functional annotation of human ion channel variants · congenital long qt syndrome clinical...

Functional Annotation of Human

Ion Channel Variants

Alfred L. George, Jr., M.D. Department of Pharmacology

Genetic Arrhythmia Syndromes

Congenital Long QT Syndrome KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, ANK2, SCN4B, CAV3, AKAP9, SNTA1

Brugada Syndrome SCN5A, GPD1L, CACNA1C, CACNB2, SCN1B, KCNE3, SCN3B

Andersen Syndrome KCNJ2

Timothy Syndrome CACNA1C

Familial Cardiac Conduction Diseases SCN5A

Short QT Syndrome KCNQ1, KCNH2, KCNJ2

Catecholaminergic Polymorphic VT RYR2, CASQ2, TRND

Familial and Lone Atrial Fibrillation KCNQ1, KCNE2, KCNA5, SCN5A, SCN10A, NPPA

Congenital Long QT Syndrome

Clinical Features • Congenital prolongation of the rate-corrected QT interval QTc 440 msec in symptomatic individuals or QTc 460 in asymptomatic individuals • Recurrent syncope • Cardiac arrest or torsade de pointes • Family history of unexplained and premature sudden death • Incomplete penetrance and variable expressivity • Congenital deafness (recessive Jervell, Lange-Nielson)

• Molecular diagnostics - technically challenging (13 genes)

QT > 440 ms

QT < 440 ms

P

Q

R

S

T

P

Q

R

S

T

Normal ECG

Congenital LQTS

Congenital Long QT Syndrome (LQTS)

Torsades de pointes

Congenital Long QT Syndrome

Genetic Heterogenity in Congenital LQTS

Gene Current Frequency Trigger

LQT1 KCNQ1 ↓ IKs 45% Exercise, emotion

LQT2 HERG ↓ IKr 40% Sudden auditory stimuli

LQT3 SCN5A ↑ INa 10% Sleep

LQT4 Ank2 complex Rare

LQT5 KCNE1 ↓ IKs Rare

LQT6 KCNE2 ↓ IKr Rare

LQT7 KCNJ2 ↓ IK1 Rare

LQT8 CACNA1C ↑ ICa Rare

I Na

I Ca

I Ks

I Kr

I to

I K1

Na +

Ca 2+

K +

extracellular

intracellular

KCNQ1 and KCNE1 Generate IKs

IKs

Slow delayed rectifier current

in heterologous cells 11

KCNQ1

11p15.5

LQT1

21 KCNE1

21q22.1

LQT5

7

Mutations in Genetic Arrhythmias

Gene Mutations in HGMD

KCNQ1 602

KCNH2 910

SCN5A 802

RYR2 308

CACNA1C 57

Genetic Testing for LQTS

• Commercial genetic testing for LQTS has become standard-of-care

• Most variants discovered in LQTS cases are missense

• Most new variants have never been seen before (‘private mutations’)

• Most variants have uncertain functional consequences

• Diagnostic labs reports many ‘variants of uncertain significance’

• No reliable methods to distinguish benign from pathogenic variants

9

Classification of Mutations

Variant of Unknown

Significance Pathogenic

Likely

Pathogenic Benign

Likely

Benign

Strategies for Variant Annotation

Computational (in silico)

Experimental (in vitro)

Experimental (in vivo)

Variants without clear pathogenicity confound genetic diagnosis

2015

Strong evidence for pathogenicity:

Conventional patch clamp:

- 16 measurements/person/day

- days-weeks per mutation

Functional Annotation of Ion Channels Variants

Patch clamp electrophysiology is the gold standard

Functional Annotation of Ion Channels Variants

Automated patch clamp:

- 768 measurements per hour

- multiple variants per day

SyncroPatch 768PE

Patch clamp electrophysiology is the gold standard

Electroporation Automated patch-clamp

Functional Analysis of KCNQ1 Variants by Automated Electrophysiology

Overcoming need for stable cell lines

Electroporation of KCNQ1 and KCNE1 evaluated by flow cytometry

Transient Co-expression of KCNQ1 and KCNE1

Viability: 84.9% Percent Co-transfected: 79.1%

CMV

KCNQ1

IRES2EGFP

Kan R

KCNQ1

CMV

KCNE1

IRES2

DsRed2Kan R

KCNE1

Automated Electrophysiology of IKS (Transient transfection of CHO with KCNQ1/KCNE1)

500 ms

Manual Patch SyncroPatch

Normalized to peak current measured at +60 mV

Automated Electrophysiology of IKS (Transient transfection of CHO with KCNQ1/KCNE1)

Manual Patchn = 18

-50-60 -40 -20 20 40 60

I, pA/pF

Vm, mV

500

400

200

300SyncroPatch

n = 153

-80

100

Vm, (mV)I

/ I m

ax

1.0

0.8

0.6

0.4

0.2

0.0

0 40 6020-20-40-60-80

Manual Patchn = 18

V1/2

= 22.5 +/- 0.8 mV

SyncroPatchn = 153

V1/2

= 27.8 +/- 1.4 mV

Automated Electrophysiology of IKS (Transient transfection of CHO with KCNQ1/KCNE1)

Automated Electrophysiology of IKS (Transient transfection of CHO with KCNQ1/KCNE1)

Au

tom

ated

Man

ual

I132L

WT

C122Y

K196T

R174C

G179S

Automated vs Manual Electrophysiology

I132L

WT

C122Y

K196T

R174C

G179S

Au

tom

ated

Man

ual

Au

tom

ated

Man

ual

Criteria to Classify KCNQ1 Variant Function

Values Relative

to WT IKs

Normal Near

Normal

Mild

Loss of

Function

Severe

Loss of

Function

Severe

Gain of

Function Ipeak

@ +60 mV

90-110% 75-90 &

110-125% 25-75% <25% >150%

or

Activation V½ (mV)

<5 mV depolarization/

hyperpolarization

10-5 mV depolarization/

hyperpolarization

10-20 mV depolarization

>20 mV depolarization

>15 mV hyperpolarization

plus 120-150% Ipeak

Developed from a curated list of high-quality publications

on 107 variants with functional characterization

ClinVar Assertions for 36 KCNQ1 Variants

VUS Conflicting

Interpretation

Likely benign

Likely

Pathogenic

Pathogenic

No assertion

16/36 (44%) have clear classification

Original classifications

Reclassifiying 36 KCNQ1 Variants

Original Functional

Likely pathogenic Severe LOF

VUS Severe LOF

No assertion Severe LOF

No assertion Severe GOF

No assertion Normal

Likely benign Normal

Variant of Unknown

Significance Pathogenic

Likely

Pathogenic Benign

Likely

Benign

Reclassifiying 36 KCNQ1 Variants

Original Functional Reclassification

Likely pathogenic Severe LOF Pathogenic

VUS Severe LOF Likely pathogenic

No assertion Severe LOF Likely pathogenic

No assertion Severe GOF Likely pathogenic

No assertion Normal Likely benign

Likely benign Normal Benign

Variant of Unknown

Significance Pathogenic

Likely

Pathogenic Benign

Likely

Benign

ClinVar Assertions for 36 KCNQ1 Variants

VUS Conflicting

Interpretation

Likely benign

Likely

Pathogenic

Pathogenic

No assertion

16/36 (44%) have clear classification

VUS

Likely benign

Likely

Pathogenic

Pathogenic

30/36 (83%) have clear classification

Original classifications Revised classifications

Automated Electrophysiology of Human

Ion Channels

Sodium channels SCN1A Epilepsy, Dravet syndrome SCN2A Epilepsy, autism spectrum disorder SCN3A Epilepsy SCN5A Cardiac arrhythmia SCN8A* Epileptic encephalopathy SCN9A* Genetic pain disorders SCN10A* Genetic pain disorders SCN11A* Genetic pain disorders Potassium channels KCNE1 Cardiac arrhythmia KCNJ2 Anderson syndrome KCNB1 Epileptic encephalopathy KCNT1* Epileptic encephalopathy * Work in progress

epilepsy, familial migraine

epilepsy, autism

epilepsy

periodic paralysis, paramyotonia

epilepsy

painful neuropathy, congenital insensitivity to pain

LQTS, Brugada syndrome, cardiomyopathy

painful neuropathy, Brugada syndrome

painful neuropathy, congenital insensitivity to pain

>2,100 variants/mutations in HGMD

Sodium Channelopathies

SyncroPatch Recordings of NaV1.1 (stable expression in HEK293)

Holding potential -120 mV; single hole recordings

SyncroPatch Recordings of NaV1.1 (stable expression in HEK293)

SyncroPatch Recordings of NaV1.1 Persistent Current

NaV1.1-R1648H NaV1.1-WT

Pe

rsis

ten

t I N

a (

% P

ea

k)

NaV1.1-R1648H NaV1.1-WT NaV1.1-WT

NaV1.1-R1648H NaV1.1-WT

SyncroPatch Recordings of NaV1.1 Persistent Current

SCN1A Variants of Unknown Significance associated with Dravet syndrome

Transient expression in HEK293

Variants reported by Wu, et al. Pediatrics 2015

Automated Electrophysiology of NaV1.2*

Comparison of manual with automated patch clamp

*NaV1.2 is the sodium channel encoded by SCN2A

p.Glu430Ala

p.Tyr816Phe

p.Arg853Gln

p.Gly879Arg

p.Ala880Ser

p.Gly882Glu

p.Glu999Lys

p.Met1879Thr

p.Arg1882Gln

p.Arg188Trp p.Arg379His p.Arg937Cys p.Arg937His

p.Cys1386Arg p.Thr1420Met p.Lys1422Glu p.Glu1880Lys p.Arg1882Leu

Variants associated with epileptic encephalopathy or autism

Automated Electrophysiology of NaV1.2

Variants associated with epileptic encephalopathy or autism

Automated Electrophysiology of NaV1.2

Variants associated with epileptic encephalopathy or autism

Automated Electrophysiology of NaV1.2

Creation of Neuronal Cell Line (ND7/no-Nav) with suppressed endogenous Na current

Non-transfected

+NaV1.7 siRNA

+CRISPR_NaV1.7

5 ms

100 pA/pF

5 ms

100 pA/pF

ND7/23 ND7/no-Nav

Creation of Neuronal Cell Line (ND7/no-Nav) with suppressed endogenous Na current

Pharmacological Profiling of Sodium

Channel Mutations in Epilepsy

+

Acknowledgments

Northwestern Univesity

Carlos Vanoye, Ph.D.

Christoher Thompson, Ph.D.

Reshma Desai, M.S.

Katarina Fabre, M.S.

Jean-Marc DeKeyser, M.S.

Tatiana Abramova, M.S.

Vanderbilt University

Chuck Sanders, Ph.D.

Jens Meiler, Ph.D.

GeneDx, Inc.

Daniela Macaya, Ph.D.