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Genetics 101/Clinical Significance Camp Sunshine July 22, 2013 Diamond Blackfan Anemia Foundation Diamond Blackfan Anemia Canada
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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.
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HUMAN CHROMOSOMES
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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.
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GENETICS 101
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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
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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
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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.
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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.
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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
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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
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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
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Plant 1- pollen Plant 2 – egg T T t t T/T T/t t/t MENDEL’S CROSS: THEORY
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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.
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Plant 1- pollen Plant 2 – egg T T t t T / T T / t t / t MENDEL’S CROSS: REALITY T / t
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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.
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Attached ears INHERITANCE OF A HUMAN TRAIT
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Attached ears 1. Non-attached ears does not breed true in this family INHERITANCE OF A HUMAN TRAIT
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Attached ears 1. Non-attached ears does not breed true in this family 2. Attached ears does not breed true in this family INHERITANCE OF A HUMAN TRAIT
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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
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Attached ears 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 non- attached children INHERITANCE OF A HUMAN TRAIT Is Attached ears inherited as a Dominant or Recessive trait?
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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.
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Attached ears a/a A/a INHERITANCE OF A HUMAN TRAIT Dominant! a/a
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Willig T et al. Blood 1999;94:4294-4306 ©1999 by American Society of Hematology DBA IS MORE COMPLICATED: AUTOSOMAL DOMINANT WITH INCOMPLETE PENETRANCE
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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.
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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.
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Ribosome 28S rRNA 18S rRNA Small Subunit 40S Large Subunit 60S
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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
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Willig T et al. Blood 1999;94:4294-4306 ©1999 by American Society of Hematology THE IMPORTANCE OF GENOTYPING
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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
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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.
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HUMAN CHROMOSOMES
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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
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DETECTION TECHNOLOGY
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DECIPHERING THE DATA
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RPS19 DELETIONS
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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 RPS19 RPL5 RPL11 RPS26 RPS24 RPL35a RPS10 RPS17 RPS7 other deletions unknown North American DBA Registry
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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 DBA
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SELECTION OF PATIENTS FOR SEQUENCING Resequencing patients without mutations SNP array analysis for Copy Number Variants New patients (screened) negative Family 1 Family 2 Family 3 Family 4 Informed consent for exome capture sequencing North American DBA Registry
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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: 108x10 6 ; 73X; 90.5% Family 4: 103x10 6 ; 82X; 90.8% WHOLE EXOME SEQUENCING 1 Enrich Sequence Biotinylated probes 29 x 10 6 bp (85% of coding sequence) Select Exome with Streptavidin beads Elute Exome for sequencing NIH Intramural Sequencing Center (NISC)
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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 CATGGTGTCTGTTTGAGGTTGCTA MPG: CATGGTGTCTGTTTGAGGTTGCTA A 8-10,000 variants/patient AlignFilter Variants in 1000 Genomes Variants in ClinSeq <100 variants per patient NIH Intramural Sequencing Center (NISC)
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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.
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WHOLE EXOME SEQUENCING 3 VarSifter Analysis of inheritance CDPred score (severity) Erythroid expression Gene function De novo X-linked Aut. Dom. Aut. Rec. ~5 candidate mutations/family Prioritize Functional Validation Frequency Validation
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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 shRNA
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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.
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