ldl receptor genetics - lipid · ldl receptor gene • structure • mutations related to fh •...
TRANSCRIPT
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LDL receptor genetics
Nathan Stitziel, MD, PhDAssistant Professor of Medicine and GeneticsDirector, Center for Cardiovascular Genetics
Assistant Director, McDonnell Genome Institute
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DisclosuresNone
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“Breaking” a gene: a review of terminology
LDL receptor gene• Structure• Mutations related to FH• Finding mutations related to FH
Beyond LDLR: Other genetic causes of hypercholesterolemia
Putting it all together: genetic testing in FH
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DNA(Gene)
What is a Gene? A review of terminologyExons
Introns
pre-mRNA
Transcription
Splicing
mRNA
mRNA
Translation
Met
A U G C G G U G G U A G
Promotor
Arg TrpPolypeptide STOP
Post-translational modifications
Active protein
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Breaking a gene: A review of terminologyDNA
(Gene)mRNA
Met
A U G U G G C G G U A G
Trp ArgPolypeptide STOP
Missense
Met
A U G U G C C G G U A G
Cys Arg STOP
Nonsense
Met
A U G U G A C G G U A G
STOP
Splice-site
Aberrant Splicing
Single nucleotide changes in DNA
pre-mRNA
mRNA
Most missense changes tolerated
Most nonsense changes nottolerated
Most splice-site changes nottolerated
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Breaking a gene: A review of terminologyDNA
(Gene)mRNA
Met
A U G U G G C G G U A G
Trp ArgPolypeptide STOP
Single nucleotide changesMissenseNonsenseSplice-siteStartlossStoploss
Small insertions or deletions in DNA
Met
A U G C U G G C G G U A G
Leu Ala
Frameshift
Val Met
A U G C G G U A G
Arg
Inframe
STOP
pre-mRNA
mRNA
Most frameshift changes nottolerated
Some inframe changes tolerated
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Breaking a gene: A review of terminologyDNA
(Gene)mRNA
Met
A U G U G G C G G U A G
Trp ArgPolypeptide STOP
Single nucleotide changesMissenseNonsenseSplice-siteStartlossStoploss
Small insertions or deletionsFrameshiftInframe
Large structural variants in DNA
pre-mRNA
mRNA
Deletion
DuplicationMost large SVs not tolerated
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Breaking a gene: A review of terminologyDNA
(Gene)mRNA
Met
A U G U G G C G G U A G
Trp ArgPolypeptide STOP
Single nucleotide changesMissenseNonsenseSplice-siteStartlossStoploss
Small insertions or deletionsFrameshiftInframe
Large structural variants (SVs)DeletionDuplicationTranslocation/inversion
pre-mRNA
mRNA
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Breaking a gene: A review of terminologySingle nucleotide changes
MissenseNonsenseSplice-siteStartlossStoploss
Small insertions or deletionsFrameshiftInframe
Large structural variants (SVs)DeletionDuplicationTranslocation/inversion
Heterozygous
Homozygous
Compoundheterozygous
Doubleheterozygous
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Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
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Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
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Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
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Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
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LDLR mutations causing FH
Over 1,000 LDLR mutations identified for FH
Receptor negativeNo residual LDLR function (
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LDLR mutation type affects LDL level
Bertolini et al ATVB 1999
N=32 HoFH
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LDLR mutation affects drug response
HMG-CoA
HMG-CoA Reductase
Mevalonate
Cholesterol
Statins
LDL receptors
Efficacy in HoFH varies substantially:
LDLR receptor defective HoFH patients respond much better than LDLR receptor negative patients
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LDLR mutation affects drug response
LDLR receptor negative HoFH patients have little
response to PCSK9 inhibition
Mullard Nat Rev Drug Discovery 2012
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FH: a co-dominant condition
Adapted from Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
LDL=413 mg/dL
LDL=347 mg/dL
LDL=154 mg/dL
LDL=301 mg/dL
LDL=278 mg/dLLDL=204 mg/dL
LDL=695 mg/dL
When possible, genetic testing
should be pursued in
family members with
the most extreme
phenotype
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Beyond LDLR: other FH genes• Autosomal-dominant (or co-dominant)
hypercholesterolemia caused by:• LDLR• APOB• PCSK9
• Autosomal-recessive hypercholesterolemia caused by:• LDLRAP1
• Other notable recessive genes:• ABCG5, ABCG8, LIPA
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Finding mutations causing FHHistorical “Gold standard”: Sanger sequencing
Identifies all mutated basesIdentifies small insertions and deletions
Homozygousreference Heterozygous Homozygous
Heterozygous for frameshift
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Finding mutations causing FHHistorical “Gold standard”: Sanger sequencing
Identifies all mutated basesIdentifies small insertions and deletionsCostly and labor-intensive
Additional screening techniques previously used:High-resolution Melting Analysis (HRM)Single-Strand Conformation Polymorphism (SSCP)Denaturing Gradent Gel Electrophoresis (DDGE)and others
Goal of these techniques is to detect a region of the gene with possible sequence variant to focus subsequent Sanger sequencing
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Finding mutations causing FHHistorical “Gold standard”: Sanger sequencing
Identifies all mutated basesIdentifies small insertions and deletionsCostly and labor-intensive
Multiplex Ligation-dependent Probe Amplification (MLPA) used historically and typically today to detect large deletions and duplications
Next-generation sequencing becoming standard for many sequencing-based diagnostic tests
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How common are FH mutations?
What is the impact of FH?
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• 10,094 children, screened in early childhood (~1 year age)
• Screened all children for 48 most common FH mutations
• Full genetic evaluation in children with total cholesterol >95%
• Mutation carrier prevalence 1:273
Wald et al, NEJM 2016
Prevalence of FH: ~1:250
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Clinical impact of FH mutations?
OR for CAD
OR of CAD adjusted for LDL
LDL > 190 mg/dLwithout FH mutation
6 1.6
LDL > 190 mg/dLwith FH mutation 22 4.2
Diagnostic Yield and Clinical Utility of Sequencing Familial
Hypercholesterolemia Genes in Patients with Severe
Hypercholesterolemia
Khera et al, JACC 2016
OR for CAD
LDL > 190 mg/dLwithout FH mutation
6
LDL > 190 mg/dLwith FH mutation 22
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Beyond FH genes: other causes of hypercholesterolemia
41 families with monogenic dyslipidemia
13 with hypercholesterolemia
No mutations in LDLR, APOB, PCSK9 found clinically
3 families (of 13) harbored causal FH mutations: 23%
Stitziel et al, Circ Cardiovasc Genet 2015
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Beyond FH genes: other causes of hypercholesterolemia
Exome sequencing in 125 unrelated FH patients (clinical testing negative)
20% (25/125) had FH mutation previously undiscovered
Futema et al, J Med Genet 2014
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Beyond FH genes: other causes of hypercholesterolemia
Population lipid values
Three possibilities:
1. Single gene with large effect(monogenic i.e. LDLR)
2. Multiple genes of small effect (polygenic)
3. Environment
How does one get to the 1% tail?
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Beyond FH genes: other causes of hypercholesterolemia
Multiple genes of small effect (polygenic)
Common genetic variation explains ~20% of population heritability for lipid levels.
Can common variation explain some individual’s extreme LDL value?
nn SNPSNPSNPLDL *...** 22110 ββββ ++++=
Population polygenic LDL score
Likely monogenic etiology
Likely polygenic etiology
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Beyond FH genes: other causes of hypercholesterolemia
Multiple genes of small effect (polygenic)
Approximately 15-20% of individuals with severe hypercholesterolemia likely have a polygenic etiology
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Putting it all together: clinical genetic testing in FHClinical genetic testing for FH is available from several molecular diagnostic laboratories
Most labs perform Sanger or next-generation sequencing of LDLR, APOB, PCSK9, and/or LDLRAP1. Polygenic scores not available clinically.
Some offer single gene testing (LDLR) with reflex to deletion/duplication and/or other FH genes
Mostly covered by insurance
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Putting it all together: a practical approach to clinical genetic testing in FH
1) If previous FH genetic evaluation negative and done using “older” technology, can consider repeat testing
2) Some individuals have more than 1 mutation.
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Putting it all together: a practical approach to clinical genetic testing in FH
1) If previous FH genetic evaluation negative and done using “older” technology, can consider repeat testing
2) Some individuals have more than 1 mutation. Full FH panel in the most affected individual offers the most comprehensive genetic evaluation.
3) FH is common (~1:200-250) and has significant impact.
4) FH genetic testing can identify causal mutation in ~80% of individuals. Other causes include polygenic inheritance, environment, and unidentified monogenic causes.
LDL receptor genetics��Nathan Stitziel, MD, PhD��Assistant Professor of Medicine and Genetics�Director, Center for Cardiovascular Genetics�Assistant Director, McDonnell Genome Institute�DisclosuresSlide Number 3What is a Gene? A review of terminologyBreaking a gene: A review of terminologyBreaking a gene: A review of terminologyBreaking a gene: A review of terminologyBreaking a gene: A review of terminologyBreaking a gene: A review of terminologySlide Number 10Slide Number 11Slide Number 12Slide Number 13LDLR mutations causing FHLDLR mutation type affects LDL levelLDLR mutation affects drug responseLDLR mutation affects drug responseFH: a co-dominant conditionBeyond LDLR: other FH genesFinding mutations causing FHFinding mutations causing FHFinding mutations causing FHHow common are FH mutations?�� What is the impact of FH?Prevalence of FH: ~1:250Clinical impact of FH mutations?Beyond FH genes: other causes of hypercholesterolemiaBeyond FH genes: other causes of hypercholesterolemiaBeyond FH genes: other causes of hypercholesterolemiaBeyond FH genes: other causes of hypercholesterolemiaBeyond FH genes: other causes of hypercholesterolemiaPutting it all together: clinical genetic testing in FHPutting it all together: a practical approach to clinical genetic testing in FHSlide Number 33Putting it all together: a practical approach to clinical genetic testing in FH