Genetics 101/Clinical Significance Camp Sunshine

July 22, 2013

Diamond Blackfan Anemia FoundationDiamond Blackfan Anemia Canada

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

HUMAN CHROMOSOMES

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

GENETICS 101

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Mendel’s tall pea plants and short pea plants make similar offspring: breed true

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Tall pea plants crossed to short pea plants make tall offspring

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Crossing the tall progeny of the original cross gives 3 tall plants to every 1 short plant.

Mendel’s conclusions: The tall trait masks the short trait, but the short trait is present in the parents.The tall and short traits segregate independently of each other.

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair. (Peas have 7 pairs).

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father. (Peas have 10s of thousands of genes too).

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Mendel’s tall pea plants and short pea plants breed true because they are homozygous (2 copies) for the tall or short trait.

T/T T/T

T/T T/T T/T T/T

t/t t/t

t/t t/t t/t t/t

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Tall pea plants crossed to short pea plants makes heterozygous offspring. The heterozygous offspring are tall because tall is a dominant trait.

T/T

T/t T/t T/t T/t

t/t

T/t T/t

GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS

YOU WANT!

Crossing the tall progeny of the original cross gives 3 tall plants to every 1 short plant ON AVERAGE.

T/TT/t T/t T/t T/t t/t

Plant 1- pollen

Plant 2 – egg

T

T

t

t

T/T T/t

T/t t/t

MENDEL’S CROSS: THEORY

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

Plant 1- pollen

Plant 2 – egg

T

T

t

t

T / T T / t

t / t

MENDEL’S CROSS: REALITY

T / t

PATTERNS OF INHERITANCE

Autosomal Dominant: Homozygous and Heterozygous individuals show the trait.

Autosomal Recessive: Only Homozygous individuals show the trait.

X-Linked: The trait is only seen in males.

Attached ears

INHERITANCE OF A HUMAN TRAIT

Attached ears

1. Non-attached ears does not breed true in this family

INHERITANCE OF A HUMAN TRAIT

Attached ears

1. Non-attached ears does not breed true in this family2. Attached ears does not breed true in this family

INHERITANCE OF A HUMAN TRAIT

Attached ears

INHERITANCE OF A HUMAN TRAIT

1. Non-attached ears does not breed true in this family2. Attached ears does not breed true in this family

3. Attached ears can be inherited from one parent

Attached ears

1. Non-attached ears does not breed true in this family2. Attached ears does not breed true in this family

3. Attached ears can be inherited from one parent4. A parent with attached ears can have non-attached children

INHERITANCE OF A HUMAN TRAIT

Is Attached ears inherited as a Dominant or Recessive trait?

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

Attached ears

a/a

a/a

a/aa/aa/a

a/a a/a

A/a

A/a

A/a

INHERITANCE OF A HUMAN TRAIT

Dominant!

a/a a/a

Willig T et al. Blood 1999;94:4294-4306

©1999 by American Society of Hematology

DBA IS MORE COMPLICATED: AUTOSOMAL DOMINANT WITH

INCOMPLETE PENETRANCE

MUTATIONS

Mutations are changes in the DNA that are inherited.

Deletion: complete loss of a segment of DNA containing multiple gene or part of a gene.

Point Mutation: A change in the DNA sequence the amino acid sequence of a protein.

Indel: The addition or loss of a base into a sequence that alters the amino acid sequence of a protein.

Genes make RNA which is translated into proteins.

RPS19RPL5RPL11RPS26RPS24RPL35aRPS10RPS17RPS7othersunknown

DIAMOND BLACKFAN ANEMIA MUTATIONS

RPS19: Draptchinskaia et al. Nat Genet. 1999. 21: 169-175.

RPS24: Gazda et al.. Am J Hum Genet. 2006. 79: 1110-1118.

RPS17: Cmejla et al. Hum Mutation 2007. 28: 1178-1182.

RPL35a: Farrar et al. Blood, 2008. 112: 1582-1592.

RPL11/RPL5: Gazda et al. Am J Hum Genet, 2008. 83: 769-780.

RPS10/26: Doherty et al. Am J Hum Genet, 2010. 86: 222-228.

Ribosome

28S rRNA

18S rRNA

Small Subunit40S

Large Subunit60S

WHY IS IT IMPORTANT TO IDENIFY THE MUTATIONS IN THE REMAINING DBA

PATIENTS?

Improve clinical opportunitiesStem cell transplant from matched sibling donor

WITHOUT the mutation – incomplete penetrance

Genotype/phenotype correlationsRemission ~15% of patientsSteroid responsiveness ~40% of patients

Willig T et al. Blood 1999;94:4294-4306

©1999 by American Society of Hematology

THE IMPORTANCE OF GENOTYPING

RPS19RPL5RPL11RPS26RPS24RPL35aRPS10RPS17RPS7othersunknown

DIAMOND BLACKFAN ANEMIA MUTATIONS

RPS19: Draptchinskaia et al. Nat Genet. 1999. 21: 169-175.

RPS24: Gazda et al.. Am J Hum Genet. 2006. 79: 1110-1118.

RPS17: Cmejla et al. Hum Mutation 2007. 28: 1178-1182.

RPL35a: Farrar et al. Blood, 2008. 112: 1582-1592.

RPL11/RPL5: Gazda et al. Am J Hum Genet, 2008. 83: 769-780.

RPS10/26: Doherty et al. Am J Hum Genet, 2010. 86: 222-228.

Hypothesis: Deletions and copy number variations cause DBA

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

HUMAN CHROMOSOMES

HUMAN CHROMOSOMES

SINGLE NUCLEOTIDE POLYMORPHISMS

• 99.9% of the bases are identical in all people – but that leaves 3 MILLION bases that can be different between any two people

• ~ 8 million validated human SNPs identified to date– Represent individual point mutations– 1 SNP per 500-1000 bp– Human genome may contain as many as

12 million SNPs– Over 200,000 SNPs may be present within genes

• Provides a means to identify individual parts of a genome

DETECTION TECHNOLOGY

DETECTION TECHNOLOGY

DECIPHERING THE DATA

RPS19 DELETIONS

High resolution SNP array genotyping

n=106

Deletions of known DBA genes

(14 + 2 new genes)

Variable, mosaic copy number loss of

3q (2), 13q, 15q (2), 19q

Mosaic copy loss of 5q33 (RPS14)

(2)

DBA COPY NUMBER VARIANT DETECTION

~10% of DBA patients have a deletion of a DBA gene

RPS19RPL5RPL11RPS26RPS24RPL35aRPS10RPS17RPS7other

deletions

unknown

North American DBA Registry

RPS19

RPL5

RPL11RPS26

RPS24

RPL35aRPS10

RPS17

RPS7

other

deletions

unknown

DIAMOND BLACKFAN ANEMIA MUTATIONS

~30-35% of patients do not have a molecular diagnosis

Hypothesis: Mutations in non-ribosomal genes cause DBA

SELECTION OF PATIENTS FOR SEQUENCING

Resequencing patients without

mutations

SNP array analysis for Copy Number Variants

New patients

(screened)

negative

negative

Family 1 Family 2 Family 3 Family 4

Informed consent for exome capture sequencing

North American DBA Registry

Minimum requirements:>50 x 106 100 bp reads

30X coverage; 85% of exome

Family 1: 112x106; 85X; 91.5% Family 2: 109x106; 79X; 91.2%Family 3: 108x106; 73X; 90.5%Family 4: 103x106; 82X; 90.8%

WHOLE EXOME SEQUENCING 1Enrich Sequence Biotinylated probes

29 x 106 bp (85% of coding sequence)

Select Exome with Streptavidin beads

Elute Exome for sequencingNIH Intramural Sequencing Center (NISC)

WHOLE EXOME SEQUENCING 2

ref. CATGGTGTCTGTTTGAGGTTGCTA 1 CATGGTGTCTGTTTGAGGTTGCTA 2 CATGGTGTCTGTTTGAGGATGCTA 3 CATGGTGTCTGTTTGAGGTTGCTA 4 CATGGTGTCTGTTTGAGGATGCTA 5 CATGGTGTCTGTTTGAGGATGCTA 6 CATGGTGTCTGTTTGAGGATGCTA 7 CATGGTGTCTGTTTGAGGTTGCTA 8 CATGGTGTCTGTTTGAGGTTGCTA 9 CATGGTGTCTGTTTGAGGATGCTA … n CATGGTGTCTGTTTGAGGTTGCTAMPG: CATGGTGTCTGTTTGAGGTTGCTA A

8-10,000 variants/patient

AlignFilter

Variants in 1000 Genomes

Variants in ClinSeq

<100 variantsper patient

NIH Intramural Sequencing Center (NISC)

THE FUNDAMENTALS• Humans have 46 chromosomes in each cell: 23 pairs.

Sperm and egg cells have 23 chromosomes, 1 of each pair.

• Genes are segments of DNA that tell your body what proteins to make. There are over 40,000 genes in a human cell: 20,000 on the chromosomes from your mother and a matching set of 20,000 on the chromosomes from your father.

• Changes in the sequence of the DNA in a gene can alter the function of the protein, causing disease.

WHOLE EXOME SEQUENCING 3

VarSifter Analysis of inheritance

CDPred score(severity)

Erythroidexpression

Genefunction

De novo

X-linked

Aut. Dom.

Aut. Rec.

~5 candidate mutations/family

Prioritize

Functional Validation

Frequency Validation

KNOCKDOWN OF RPL15 AND RPL31 INHIBITS RED CELL PRODUCTION IN VITRO

∂

Luc shRNA

RPL15 shRNA

∂RPL31 shRNA

∂

∂

CD

41

CD235

RPL15RPL31

Rel

ativ

e m

RN

A L

evel

Luc L15 L31 shRNA

FINAL THOUGHTS

Thanks to all of you who took the time and made the effort to be here. You are providing the most valuable

contributions of all: Hope, Support and a Sense of Community.

No DBA patient or family could feel alone in the presence of people like you.

Any researcher would feel inspired by all of you.