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 ID: 136063
Download Presentation The PPT/PDF document "Genetics 101/Clinical Significance" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Genetics 101/Clinical Significance
Camp Sunshine
July 22, 2013
Diamond
Blackfan
Anemia
Foundation
Diamond
Blackfan
Anemia CanadaSlide2
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. Slide3
HUMAN CHROMOSOMESSlide4
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. Slide5
GENETICS 101Slide6
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 trueSlide7
GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS YOU WANT!
T
all pea plants crossed to short pea plants make tall offspringSlide8
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.Slide9
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. Slide10
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
/
tSlide11
GENETICS IN PEA PLANTS AND OTHER MODELS: YOU CAN MAKE THE MATINGS YOU WANT!
T
all 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/tSlide12
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/T
T/t
T/t
T/t
T/t
t
/
tSlide13
Plant 1- pollen
Plant 2 –
egg
T
T
t
t
T/T
T/t
T/t
t/t
MENDEL’S CROSS: THEORYSlide14
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. Slide15
Plant 1- pollen
Plant 2 –
egg
T
T
t
t
T / T
T / t
t
/ t
MENDEL’S CROSS: REALITY
T / tSlide16
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.Slide17
Attached ears
INHERITANCE OF A HUMAN TRAITSlide18
Attached ears
1. Non-attached ears does not breed true in this family
INHERITANCE OF A HUMAN TRAITSlide19
Attached ears
1. Non-attached ears does not breed true in this family
2.
A
ttached ears does not breed true in this family
INHERITANCE OF A HUMAN TRAITSlide20
Attached ears
INHERITANCE OF A HUMAN TRAIT
1. Non-attached ears does not breed true in this family
2.
A
ttached ears does not breed true in this family
3.
A
ttached ears can be inherited from one parentSlide21
Attached ears
1. Non-attached ears does not breed true in this family
2.
A
ttached ears does not breed true in this family
3.
A
ttached ears can be inherited from one parent
4. 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?Slide22
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. Slide23
Attached ears
a/a
a/a
a/a
a/a
a/a
a/a
a/a
A/a
A/a
A/a
INHERITANCE OF A HUMAN TRAIT
Dominant!
a/a
a/aSlide24
Willig
T et al. Blood 1999;94:4294-4306
©1999 by American Society of Hematology
DBA IS MORE COMPLICATED: AUTOSOMAL DOMINANT WITH INCOMPLETE PENETRANCESlide25
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.Slide26
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. Slide27
Ribosome
28S
rRNA
1
8S
rRNA
Small Subunit
40S
Large Subunit
60SSlide28
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 patientsSlide29
Willig
T et al. Blood 1999;94:4294-4306
©1999 by American Society of Hematology
THE IMPORTANCE OF GENOTYPINGSlide30
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 Slide31
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. Slide32
HUMAN CHROMOSOMESSlide33
HUMAN CHROMOSOMESSlide34
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 genomeSlide35
DETECTION TECHNOLOGYSlide36
DETECTION TECHNOLOGYSlide37
DECIPHERING THE DATASlide38
RPS19 DELETIONSSlide39
High resolution SNP array genotyping
n=106
D
eletions 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
RPS19
RPL5
RPL11
RPS26
RPS24
RPL35a
RPS10
RPS17
RPS7
other
deletions
unknown
North American DBA RegistrySlide40
RPS19
RPL5
RPL11
RPS26
RPS24
RPL35a
RPS10
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 DBASlide41
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 RegistrySlide42
Minimum requirements:
>50 x 10
6
100
bp
reads
30X coverage; 85% of
exome
Family 1: 112x10
6
; 85X; 91.5%
Family 2: 109x10
6
; 79X; 91.2%
Family 3: 108x106; 73X; 90.5%
Family 4: 103x106; 82X; 90.8%
WHOLE EXOME SEQUENCING 1
Enrich Sequence
Biotinylated
probes
29 x 106
bp (85% of coding sequence)
Select
Exome
with Streptavidin beads
Elute
Exome
for sequencing
NIH Intramural Sequencing Center (NISC)Slide43
WHOLE EXOME SEQUENCING 2
ref. CATGGTGTCTGTTTGAGG
T
TGCTA
1
CATGGTGTCTGTTTGAGG
T
TGCTA
2 CATGGTGTCTGTTTGAGG
A
TGCTA
3 CATGGTGTCTGTTTGAGG
T
TGCTA
4 CATGGTGTCTGTTTGAGG
A
TGCTA
5 CATGGTGTCTGTTTGAGG
ATGCTA 6 CATGGTGTCTGTTTGAGG
ATGCTA 7 CATGGTGTCTGTTTGAGGT
TGCTA
8 CATGGTGTCTGTTTGAGG
TTGCTA 9 CATGGTGTCTGTTTGAGG
ATGCTA …
n CATGGTGTCTGTTTGAGGTTGCTAMPG: CATGGTGTCTGTTTGAGG
T
TGCTA
A
8-10,000 variants/patient
Align Filter
Variants in
1000 Genomes
Variants in
ClinSeq
<100 variants
p
er patient
NIH Intramural Sequencing Center (NISC)Slide44
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. Slide45
WHOLE EXOME SEQUENCING 3
VarSifter
Analysis
of inheritance
CDPred
score
(severity)
Erythroid
e
xpression
Gene
function
De novo
X-linked
Aut
. Dom.
Aut
. Rec.
~
5 candidate mutations/family
Prioritize
Functional
Validation
Frequency
ValidationSlide46
KNOCKDOWN OF RPL15 AND RPL31
INHIBITS RED CELL PRODUCTION IN VITRO
∂
Luc
shRNA
RPL15
shRNA
∂
RPL31
shRNA
∂
∂
CD41
CD235
RPL15
RPL31
Relative mRNA Level
Luc L15 L31
shRNASlide47
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.