Genetics 101/Clinical Significance
Camp Sunshine
July 22, 2013
Diamond Blackfan Anemia Foundation
Diamond 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!
T/T
T/T
T/T
T/T
T/T
T/T
t/t
t/t
t/t
t/t
t/t
t/t
Mendel’s tall pea plants and short pea plants breed true because they are
homozygous (2 copies) for the tall or short trait.
GENETICS IN PEA PLANTS AND OTHER
MODELS: YOU CAN MAKE THE MATINGS
YOU WANT!
T/t
T/t
T/T
t/t
T/t
T/t
T/t
T/t
Tall pea plants crossed to short pea plants makes heterozygous offspring.
The heterozygous offspring are tall because tall is a dominant trait.
GENETICS IN PEA PLANTS AND OTHER
MODELS: YOU CAN MAKE THE MATINGS
YOU WANT!
T/t
T/t
T/T
T/t
T/t
t/t
Crossing the tall progeny of the original cross gives 3 tall plants to every 1
short plant ON AVERAGE.
MENDEL’S CROSS: THEORY
Plant 1- pollen
T
t
T
T/T
T/t
t
T/t
t/t
Plant 2 –
egg
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.
MENDEL’S CROSS: REALITY
Plant 1- pollen
T
t
T
T / T
T / t
t
T / t
t / t
Plant 2 –
egg
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.
INHERITANCE OF A HUMAN TRAIT
Attached ears
INHERITANCE OF A HUMAN TRAIT
1. Non-attached ears does
not breed true in this family
Attached ears
INHERITANCE OF A HUMAN TRAIT
1. Non-attached ears does
not breed true in this family
2. Attached ears does not
breed true in this family
Attached ears
INHERITANCE OF A HUMAN TRAIT
1. Non-attached ears does
not breed true in this family
2. Attached ears does not
breed true in this family
3. Attached ears can be
inherited from one parent
Attached ears
INHERITANCE OF A HUMAN TRAIT
1. Non-attached ears does
not breed true in this family
2. Attached ears does not
breed true in this family
3. Attached ears can be
inherited from one parent
4. A parent with attached
ears can have nonattached children
Attached ears
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.
INHERITANCE OF A HUMAN TRAIT
A/a
a/a
a/a
a/a
A/a
a/a
a/a
A/a
a/a
a/a
Attached ears
a/a
a/a
Dominant!
DBA IS MORE COMPLICATED:
AUTOSOMAL DOMINANT WITH
INCOMPLETE PENETRANCE
Willig T et al. Blood 1999;94:4294-4306
©1999 by American Society of Hematology
MUTATIONS
Genes make RNA which is translated into proteins.
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.
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.
RPS19
RPL5
RPL11
RPS26
RPS24
RPL35a
RPS10
RPS17
RPS7
others
unknown
Ribosome
28S rRNA
Small Subunit
40S
Large Subunit
60S
18S rRNA
WHY IS IT IMPORTANT TO IDENIFY THE
MUTATIONS IN THE REMAINING DBA
PATIENTS?
Improve clinical opportunities
Stem cell transplant from matched sibling donor
WITHOUT the mutation – incomplete penetrance
Genotype/phenotype correlations
Remission ~15% of patients
Steroid responsiveness ~40% of patients
THE IMPORTANCE OF GENOTYPING
Willig T et al. Blood 1999;94:4294-4306
©1999 by American Society of Hematology
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
RPS19
RPL5
RPL11
RPS26
RPS24
RPL35a
RPS10
RPS17
RPS7
others
unknown
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
DBA COPY NUMBER VARIANT DETECTION
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
North American
DBA Registry
~10% of DBA patients have a
deletion of a DBA gene
Mosaic copy loss of
5q33 (RPS14)
(2)
RPS19
RPL5
RPL11
RPS26
RPS24
RPL35a
RPS10
RPS17
RPS7
other
unknown
deletions
DIAMOND BLACKFAN ANEMIA MUTATIONS
RPS19
RPL5
RPL11
RPS26
~30-35% of patients do not
have a molecular diagnosis
RPS24
RPL35a
RPS10
RPS17
RPS7
other
unknown
deletions
Hypothesis: Mutations in non-ribosomal genes cause DBA
SELECTION OF PATIENTS FOR SEQUENCING
Resequencing
patients without
mutations
North American
DBA Registry
New
patients
(screened)
negative
SNP array analysis for Copy
Number Variants
negative
Family 1
Family 2
Family 3
Informed consent for exome capture sequencing
Family 4
WHOLE EXOME SEQUENCING 1
Enrich
Sequence
Biotinylated probes
29 x 106 bp (85% of coding sequence)
Select Exome with Streptavidin beads
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%
Elute Exome for sequencing
NIH Intramural Sequencing Center (NISC)
WHOLE EXOME SEQUENCING 2
Align
Filter
Variants in
Variants in
1000 Genomes ClinSeq
ref.
1
2
3
4
5
6
7
8
9
…
n
MPG:
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGATGCTA
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGATGCTA
CATGGTGTCTGTTTGAGGATGCTA
CATGGTGTCTGTTTGAGGATGCTA
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGATGCTA
<100 variants
per patient
CATGGTGTCTGTTTGAGGTTGCTA
CATGGTGTCTGTTTGAGGTTGCTA
A
8-10,000 variants/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
Prioritize
CDPred score Erythroid
Gene
(severity) expression function
Functional
Validation
De novo
X-linked
Aut. Dom.
Aut. Rec.
~5 candidate
mutations/family
Frequency
Validation
KNOCKDOWN OF RPL15 AND RPL31
INHIBITS RED CELL PRODUCTION IN VITRO
RPL31
shRNA
CD235
∂
Relative mRNA Level
∂
∂
RPL15
shRNA
∂
CD41
Luc
shRNA
RPL15
RPL31
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.