can vary in

disease presence and severity.

As shown in the graphic, a mother’s primordial germ cell can result in mature oocytes with

varying degrees of mutant mitochondria.

As all mitochondria are derived from the ovum, a mother carrying abnormal mitochondria will pass the abnormal mitochondria to all of her children, but only her female offspring can transmit the abnormal mitochondria to further generations. Affected males do not pass on abnormal mitochondria.