Transcript usmle

APPLIED MOLECULAR GENETICS
AND MENDELIAN INHERITANCE
GENETICS 202
Jon Bernstein
Department of Pediatrics
October 1st, 2015
Learning Goals
Understand that different molecular testing methodologies
detect different types of mutation
 Understand in conceptual terms how each of the following
diagnostic methods works
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Sanger sequencing (Short Video)
Deletion/duplication testing (Short Video)
Next generation sequencing (Short Video)
Trinucleotide repeat sizing
Mutation screening
Learning Goals
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Gain familiarity with Mendelian modes of inheritance and
associated recurrence risks (Short Video)
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Autosomal dominant
Autosomal recessive
X-linked recessive
X-linked dominant
Understand how molecular mechanisms relate to modes of
inheritance
Scales of Genetic Variation
ACTGATCCG
ACTGATAACCCG
ACTGCG
Structural Variant (SV)
ACTGATCCG
ACTGGTCCG
Small insertion or deletion (indel) Single nucleotide variant (SNV)
Nature Reviews Genetics 12, 363-376 (May 2011)
Gallery of molecular tests
or
Lodish et al., Molecular Cell Biology, 1995.
Nat Rev Genet. 2010 Jan;11(1):31-46. PMID: 19997069
Sanger sequencing
Next generation sequencing Deletion-duplication testing
Lecture Outline

Clinical cases
◦ Autosomal dominant
◦ Autosomal recessive
 Interpreting the significance of novel variants
◦ X-linked recessive
 Penetrance
 Expressivity
 Anticipation
◦ X-linked dominant
 Limitations of sequencing
Case #1 Autosomal Dominant
The parents of a 12 year old girl with
autism are interested to know what may
have caused her condition.
◦ Walked at 18 months
◦ No spoken words until age 2.5
◦ Does not readily make eye contact with
others
Van Bon, B. W. M., et al. Molecular psychiatry (2015).

Family history notable for late speech
in father
Many genes and loci associated with autism
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22q13, 15q11, 15q13.3, 16p11.2….
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CHD8, DYRK1A, SHANK3, MECP2, PTEN, SCN2A,
GRIN2A, GRIN2B, SHANK2, SYNGAP1, FMR1….
Array CGH with paternally inherited deletion of
unclear significance
 Whole exome sequencing

Clinical Case – Autosomal Dominant
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Patient is heterozygous
for a an apparently de
novo point mutation in
intron 9 of DYRK1A
(c.1098+1G>A)
Exon
Exon
Exon
G>A
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How do we know this is a
pathogenic mutation
rather than a variant?
Both gain of function and loss of function
mutations can be seen in dominant inheritance

In this case one heterozygous predicted loss of function
allele can cause disease
◦ Haploinsufficiency *
◦ Dominant negative
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In other diseases one gain of function allele can cause
disease
◦ Examples – Osteogenesis imperfecta type 1 (structural protein),
the Rasopathies (signaling molecules)
Autosomal Dominant – Recurrence Risk -Offspring
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Offspring of an affected
individual – 50%
recurrence risk
?
= Autism
Autosomal Dominant – Recurrence Risk -- Siblings
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Siblings of an affected
individual with clinically
unaffected parents – low,
but not zero risk due to
possibility of mosaicism.
Recurrence risk is not 50%
in this case.
?
= Craniosynostosis
Somatic and Germline Mosaicism

Somatic Mosaicism – Two genetically different cell lines in the
same person.
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Germline Mosaicism – Two genetically different cell lines in the
reproductive cells (or their precursors) of the same person.
http://dermatlas.med.jhmi.edu
◦ Based upon empiric data the risk of this is commonly estimated to be
around 1% in families with apparently de novo dominant conditions. The
risk may be higher or lower in specific disorders. When present the risk
of recurrence in future pregnancies of the mosaic parent can be up to
50%.
Case #1 Revisited – Autosomal Recessive
The parents of a 12 year old
girl with autism are interested
to know what may have
caused her condition.
Array CGH normal
Whole exome sequencing
The mutation and the variant
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BCKDK
◦ c. c.671G>C (p.Arg224Pro)
 “Probably damaging”
◦ c.466C>T (p.Arg156X)
Assessing significance of missense variants
Inherited versus de novo (if dominant)
 Segregating with or associated with disease
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◦ Literature/Databases
 dbSNP - http://www.ncbi.nlm.nih.gov/projects/SNP/
 ExAC (Exome aggregation consortium) database - http://exac.broadinstitute.org/
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Predicted impact on protein
◦ PolyPhen2 - http://genetics.bwh.harvard.edu/pph2/
 Polymorphism Phenotyping
 Sequence alignment
 Protein structure
◦ SIFT - http://sift.jcvi.org/
 Sorting Intolerant from Tolerant
 Sequence homology
◦ Mutation Taster - http://www.mutationtaster.org/
 Similarity in features to other pathogenic changes
The mutation and the variant

BCKDK
◦ c. c.671G>C (p.Arg224Pro)
 “Probably damaging”
◦ c.466C>T (p.Arg156X)
 Conserved in human, chimp, rat, zebrafish
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If we have two mutations, is that a diagnosis
◦ What else do we need to know
 Cis versus trans configuration
Cis versus trans configuration of mutations in a
gene
Allele 1
A
Trans
Allele 2
Allele 1
Allele 2
B
A
B
Cis
How to determine cis versus trans configuration
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Test the parents
◦ If one mutation comes
from each parent they
are in trans.
a
b
or
a
b
Loss of function mutations can lead to autosomal
recessive disease
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This patient has a nonsense
mutation and a missense
mutation predicted to result
in loss of function.
◦ Do nonsense mutations always
result in loss of function?
 Nonsense mediated decay not
triggered if mutation is in the last
exon.
http://www.nature.com/horizon/rna
Autosomal Recessive – Recurrence Risk
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Chance that each parent
is a carrier and passes on
the mutation
Aa
(_ x _) x( _ x _)
Aa
?
aa
(1 x 1/2) x( 1 x 1/2)=1/4
??
Autosomal Recessive Recurrence Risk
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What are the chances the
patient’s unaffected brother is a
carrier?
There are four possible pairs of
alleles he could inherit.
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AA
Aa
aA
aa
But we know that he is not aa,
so 2/3rds risk of being a carrier
Aa
Aa
?
aa
??
Autosomal Recessive Recurrence Risk
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What are the chances that
the patient’s aunt is a
carrier?
◦ Mutations in autosomal
recessive conditions are
typically old
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??
Aa
One of the maternal
grandparents is very, very
likely a carrier.
◦ The aunt has a 50% chance
of being a carrier
aa
Aa
Case #3 – X-linked Recessive
A patient is noted to have
a cardiac murmur. His
echocardiogram shows
prolapse of multiple
cardiac valves.
 He is otherwise healthy.
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Bernstein et al., AJMG, 2011.
Clinical Case – X-linked Recessive
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Review of the family history
reveals that his younger
brother also has valvular
disease.
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What mode(s) of
inheritance could this be?
Literature and Database Search
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There appear to be multiple Mendelian forms of
valvular disease, primarily dominant or X-linked
◦ OMIM Entry #314400
 X-linked form due to mutations in the gene encoding Filamin A
Clinical Case – X-linked Recessive
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The lab reports a missense variant in exon 5 of
FLNA (GGG>AGG) predicted to result in G288R;
the significance of this is unclear. Family studies
to assess segregation are recommended.
Is the variant detected pathogenic?
Inherited or de novo?
 Seen in healthy individuals?
 Previously reported?
 In databases?
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Predicted impact on protein function?
◦ Polyphen2, SIFT
Is the variant pathogenic?
Inherited from clinically asymptomatic mother
 Also found in symptomatic brother and asymptomatic
grandmother
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Not reported as a variant in dbSNP
 Laboratory performing testing has not observed the
variant in 234 chromosomes.
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Is the variant pathogenic?
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Grandmother’s
echocardiogram shows
evidence of valvular
thickening
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This mutation has
previously been reported in
a British family with Xlinked valvular disease.
Penetrance and Expressivity
Penetrance and Expressivity
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Penetrance
◦ The proportion of individuals with a mutation that show
any evidence of disease (the mutation associated
phenotype).
 Incomplete penetrance
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Expressivity
◦ The degree of severity or extent of symptoms in
individuals with a genetic disease
 Variable expressivity
One Gene Many Diseases/Phenotypes
X-linked Valve Disease
Periventricular Nodular
Heterotopia
Melnick-Needles
Syndrome
Filamin A
Otopalataldigital
Syndrome
Robertson, 2005,
Curr Opinion in
Genet and Dev
Sheen et al. 2005,
Neurology
Periventricular Nodular
Heterotopia, EhlersDanlos Variant
Frontometaphyseal
dysplasia
Loss of Function
Gain of Function
X-linked recessive – Recurrence Risk
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The patient’s parents are
expecting another child.
What is the probability
that the infant will be
affected by the familial
valvular disease?
X-linked Recessive – Recurrence Risk
?
= Duchenne Muscular Dystrophy
X-linked Recessive – Recurrence Risk
?
= Duchenne Muscular Dystrophy
Case #4 – X-linked Dominant
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An infant girl is
brought to your office
due to history of an
unusual skin rash.
Differential diagnosis
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You suspect an X-linked dominant or mosaic
autosomal condition due to the linear and
whorled pattern of the skin findings.
◦ Follows the lines of Blaschko
Interesting Review Article on Mosaicism:
Biesecker LG, Spinner NB. A genomic view of
mosaicism and human disease. Nat Rev Genet.
2013 May;14(5):307-20. PMID: 23594909
Clinical Case – X-linked Dominant
After consulting the literature you consider the
possible diagnosis of Incontinentia Pigmenti.
 Family History
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◦ The patient's mother has a history of congenitally
absent teeth and areas of sparse scalp hair. She also
has a history of multiple miscarriages.
Clinical Case – X-linked Dominant
= Incontinentia Pigmenti
Clinical Case – X-linked Dominant
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You would like to perform genetic testing to
confirm the suspected diagnosis
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DNA sequencing of the coding regions and
flanking intronic regions of IKBKG was normal.
Allele drop-out
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Conventional sequencing in the
clinical lab is dependent on PCR
amplification of the exon and
primer binding to target DNA.
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If amplification or primer
binding only occurs for 1 of 2
alleles the other will not be
“seen” in the sequencing
reaction.
Lodish et al., Molecular
Cell Biology, 1995.
How can a deletion be detected?
aCGH – if deletion large enough or array dense enough
 MLPA – Multiplex Ligation dependent Probe Amplification
 Next generation sequencing – if coverage and depth adequate
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Assessment for heterozygosity by genotyping or sequencing
 FISH – if large enough and you know exactly where to look (note
limited ability of metaphase FISH to detect duplications)
 Southern blot
 PCR based methods
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Detecting SVs with NGS
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Read depth analysis
Analysis of split reads
Mapping of paired-end reads
Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet. 2010 Jan;11(1):31-46. PMID: 19997069
Read-depth analysis
Sensitive and accurate detection of copy number variants using read depth of coverage. Yoon S, Xuan Z, Makarov V, Ye K, Sebat J.
Genome Res. 2009 Sep;19(9):1586-92 PMID: 19657104
Analysis of split reads
Karakoc E et al. Detection of structural variants and indels within exome data.Nat Methods. 2011 Dec 18;9(2):176-8. PMID: 22179552
Mapping of Paired-End Reads
Korbel JO et al. Paired-end mapping reveals extensive structural variation in the human genome. Science. 2007 Oct 19;318(5849):420-6.
PMID: 17901297
http://www.ncbi.nlm.nih.gov/dbvar/content/overview/
Clinical Case #5
Fragile X - Syndrome
 X-linked Intellectual
Disability Syndrome
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Affected individuals
have > 200 CGG
repeats
Garber et al., EJHG, 2008, PMID: 18398441
Anticipation

Definition – When a
dominant condition becomes
more severe in subsequent
generation of the same
family.
◦ Usually associated with
trinucleotide repeat disorders
Nussbaum and Ledbetter, Ann Rev Genet, 1986, 20:109-145.
Sequencing is not a good test for trinucleotide
repeat disorders
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Current sequencing
methods do not accurately
quantify repeat length,
especially long repeats
PCR Study of Fragile X Repeats
(CGG)n
Other methods are used
◦ PCR
◦ Southern blot
Chong et al., 1994, AJMG
Lecture Summary

The significance of variants can be assessed by
◦ Segregation analysis
◦ Association with disease in other families
◦ Modeling of impact on protein structure
◦ Type of impact on protein function is consistent with
molecular mechanism of disease – will this variant
act like other known mutations
Lecture Summary

Common Mendelian modes of inheritance and
associated recurrence risks
◦ Autosomal dominant
◦ Autosomal recessive
◦ X-linked recessive
◦ X-linked dominant
Lecture Summary

Strengths and limitations of commonly used molecular diagnostics
◦ Sanger sequencing
 Allele drop out
 Trouble with long repetitive sequences
 Cis vs trans
◦ Next-Generation sequencing
 Trouble with long repetitive sequences
 Cis vs trans
◦ Deletion-duplication testing
◦ Trinucleotide repeat sizing
Review Question

You are attempting to confirm a clinical diagnosis of a
recessive condition by DNA testing. You identify one
nonsense mutation in the affected patient in gene XXXZ.
What might you consider next?
◦ A) An alternative diagnosis
◦ B) Deletion-duplication testing of XXXZ
◦ C) Karyotype
◦ D) Investigating if another gene can cause this condition