A New Era is Emerging for and Research

Alfred / *eorge -r 0' Alfred L. George, Jr., MD is the 0agerstadt Professor and Chair of featured Ey the 'epartment of Pharmacology and 'irector of the Center for Nanion Technologies Pharmacogenomics at Northwestern University )einEerg School of 0edicine +is work has contriEuted The channelopathy field is an e[citing Eranch of ion greatly to understanding the channel research that comEines human genetics mechanisms Ey which ion channel cellular electrophysiology and pharmacology mutations cause a variety of inherited disorders of memErane The main goals of this field are to elucidate the e[citaEility molecular mechanisms of diseases caused Ey ion channel dysfunction and to develop more personali]ed treatment strategies Eased on an the interpretation Unfortunately for most individual’s 'NA sequence genes functional evidence to support or refute pathogenicity is challenging to oEtain )ortunately Ion channels are encoded Ey specific genes that this is not the case for genes encoding ion channels e[hiEit nucleotide and amino acid sequence which can Ee assayed with well-estaElished variation among individuals in the general e[perimental paradigms and methodologies The population In rare cases specific mutations gold standard approach for assessing functional cause altered function or impaired trafficking of effects of ion channel variants is patch clamp an ion channel protein Channelopathy is the term electrophysiologyphysiology But ththisis approacapproachh is ttediouse  used to descriEe those disorders associated with time- andnd laEor-or-intensiveintensive and not scalaEle to mutations in ion channel genes In recent years match thehe ramrampantpant ddiscoveryiscovery ooff new iionon cchannelh there has Eeen accelerated effort to sequence variants genes and genomes in persons with all forms of genetic diseases as well as healthy people and this has led to an e[plosion in the numEer of genetic variants discovered +owever the interpretation of findings from genetic studies can Ee difficult when variants are classified Ey the designation ¶variant of unknown significance’ (VUS) which is often the case when insufficient data e[ist to distinguish Eenign from pathogenic changes This distinction is critically important in clinical medicine for making a proper diagnosis planning appropriate genetic and reproductive counseling and in some cases for devising a clinical management plan

Nanion´s SyncroPatch 384i platform (successor of SyncroPatch A widely used approach for classifying genetic 384PE) allows effortless high-throughput ion channel screening variants enaEles inclusion of functional evidence coupled with high Áe[iEility and reliaEility due to the comEination of its patch clamp module with Beckman Coulter’s new i-Series demonstrating a damaging effect of a variant into liquid handling roEot Biomek i In the past few years an emerging group of pio- Eeen 'r Al *eorge 0agerstadt Professor Chair of neering researchers have utilized high throughput the 'epartment of Pharmacology and the 'irector automated patch clamp instrumentation originally of the Center for Pharmacogenomics Northwestern designed for drug discovery to study ion channel University )einEerg School of 0edicine +e variants at an unprecedented scale The SyncroPa- recognized the great potential of high throughput tch 384PE system has emerged as the automated automated electrophysiology to determine patch clamp system of choice among these groups the functional consequences of ion channel A pioneer in the study of ion channel variants has variants +is laE had the foresight to purchase a SyncroPatch 384PE system in 4 to characterize cardiac and Erain ion channel variants that were associated with congenital long-4T syndrome or genetic  respectively 2ne year later “... the SyncroPatch has they upgraded the instrument to have a second revolutionized our ability to 384-well module (SyncroPatch 8PE) Eecause of the tremendous initial success with this system determine the functional consequences of hundreds To date his laE has characterized the of human ion channel electrophysiological properties of more than  variants in voltage-gated sodium and variants, which could be potassium channels using the SyncroPatch considered one of the most 8PE system-4 with many more in the pipeline siJniÀcant recent advances 'etermining the functional effects of ion in channelopathy research.” channel variants can help with classification as potentially disease causing or not In their recent study of long 4T syndrome type  the *eorge

7KH6\QFUR3DWFK3(UHFRUGLQJVRIWKHFDUGLDFVORZGHOD\HGUHFWLÀHUFXUUHQW ,.V IURP&+2FHOOVH[SUHVVLQJ.&14DQG.&1( The Àgure shows a snapshot of SyncroPatch 8PE software measurements made at Northwestern University *reen Àelds represent successful recordings indicating a success rate of appro[  remarkaEle and enaEles a new way of looking at ion channel diseases and their treatment

While this pioneering work is still emerging and its potential is Eeing rapidly realized other laEs have also emEraced the SyncroPatch 384PE to perform similar studies -en Pan Ph' 'irector Translational NeuroEiology at the Stanley Center of the Broad Institute is another early adopter of 'U &DUORV 9DQR\H H[SODLQV WKH 1RUWKZHVWHUQ 8QLYHUVLW\ 6\QFUR3DWFK 3( WR :LOO +XWVRQ DQG /HDK 6FKXVW IURP WKH the SyncroPatch platform working with it since )DPLOLH6&1$ )RXQGDWLRQ Together with the memEers of early - +er laEoratory and collaEorators the foundation researchers at Northwestern University are collaEorating with focus on a sodium channelopathy involved are using SyncroPatch recordings to study the in neuronal disorders (SCNA encodes one of the Erain mechanism of action of ion channel modulators sodium channels involved with epilepsy and autism) )or more information visit wwwscnaorg identified from high throughput )/IPR assays In addition her group characterized genetic variants of genes that encode voltage-gated sodium and calcium channels that are implicated in schizophrenia and neurological disorders They “... we believe the Syncro- developed novel methods for noise analyses using Patch platform will transform the SyncroPatch that can estimate single channel our approach to determin- conductance and surface e[pression (Nanion User meeting ) They presented a methodology to ing the most effective phar- predict gain-of-function (*2)) and loss-of-function macological remedy for cer- (/2)) and then validated the model Ey studying tain , and  variants on the SyncroPatch 384 system enable a precision medicine Researchers at the Victor Chang institute in approach to these rare ge- Australia puElished a paper recently assessing the netic conditions.” functional properties of 3 variants of the .CN+

gene encoding the .V  (or hER*) channel which is a maMor gene responsiEle for congenital long 4T syndrome8 Using the SyncroPatch 384PE system they e[amined the full range of functional (cont) laEoratory was aEle to reclassify ! of attriEutes of heterologously e[pressed hER*

.CN4 (encoding .V ) variants of uncertain channels and were aEle to distinguish Eenign from significance as likely pathogenic 0ost recently pathogenic variants they presented a study of 8 .CN4 variants at the annual meeting of the American Epilepsy Society In another recent paper researchers at VanderEilt University studied cardiac (SCNA

2nce functional defects in ion channel variants gene encoding NaV ) variants associated with are identified researchers can evaluate whether Brugada and long 4T syndromes They studied e[isting drugs or investigational compounds can 83 SCNA variants including  positive and  correct the dysfunction and potentially translate negative controls They identiÀed 4 new or partial this information to more precise pharmacotherapy loss-of-function variants and were aEle to reclassify in the clinic The potential for this strategy to  of  variants of uncertain signiÀcance using the change the treatment of human channelopathies is SyncroPatch 384PE system 0any more variants associated with ion channel References: diseases are identiÀed every year through clinical  9anoye C* 'esai RR )aEre ./ *allagher S/ Potet ) 'e.eyser -0 0acaya ' 0eiler - Sanders CR *eorge A/ -r +igh-Throughput )unctional Evaluation of genetic testing and genomic research studies .CN4 'ecrypts 9ariants of Unknown Significance Circ *enom Precis 0ed A large proportion of the identiÀed variants get 8 :e34  .ang S. 9anoye C* 0isra SN Echevarria '0 Calhoun -' 2’Conner -B )aEre classiÀed as variants of unknown signiÀcance ./ 0c.night ' 'emmer / *oldenEerg P *rote /E Thiffault I Saunders C Strauss .A Torkamani A van der Smagt -- van *assen ./ Carson RP 'ia] - /eon E Eut the use of automated electrophysiology on -acher -E +anniEal 0C /itwin - )riedman NR SchreiEer A /ynch B Poduri A

0arsh E' *oldEerg E0 0illichap -- *eorge A/ -r .earney -A Spectrum of .V platforms such as the SyncroPatch 384PE can  dysfunction in .CNB-associated neurodevelopmental disorders Ann Neurol provide much needed functional data to Eetter  8:8- 3 Calhoun -' Vanoye C* .ok ) *eorge A/ -r .earney -A Characteri]ation of classify these variants a .CNB variant associated with autism intellectual disaEility and epilepsy Neu- rol *enet  3:e8 4 *ertler TS Thompson C+ Vanoye C* 0illichap -- *eorge A/ -r )unctional Automated patch clamp systems such as the consequences of a .CNT variant associated with status dystonicus and early- onset infantile encephalopathy Ann Clin Transl Neurol  :- SyncroPatch 384PE are poised to further transform  Pan -4 Bae]-Nieto ' Allen A :ang +R Cottrell -R 'eveloping +igh-Throughput Assays to Analy]e and Screen Electrophysiological Phenotypes 0ethods 0ol Biol the channelopathy Àeld enaEling researchers the 8 8:3- throughput needed to determine the functional  +eyne +2 Bae]-Nieto ' IqEal S Palmer ' Brunklaus A Epi CollaEorative -ohannesen .0 /au[mann S /emke -R 0oller RS Pere]-Palma E Scholl U SyrEe consequences of newly discovered ion channel S /erche + 0ay P /al ' CampEell A- Pan - :ang +R 'aly 0- Predicting )unc- tional Effects of 0issense Variants in Voltage-*ated Sodium and Calcium Chan- variants collect novel data on structure-function nels EioR[iv  43 relationships ascertain molecular mechanisms  Bae]-Nieto ' Allen A *oshal A

Contact Information

Alfred L. George, Jr., MD Jen Pan, Ph.D. Chair 'epartment of Pharmacology 'irector Translational NeuroEiology 'irector Center for Pharmacogenomics Institute Scientist 0agerstadt Professor of Pharmacology Broad Institute of 0IT and +arvard Northwestern University The Ted and 9ada Stanley Building )einEerg School of 0edicine  Ames Street Searle Building Room 8- CamEridge 0A 4 3 E Superior httpswwwEroadinstituteorgEiosMen-pan Chicago I/  httpswwwpharmnorthwesternedu

Acknowledgments

:e thank Al and -en for sharing their views on the importance of ion channel and channelopathy research :e are grateful for your valuaEle insights and wonderful collaEoration

Nanion Technologies *mE+ I *anghoferstra‰e A 833 0unich *ermany I 4 8  - I info#nanionde I wwwnanionde