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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
DisclosuresNone
“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
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
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
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
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
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
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
Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
Soutar AK and Naoumova RP. Nat Clin Pract Cardiovasc Med 2007
LDLR mutations causing FH
Over 1,000 LDLR mutations identified for FH
Receptor negativeNo residual LDLR function (
LDLR mutation type affects LDL level
Bertolini et al ATVB 1999
N=32 HoFH
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
LDLR mutation affects drug response
LDLR receptor negative HoFH patients have little
response to PCSK9 inhibition
Mullard Nat Rev Drug Discovery 2012
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
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
Finding mutations causing FHHistorical “Gold standard”: Sanger sequencing
Identifies all mutated basesIdentifies small insertions and deletions
Homozygousreference Heterozygous Homozygous
Heterozygous for frameshift
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
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
How common are FH mutations?
What is the impact of FH?
• 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
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
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
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
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?
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
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
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
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.
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