Genomic
Medicine
UNIT 05
Genomic Medicine
• In announcing on June 26, 2000, that the first draft of the
human genome had been achieved, President Clinton said it
would “revolutionize the diagnosis, prevention and treatment
of most, if not all, human diseases.”
• Francis Collins (2000) - Genetic diagnosis of diseases would be
accomplished in 10 years and that treatments would start to
roll out perhaps five years after that. “Over the longer term,
perhaps in another 15 or 20 years, you will see a complete
transformation in therapeutic medicine.”
• Ok, so where is it?
Genomic Medicine
• Ok, so where is it?
• Report from NIH – 17 years from scientific discovery to clinical
implementation
• The study of genomic disease is really a study of genomic
variation in humans and other organisms
• We’ll focus on human variation
Genomic Medicine
• A history of human genomics
• 2001 – the initial draft of the human genome
• Derived from multiple individuals
• Coordinated with the HapMap and dbSNP
• Provided our first glimpse into the complex architecture
of our genome – SNPs, copy number variants (CNVs),
segmental duplications, low-copy repeats
• Details 10 million common variants
• Assembled using a hierarchical strategy and Sanger
sequencing
Genomic Medicine
• A history of human genomics
• The Venter genome (JCV)
• First genome from a single individual
• Combined shotgun and reference-mapped Sanger
sequences
• 1.2 million variants compared to the HGP reference
• Many non-SNP variants including small indels and larger
CNVs and inversions
• 95 genes associated with CNVs
• 10,208 genes had at least one heterozygous base
Genomic Medicine
• The Venter genome (2007)
• Heterozygous for several gene variants
associated with heart disease
• Also heterozygous for several gene variants
associated with protection from heart disease
• Heterozygous for GSTM1 – associated with
detoxification of xenobiotics and increases
susceptibility to environmental toxins and
increased cancer rates
Genomic Medicine
• The Watson genome (2008) (JDW)
• First genome from a single individual to be
sequenced using NGS (454)
• More deletions than insertions were identified
• Many indels within genes were multiples of three nt
• Significant enrichment for indels 300-350 bp (Alusized)
• 23 large CNVs ranging from 26 kb - 1.6M bp, likely
benign
Genomic Medicine
• African genomes
• Africa is where most genetic variation should reside
• First – a Yoruban individual (2008) (ABT)
• Illumina
• Enriched for heterozygous SNPs associated with
pharmacogenetic traits and/or complex disease
• Indels in exons of 2,241 genes
Genomic Medicine
• African genomes
• Khoisan and Bantu individuals + exomes from three
individuals from indigenous Kalahari groups (KB1,
ABT, NB1, TK1, MD8)
• KB1 was sequenced using Sanger in anticipation of
the expected increased variation
• One was from Desmond Tutu (ABT)
• On average, two Kalahari Bushmen differ from each
other at 1.2 nt/kb in exons
• Only 1 nt/kb between European individuals
• Enriched for SNPs in promoter regions
Genomic Medicine
• African genomes
• Many SNPs have functional associations
• Lactase (LCT) promoter polymorphism associated
with lactase persistence in Europeans is missing in
Bushmen
• SLC24A5 variants associated with increased melanin
were identified
• CNVs altering copy numbers of 193 genes when
comparing Khoisan and Yoruban genomes
• 5x variation among southern African mtGenomes
when compared to Caucasian mtGenomes
Genomic Medicine
• Asian genomes
• First Asian genome – Han Chinese individual (YH)
• Followed by two Korean individuals (SJK, AK1)
• Various findings/individual
• SNPs associated with tobacco addiction in YH, a
heavy smoker
• SNP associated with dry earwax in AK1
• 27 indels likely impacting gene function in SJK
• 106 CNVs potentially impacting gene function in
AK1
Genomic Medicine
Genomic Medicine
Total SNPs
From 10
personal
genomes
Genomic Medicine
• The findings of substantial variation indicated the need
to examine large numbers of genomes
• 1000 Genomes Project (TGP), 1000genomes.org
• Goal: characterize human variation by unbiased
sequencing of 1000+ human genomes from diverse
populations
• Pilot data (2012) indicates nearly 39 million SNP
variants alone
• Many of those variants are private (unique to a
population or individual)
Genomic Medicine
• Structural variation in human genomes
• Prior to large scale re-sequencing of human genomes,
structural variation was considered rare
• CNVs
• Data suggests that the average individual harbors
~1000 polymorphic CNVs ranging from 500 bp - 1.2
Mb
• ~35% of all genes are impacted by CNVs
• Inversions
Genomic Medicine
• Inversions
• One such inversion is disrupts Factor VIII and is
responsible for 20% of all hemophilia A cases
Genomic Medicine
• Structural rearrangement consequences
• Small scale – usually neutral
• Large scale – variable but can be severe
• Charcot-Marie-Tooth disease (CMT1A)
• 70-80% of cases involve duplication of a portion
of the short arm of chromosome 17
• Neuropathy leading to weakness in leg/foot
muscles, spreading to the hands, wrists and
tongue.
• Pain ranges from mild to severe
Genomic Medicine
• Structural rearrangement consequences
• Smith-Magenis syndrome (SMS)
• Deletion of 17p11.2
• Linked specifically to deletion of RAI1, retinoic acid
induced 1.
• Loss of other genes in the area influence severity and
specific symptoms
• Mild to moderate intellectual disability, delayed speech
and language skills, distinctive facial features, sleep
disturbances, and behavioral problems.
Genomic Medicine
• Structural rearrangement consequences
• Williams-Beuren syndrome (WBS)
• Deletion of 1.5-1.8 Mb (~26 genes) from 7q11.23
• Aortic problems, mental disabilities, distinct facial,
predisposition to anxiety and phobias
• Some CNVs are associated with
• Parkinson, Alzheimers, psoriasis, autism,
schizophrenia, and HIV susceptibility
Genomic Medicine
• Structural rearrangement consequences
• Not all bad
• CCR5 Δ32
• Chemokine receptor 5 – a receptor molecule on T-cells
that allows HIV (and other pathogens) to enter cells
• 32-bp deletion that inactivates the receptor
• Homozygotes are immune to HIV, heterozygotes exhibit
resistance
• Likely evolved in northeast Europe and has spread via
long-range dispersal and strong selection in European
populations
Genomic Medicine
• Structural rearrangement
consequences
• CCR5 Δ32
• The Viking hypothesis – a single
origin ~700-2000 years ago
• Increased prevelance associated
with the Black Death (1348-1350)
• The short duration of the Black
Death is probably not responsible
for continued positive selection
• Smallpox? (Galvani and Novembre
2005)
Genomic Medicine
• TE-induced structural rearrangements
• Alu, LINE1
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Venter – 1300 Alu indels, 53% not present in the reference
Watson – 900 Alu indels not present in the reference
Beck et al. 2010 – 68 LINE1 indels not present in the reference
Ewing et al. 2010 – any two individual humans likely differ at
~285 LINE1 insertion sites
• Total dimorphic human LINE1s – 3000-10,000
Genomic Medicine
• Exome sequencing
• Exome – the coding sequences
of all annotated protein coding
genes; ~1% of the genome
• Accomplished via targetcapture methods
• What’s the major potential
drawback?
Genomic Medicine
• First exome application to disease diagnosis (2010)
• Ng et al. sequenced 12 exomes, including 4 from
individuals with Freeman-Sheldon syndrome
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Symptoms include drooping of the upper eyelids, strabismus, low-set
ears, a long philtrum, gradual hearing loss, scoliosis, and walking
difficulties
Known to be caused by variants of MYH3
56,240 coding SNPs identified
Identified MYH3 as the causative locus
Genomic Medicine
• First application to syndrome with unknown cause
• Miller syndrome – thought to be recessive
• Suggests that effected individuals require two variants (one on each
chromosome)
• Exomes of four individuals sequenced including a pair of siblings
• Narrowed to a single gene, DHODH, dihydroorotate dehydrogenase,
biosynthesis of pyrimidines
• All individuals harbored compound heterozygous mutations
• All parents were carriers
Genomic Medicine
• Schinzel-Giedon syndrome
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severe mental retardation, distinctive
facial features and multiple congenital
malformations
“In almost all subjects, the disease
phenotype occurs sporadically, suggesting
heterozygous de novo mutations in a
single gene as the underlying
mechanism.”
Four affected individuals sequenced
“Heterozygous novel mutations were
found… in the SETBP1 gene. Testing the
presence of the identified variants in the
parents… showed that all mutations arose
de novo, consistent with dominant
mutations in this sporadic syndrome.”
“All changes occurred in a genomic stretch
of only 11 nucleotides, affecting three of four
consecutive amino acids (868–871:
aspartate, serine, glycine and isoleucine).”
Genomic Medicine
• Previous examples are relatively rare
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What about more common problems?
Clinical applications?
• Worthey et al. 2011
• Single male with IBD-like symptoms
• Multiple rounds of hospitalization from 15 months to four
years old
• Normal genetic screening revealed nothing
• Exome sequencing – 15,272 SNPs, 6799 of them
nonsynonymous, 706 novel, 13 produced nonsense mutations
• Researchers searched for variants that were homozygous,
hemizygous or compound heterozygous
Genomic Medicine
• Worthey et al. 2011
• Identified a single change in the X-linked inhibitor of apotosis
gene (XIAP)
• Confirmed heterozygosity in mother
Genomic Medicine
• Worthey et al. 2011
• Diagnosis led to treatment
• “The XIAP protein has a central role in the proinflammatory
response, leading to activation of NFkB and subsequent
activation of proinflammatory cytokines via the NOD signaling
pathway, as well as a crucial role in mediating programmed
cell death.”
• Hematopoietic stem cell progenitor transplantation was
implemented to reduce inflammation.
• The patient was reported to be improving and thriving
Genomic Medicine
Disease
Inheritance
Sequencing
Samples
Technology
Identified
gene
Congenital chloride AR
diarrhea
Illumina
1 affected
SLC26A3
Miller syndrome
AR
Illumina
4 affecteds (1
sib-pair)
DHODH*
Schinzel-Giedion
syndrome
AD
SOLiD
4 unrelated
affecteds
SETBP1*
Nonsyndromic
hearing loss
DFNB82
AR
Illumina
1 affected in
family
GPSM2*
Perrault syndrome AR
Illumina
Kabuki syndrome
AD
Illumina
Severe brain
malformations
AR
Illumina
1 affected in
family
10 unrelated
affecteds
1 affected in
family
Sensenbrenner
syndrome/
cranioectodermal
dysplasia (CED)
Mabry syndrome/
hyperphosphatasia
with mental
retardation
Autosomaldominant
spinocerebellar
ataxia
Mental retardation
AR
SOLiD
2 unrelated
affecteds
WDR35*
AR
SOLiD
3 affected
siblings
AD
Illumina
4 related
affecteds
AD
SOLiD
10 parent-case DYNC1H1,
trios
ZNF599*, RAB39B,
YY1, BPIL3*,
PGA5*, DEAF1,
CIC, SYNGAP1,
JARID1C
Disease
Inheritance
Mitochondrial AR
complex I
deficiency
Familial
AR
combined
hypolipidemia
Amyotrophic
AD
lateral sclerosis
Sequencing
Identified
Samples
Technology
gene
SOLiD
1 affected ACAD9*
Illumina
2 related
affecteds
ANGPTL3*
Illumina
2 related
affecteds
VCP*
Autoimmune
AR
lymphoproliferat
ive syndrome
(ALPS)
Illumina
1 affected
FADD*
HSD17B4*
Seckel syndrome AD
Illumina
1 affected
CEP152*
MLL2*
CMT1X
Illumina
WDR62*
Inflammatory
XL
bowel disease/Xlinked inhibitor
of apoptosis
deficiency
Severe skeletal AR
dysplasia
Roche 454
2 related GJB1
affecteds
1 affected XIAP
Illumina
2 affecteds
and
parents
POP1*
PIGV*
Hajdu-Cheney AD
syndrome (HCS)
Illumina
3
unrelated
affecteds
NOTCH2*
TGM6*
Osteogenesis
AR
imperfecta (OI)
SOLiD
1 affected
in family
SERPINF1*
Hereditary
hypotrichosis
simplex (HHS)
Illumina
1 affected
in family
RPL21*
XL
AD
Genomic Medicine
Disease
Inheritanc Sequencing
e
Technology
Samples Identified gene
Disease
Inheritance
Sequencing
Samples Identified gene
Technology
Acne
AD
inversa/hidradeniti
s suppurativa
Primary
AD
lymphoedema
Illumina
2 affecteds in NCSTN
family
3-M syndrome AR
SOLiD
3 affecteds CCDC8*
Illumina
1 affected in
family
Illumina
Hereditary sensory AR
neuropathy with
dementia and
hearing loss
(HSAN1)
Hereditary spastic AR
paraparesis (HSP)
Illumina/Roche 4 kindreds
454
Late-onset
AD
Parkinson
disease
Leber
AR
congenital
amaurosis (LCA)
Illumina
2 affecteds VPS35*
&2
affecteds
1 affected KCNJ13*
Illumina
1 parent-case KIF1A*
trio
Gray platelet AR
syndrome (GPS)
Illumina
4 affecteds NBEAL2*
Hereditary
AR
progeroid syndrome
Chondrodysplasia AR
and abnormal joint
development
Amelogenesis
AR
imperfecta and
gingival hyperplasia
syndrome
Hypertrophic
AR
mitochondrial
cardiomyopathy
Illumina
2 affecteds
BANF1*
KBG syndrome AD
Illumina
3 affecteds ANKRD11*
SOLiD
3 affecteds
IMPAD1*
Illumina
3 affecteds MAX*
SOLiD
1 affected
FAM20A*
Hereditary
AD
pheochromocyt
oma (PCC)
Bohring-Opitz AD
syndrome
SOLiD
3 affecteds ASXL1*
Illumina
1 affected
AARS2*
Acromicric and AR
geleophysic
dysplasias
SOLiD
2 affecteds FBN1
Mosaic variegated AR
aneuploidy
syndrome (MVA)
Autism spectrum AD
disorder (ASD)
Illumina
2 affected
siblings
CEP57*
Hajdu-Cheney AD
syndrome (HCS)
Illumina
6 affecteds NOTCH2*
Illumina
20 parent-case Potential genes
trios
identified
Illumina
1 affected
Immunodeficiency– AR
centromeric
instability–facial
anomalies
syndrome type 2
(ICF2)
High myopia
AD
Illumina
1 affected
ZBTB24*
Mitochondrial AR
cardiomyopath
y
Proteus
somatic
syndrome
Illumina
17 samples AKT1*
from 12
affecteds
Illumina
2 affecteds
ZNF644*
GJC2*
DNMT1*
MRPL3*
Genomic Medicine
• WGS for diagnosis and management
• Dopa-responsive dystonia (DRD)
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Symptoms are increased muscle tone (dystonia, such as clubfoot)
and Parkinsonian features, typically absent in the morning or after
rest but worsening during the day and with exertion.
Responds well to treatment with levodopa.
• Bainbridge et al. 2011 – fraternal twins with DRD
• 1.64 million shared SNPs, 4605 nonsynonymous
• Recessive inheritance pattern, searched for genes with two or
more variants
• List narrowed to three genes
Genomic Medicine
• Dopa-responsive dystonia (DRD)
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One of the three, SPR (sepiapterin
reductase)
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The other two, unlikely to be causative
Had previously been associated with DRD
Contained compound heterozygosity
Missense mutation R150G
Nonsense mutation K251X
Parents were heterozygous for the
variants
Genomic Medicine
• Dopa-responsive dystonia (DRD)
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Additional linked variation
Genomic Medicine
• Dopa-responsive dystonia (DRD)
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SPR is involved in tetrahydrobiopterin (BH4) biosynthesis
BH4 is a cofactor involved in metabolism of aromatic amino acids that
are also involved in the biosynthesis of dopamine and serotonin
Dopamine treatment was already being performed
Diagnosis suggested that serotonin supplements would also help
Fibromyalgia is known to respond to serotonin supplementation,
suggesting treatment for mom and grandma
• Pharmacogenomics
• The identification of potentially
clinically relevant variants related
to drug metabolism
Genomic Medicine
Genomic Medicine
• WGS/Exome workflow
Genomic Medicine
• Cancer is a “disease of the genome”
• Cancer genomics – the systematic study of the genome to
find sites of recurrent derangement in specific cancer
types
• First ‘cancer genome’ sequenced in 2008
• An acute myeloid leukemia tumor and it’s normal counterpart
• More recent work involves hundreds of samples at a time
• Late 2012 at the Broad Institute - >16,000 samples
subjected to WGS or exome sequencing
Genomic Medicine
• Mutations are numerous
and diverse in cancer
genomes
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Translocations/deletions can fuse two
genes to create an oncogene
Translocations/Deletions can inactivate a
tumor suppressor gene
Amplifications can increase oncogene
products
SBS = Single base substitutions
Genomic Medicine
• Mutation timing
• The number of mutations in certain tumors of self-renewing tissues is
directly correlated with age
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Epithelial cells are constantly creating new cells to replace linings
(gastrointestinal, urogenital, etc.)
Every time a cell divides, it’s an opportunity to make a replication mistake
Brain cells, pancreatic cells do not replicate, typically have fewer mutations
Genomic Medicine
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Detecting relevant mutations is tricky
Is the tumor tissue free of normal tissue?
Tumors often have ploidy and CNV issues.
Some tumors are heterogeneous
Driver mutations vs. passenger mutations
• Which mutations are causative and which aren’t?
• Requires determining which genes show more mutations than
expected
Genomic Medicine
• Mutations are numerous
and diverse in cancer
genomes
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Number of somatic mutations in
representative human cancers,
detected by genome-wide
sequencing studies. (A) The genomes of
a diverse group of adult (right) and pediatric
(left) cancers have been analyzed. Numbers
in parentheses indicate the median number of
nonsynonymous mutations per tumor.
Genomic Medicine
• Mutations are numerous
and diverse in cancer
genomes
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Number of somatic mutations in
representative human cancers,
detected by genome-wide
sequencing studies. (B) The median
number of nonsynonymous mutations per
tumor in a variety of tumor types. Horizontal
bars indicate the 25 and 75% quartiles. MSI,
microsatellite instability; SCLC, small cell lung
cancers; NSCLC, non–small cell lung
cancers; ESCC, esophageal squamous cell
carcinomas; MSS, microsatellite stable; EAC,
esophageal adenocarcinomas.
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Note the increased numbers in cancers
caused by mutagens
Genomic Medicine
• Mutation timing
• Tumors evolve from benign to malignant lesions by acquiring a series of
mutations over time
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‘Gatekeeper’ mutations provide a selective growth advantage
A second mutation in another gene produces additional growth
A third mutation may allow for metastasis, invasiveness, lack of response to
apoptosis signaling, etc.
Genomic Medicine
• Tumor genetic heterogeneity
• As tumors grow and become malignant, they tend to replicate more
rapidly, providing opportunities for additional mutations in subclones of
the original tumor
• It’s rare to see a solid tumor in which all of the cells display the same
karyotype
• Sequencing studies suggest the same heterogeneity
• Cells in the same tumor but far from one another will display more
differences than neighboring cells
• It is not uncommon for one metastatic lesion to have 20+ unique genetic
alterations compared to another metastatic lesion in a single patient
Genomic Medicine
• Tumor genetic heterogeneity
• Means that every tumor/cancer is, by definition, likely to be unique
• Suggests that it’s less important to identify the cell type than it is to
identify the particular mutations responsible
• Examples:
• CDKN2A mutation may occur in bladder or colon
• CDKN2A inactivation produces increased CDK4 activity, which
increases cell cycle progression, regardless of cell type
• Suggests that kinase inhibitors would help in both cases
• BRCA1/BRCA2 inactivations damage the DNA repair pathway
• Suggests therapy that induces DNA damage that can be repaired in
normal cells but not the tumor cells
Genomic Medicine
• Ethical considerations
• DRD example – discovery of the variant in children had
the potential to impact relatives
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Does the doctor have an obligation to tell the relatives?
Does the doctor have a legal right to tell the relatives?
What if the relatives don’t want to know?
What if the primary patient doesn’t want to tell them?
What if parents want to know but the child doesn’t?
What about infant screening? The child has no say in whether
they know or not.
Genomic Medicine
• Ethical considerations
• Incidental findings (IF) – previously undiagnosed
conditions that are discovered unintentionally and are
unrelated to the current patient/condition being treated
but may be of importance for a second
individual/condition
• Variants of Uncertain Significance (VUS) – An alteration in
the normal sequence of a gene whose association with
disease risk is unclear
Genomic Medicine
• Example 1
• Huntington’s Disease – a dominant progressive neurological disorder
that doesn’t typically strike until later (>30) in life
• Patient A – knows of a paternal grandmother with Huntington’s and
decides to get tested, no maternal history
• Father of A – so far is asymptomatic
• If A tests positive, this essentially diagnoses the father with
Huntington’s
• If A tests negative, father could still be positive
• Brother of A – asymptomatic, thinking of having a child
• If A tests positive, confirms father of both, increases apparent
risk to brother and possible nephew/niece
• If A tests negative, brother could still be positive
Genomic Medicine
• Example 1
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Test results for A impact several other people
To whom does the test result information belong?
What if A finds out but doesn’t want to share the results?
If the father doesn’t want to know, does the doctor still have a duty
to warn?
• Tarasoff v the Regents of the University of California, and the Health
Information Portability and Accountability Act (HIPAA).
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suggest that we hold information about patients to be private and that
disclosure without the patients' permission can only occur if the person
at-risk is identifiable, the situation at risk is severe and potentially
preventable.
the doctor-patient relationship (and therefore its privacy obligations and
other duties) extends only to the person with whom the medical
relationship exists, rather than to their family members; this means that
a patient's autonomous decision not to tell potentially at-risk family
members is typically honored.
Genomic Medicine
• Example 2
• Patient B, a 60 year old man diagnosed with hereditary nonpolyposis
colon cancer – a dominant inherited tendency toward colon cancer
• Patient B is tested and confirmed for the trait
• B doesn’t want to worry his family, which is already under stress due
to his diagnosis.
• Should he tell the family?
• Should the doctor tell the family?
Genomic Medicine
• Example 2
• Patient B, a 60 year old man diagnosed with hereditary nonpolyposis
colon cancer – a dominant inherited tendency toward colon cancer
• Patient B is tested and confirmed for the trait
• B doesn’t want to worry his family, which is already under stress due
to his diagnosis.
• Should he tell the family?
• Should the doctor tell the family?
• Tells the family
• Son considers testing but, if he tests positive, he is concerned that his
company’s insurance provider may drop his coverage or, if he
changes jobs, the new provider will consider it a pre-existing
condition and not offer coverage