MitoDx™ Complete Mitochondrial Genome Sequencing with Deep Heteroplasmy Detection

MitoActionSeptember, 2010

Steve S. Sommer, MD, PhD, FACMGPresident and Laboratory DirectorMEDomics, LLCwww.medomics.com

Steve S. Sommer, MD, PhD, FACMG

• Founding Fellow, American College of Molecular Genetics

• Double Clinical Certifications• 20+ years of experience in DNA diagnostics• Geneticist Professor in the field of DNA

diagnosis with 300+ publications• Started MEDomics in 2008 to apply “NextGen

sequencing” to clinical diagnosis

Mito Minute

• The emerging consensus is that the frequency of mitochondrial diseases in children is 1 in 500… implying that every minute, there is a baby born somewhere in the world who will develop a mitochondrial disease by age 10.

Mitochondrial Diseases

• The mitochondria contain DNA with some of the genes needed for mitochondrial function.

• Other genes needed for mitochondrial function are found on chromosomes in the nucleus of the cell.

MitoDx

• NextGen sequencing of the entire mitochondrial DNA genome thousands of times.

• In some cases, allows the diagnosis of mitochondrial diseases to be made with a blood sample rather than with painful muscle biopsies.

• A test of unprecedented power for diagnosing mitochondrial disease.

NextGen Sequencing

• Also known as massively parallel sequencing

• A revolutionary tool will drive a revolution in personalized medicine

Homoplasmy vs. Heteroplasmy(Big Picture for The Genome)

• Homoplasmy▫ There is only one type of mitochondrial DNA in an

individual.

• Heteroplasmy▫ Two or more types of mitochondrial DNA in an

individual.One type may be normal; the other type may have a mutation.

The percentage of the abnormal mitochondrial DNA in a particular tissue determines whether the individual is affected or not, with mild or severe disease.

Homoplasmy vs. Heteroplasmy(Picture at Each Nucleotide)

• When more than 99% (and often 99.9%) of the nucleotides at a site in the mitochondrial genome are of one kind, that nucleotide site is called “homoplasmic”.

• When less than 99% (and sometimes 1%) of the nucleotides at a site in the genome are of one kind, that nucleotide site is called “heteroplasmic”.

MitoDx Process Flow Chart

MitoDx Bioinformatics SNV Pipeline OverviewCollect ~5 Million 50bp Sequence Reads with Quality Data from NextGen Sequencer

Trim Sequence Ends to Remove Poor Quality Regions

Map to Primer Sequences to Deplete Primer Sequences In Silico

Map to Reference Genome

Call Single Nucleotide Variantswith High Coverage Algorithm

Analyze Coverage

Completeness and Uniformity

Review Mapping Pileups for Variant Sequence Start Point and Forward/Reverse Uniformity

Call Single Nucleotide Variantswith Low Coverage Algorithm

Apply SNV Calling Quality Filterto Select High Quality Mapped Sequences

Validate by Cross Referencing Replicate Data

Clinical Analysis of 21 Consecutive cases Referred by “Mito Docs”

8

Heteroplasmic mutation(s) unlikely to be deleterious 6

7

Heteroplasmic, putatively deleterious mutations

No evidence of mitochondrial genome disease

Patient 1 (information on patients changed to protect privacy)

• 3 year old girl with mitochondrial disease affecting Complexes I and III, developmental delay, microcephaly, cerebellar atrophy, seizures, leukoencephalopathy, and lactic acidosis.

• She has had multiple hospitalizations.• MitoDx revealed the U2e1a haplogroup• Two heteroplasmic variants detected• No obviously relevant homoplasmic variants found

09-12

Patient 1: Heteroplasmic Variants• 151524 G>A; gly1363ser in the CYTB gene▫ Heteroplasmic at 58% ▫ Novel alteration▫ Evolutionary conservation: gly in an alignment

of 23/23 vertebrates spanning 2+ billion years of evolutionary divergence

• 47323 A>G; asn883ser in the ND2 gene▫ Heteroplasmic at 2.9%▫ In homoplasmic form, variant seen at 1.1% in

mtDB▫ Asn in 7/7 non-human primates; ser in 0/23

vertebrates

09-12

Patient 2

• 8 year old girl with developmental delay, muscle weakness, ADHD, and ataxia

• Suggestive maternal inheritance• MitoDx revealed the N1B1 haplogroup• Two heteroplasmic variants detected • Two potentially relevant homoplasmic variants

analyzed• The mother was analyzed subsequently

09-11

Patient 2: Heteroplasmic Variants• 38203 C>A: leu1732ile in the ND1 gene▫ Heteroplasmic at 25%; ▫ Novel alteration▫ Evolutionary conservation: leu and met predominate

in an alignment of 23 vertebrates spanning 2+ billion years of evolutionary divergence: however, ile is found in gibbon & chicken

• 18883 G>A in the 163S ribosomal RNA gene (RNR2)▫ Heteroplasmic at 33%▫ In homoplasmic form, variant seen at 5.3% in

mtDB▫ Poorly conserved; A is found in 19 of 23 vertebrate

mtDNA sequences.

09-11

Patient 2: Homoplasmic Variants• 88363 A>G; met104val in the ATP6 gene▫ Homoplasmic, seen at 0.2% in mtDB▫ Met found in 18/18 mammals; val in 0/23

vertebrates ▫ Associated with haplotype N1b & possibly with LHON-

like optic neuropathy (Abu-Amero, KK and Bosley (2006).

• 75183 A>G at the 1st nucleotide of the tRNA-asp gene ▫ Homoplasmic▫ Novel alteration▫ Poorly conserved; A in 6/7 primates; G in 7/16

non-primate vertebrates09-11

Mother of Patient 2• 39 year old woman with mitochondrial dysautonomia,

fatigue, and reflux▫ Less severely affected than her daughter

• Supports maternal inheritance• MitoDx revealed the N1B1 haplogroup• Three heteroplasmic variants detected▫ 38204 C>A: 2.5% heteroplasmy▫ 18883 G>A: 2.7% heteroplasmy▫ 46043 C>T: 5.9% heteroplasmy (silent in the ND2

gene)• All homoplasmic variants are identical to Patient 1.

10-09

Patient 3

• 63 year old woman with multisystem disease and suspected maternal inheritance

• MitoDx revealed the H7a1 haplogroup• Two heteroplasmic variants detected • No obviously relevant homoplasmic variants

analyzed

09-28

Patient 3: Heteroplasmic Variants

• 106263 T>C: ser53pro in the ND4 gene▫ Heteroplasmic at 1.7% ▫ Novel alteration▫ Evolutionary conservation: ser, leu, and phe in

20/23 vertebrates; pro in 0/23 vertebrates

• 70284 C>T in the COX 1 gene▫ Heteroplasmic at 2.1%▫ Silent alteration

09-28

Patient 4

• 44 year old woman with suspected mitochondrial disorder

• 10 year old son: failure to thrive, GERD, diarrhea, Asperger syndrome, heat intolerance

• 6 year old daughter: failure to thrive, GERD, chronic abdominal pain

• Brother: migraines• Sister: cold intolerance, GI issues, poor weight

gain• Mother: GI issues, hearing loss, cold intolerance

09-20

Patient 4

• MitoDx found Haplogroup H1a• No heteroplasmic variants were

found.• No potentially relevant homoplasmic

variants were found.

09-20

Mitonucleome Dx

• NextGen sequencing of hundreds of nuclear genes that can cause mitochondrial diseases

• “The other half of the elephant”

• Coming soon

Clinical Analysis of 21 Consecutive cases Referred by “Mito Docs”

8

Heteroplasmic mutation(s) unlikely to be deleterious 6

7

Heteroplasmic, putatively deleterious mutations

No evidence of mitochondrial genome disease

THANK YOU• Questions?

• Suggestions?

• Other information desired on report?