MOLECULAR GENETIC DIAGNOSTICS IN LONG QT SYNDROME

Az SZTE Kutatóegyetemi Kiválósági Központ tudásbázisának kiszélesítése és hosszú távú szakmai fenntarthatóságának megalapozása

a kiváló tudományos utánpótlás biztosításával”

Genomikai Központ

„PhD szakmai találkozó, munkabeszámoló”

MOLECULAR GENETIC DIAGNOSTICS IN LONG QT SYNDROME

Lidia Haţegan Supervisors:

Miklós Szekeres, PhD, DSc; Biological Research Center Róbert Sepp, PhD; 2nd Department of Internal Medicine and

Cardiology Center, Univeristy of Szeged

2012. 05. 25.

TÁMOP-4.2.2/B-10/1-2010-0012 projekt

MOLECULAR GENETIC DIAGNOSTICS IN LONG QT SYNDROME

Lidia Haţegan

Supervisors:

Miklós Szekeres, PhD, DSc; Biological Research Center

Róbert Sepp, PhD; 2nd Department of Internal Medicine and Cardiology Center, Univeristy of Szeged

Long QT Syndrome

• delayed cardiac repolarization – QT prolongation in ECG

• increased risk for sudden cardiac death

• autosomal dominant disorder

• incomplete penetrance and variable expressivity

•  -blockers – protective effect, defibrillator- profilactic effect

• mutation in 3 major genes

LQTS locus Localization Affected gene Affected ion

channel Prevalence LQT1 11p15.5 KCNQ1 (KvLQT1) potassium (IKs) ~50%

LQT2 7q35-36 KCNH2 (HERG) potassium (IKr) ~35-40%

LQT3 3p21-24 SCN5A sodium (INa) ~10-15%

LQT4 4q25-27 ankyrin B (ANKB) ?

LQT5 21q22 KCNE1 (minK) potassium (IKs) 2-3%

LQT6 21q22 KCNE2 (MiRP1) potassium (IKr) <1%

LQT7 13q22 KCNJ2 potassium (Kir2.1) ?

LQT8 12p13.3 CACNA1C calcium (ICa) <1%

LQT9 3p25 caveolin-3 (CAV3) <1%

LQT10 11q23.3 SCN4B sodium (INa) <1%

JLNS1 11p15.5 KCNQ1 (KvLQT1) potassium (IKs) ? JLNS2 21q22 KCNE1 (minK) potassium (IKs) ?

LQTS: causative genes

Clinical evaluation of Andersen-Tawil Syndrome (LQT7)

• periodic paralysis

• ventricular arrhythmias

• dysmorphic features

• different from „usual” LQTS:

– extracardiac manifestations – QT prolongation is mild

– characteristic bidirectional VT

Molecular basis of Andersen-Tawil Syndrome

• autosomal dominant inheritance

• KCNJ2 gene – inward rectifier K⁺ channel Kir2.1 protein

• I_K1 – terminal phase of repolarization

LQTS mutations in Hungarian patients

Proband Causative gene Mutation Localisation LQTS subtype

L 1.0 KCNQ1 Tyr315His pore LQT1

L 2.5 KCNQ1 Arg259Cys S4/S5 LQT1

L 18.0 KCNE1 Val109Ile C-terminal LQT5

L 20.0 KCNH2 Thr162+6X N-terminal LQT2

D 190.0 KCNH2 Trp568Cys S5 LQT2

L 64.0 SCN5A Glu1784Lys C-terminal LQT3

Aim

• Genetic screening for mutations in LQTS

patients in the Hungarian population, especially

in those showing phenotype of Andersen sy.

Patients

• 4 female patients

• average age: 28±10

• frequent ventricular extrasystolia on ECG

• mild QT prolongation

Materials and methods

Blood sampling

(usually 2x7 ml of blood, lavender top tube

DNA isolation from whole blood

Direct sequencing of PCR products on a ABI 310 Genetic Analyser

PCR amplification of all coding regions of the KCNJ2 gene

KCNJ2 gene analysis strategy

• Location: 17q24.3

• 2 exons, transcript length: 1685 bp, translation length: 427 aa

• Direct sequencing the whole coding sequence in 4 overlapping fragments (396 bp, 400 bp, 385 bp, 485 bp)

Results: case no.1.

ATA CTG GAA GGC ATG GTG

exon 2 normal

mutant

Ile Leu Glu Gly Met Val Glu Ala

KCNJ2 heterozygous mutation:

del1288-1290TGG (del302V)

GAA GCC ACT GCC

Thr Ala

Ile Leu Glu Gly Met Ala Thr Ala

ATA CTG GAA GGC ATG GAA GCC ACT GCC

Glu

KCNJ2 del302V mutation: novel

Tristani-Firouzi M et al. J Clin Invest, 2002

KCNJ2 V302M mutation: cellular electrophysiology

• Mutant subunits were unable to form functional homomultimetric channels

• When coexpressed with WT subunits, current amplitude was reduced indicating a dominant negative effect

Case history

• 15-year-old female

• ventricular arhythmias, mainly manifesting as frequent

ventricular premature beats or short runs of non-sustained ventricular tachycardia

• typical bidirectional ventricular tachycardia was not recorded

• the arrhythmia did not respond to beta blockers or sotalol, amiodarone was somehow effective but it had to be stopped because of thyreotoxicosis

• Echocardiography: normal cardiac parameters and function

• leg pain or numbness, lasting for several days

• facial features including low-set ears, hypertelorism and micrognathia

Future plans

• To exclude the possibility that the del302V mutation is a rare polimorphism (check 100-200 controls)

• Family screening (clinical and genetic)

• To finalize the sequencing of the KCNJ2 gene in the remaining patients

• Functional analysis of the del302V mutation:

expression in a cell line and ion current recordings (in collaboration with the Department of Pharmacology and Pharmacotherapy)

• Screening our LQTS patient cohort for major LQTS

genes (KVQT1, KCNH2, possibly SCN5A genes)

Presentations

• Hategan L. Molecular genetic diagnostics in long QT syndrome. Sudden cardiac death: from bench to

bedside. Seminar for young researchers. 2012. May 4.

Timisoara, Romania.

• Hategan L, Katona M, Környei L, Rácz K, Csanády M, Forster T, Sepp R. Identification of a novel KCNJ2

mutation causing Andersen-Tawil syndrome in a

Hungarian patient. 13th Alpe-Adriatic Cardiology

Congress. 2012. June 7-9, Bratislava, Slovakia.

Thank you for your attention!