LDL receptor genetics - Lipid · LDL receptor gene • Structure • Mutations related to FH •...

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


(Gene)mRNA

Met



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


(Gene)mRNA

Met




Small insertions or deletionsFrameshiftInframe

Large structural variants in DNA

pre-mRNA

mRNA

Deletion

DuplicationMost large SVs not tolerated


(Gene)mRNA

Met





Large structural variants (SVs)DeletionDuplicationTranslocation/inversion

pre-mRNA

mRNA

Breaking a gene: A review of terminologySingle nucleotide changes

MissenseNonsenseSplice-siteStartlossStoploss


Large structural variants (SVs)DeletionDuplicationTranslocation/inversion

Heterozygous

Homozygous

Compoundheterozygous

Doubleheterozygous

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


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


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


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


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?


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


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

LDL receptor genetics - Lipid · LDL receptor gene • Structure • Mutations related to FH •...

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Transcript of LDL receptor genetics - Lipid · LDL receptor gene • Structure • Mutations related to FH •...