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4.1.2.1.1 Control Cases

Neurologically normal, healthy control LCL’s (n=100) [64-M, 36-F] were largely re- cruited to the ECACC replication cohort from patient partners and their unrelated carersintheNationalMNDADNABank(Section 2.1.2). These were comprised of 31

Birmingham London Sheffield ALL Familial SOD1 exon 4 p.D102G p.C111Y p.I114T 0 1 0 0 0 0 1 0 2

not known not known 0 1 0 4.4%

TARDBP exon 6 p.G348V

p.M337V 0 0 1 0 1 2 1.2%

FUS exon 15 p.Q519E

p.R521H p.R522G 0 0 0 1 1 1 0 0 0 3.0%

C9ORF72 non-coding G4C2 repeat 15 19 15 40.8%

C9ORF72+OPTN exon 10 p.E322K 0 0 1 0.8%

Total 16 24 21 50.8%

Sporadic

ANG exon 2 p.K54E 0 0 1 0.4%

C9ORF72 non-coding G4C2 repeat 2 2 21 10.0%

CHMP2B exon 1

exon 3 p.I29V-SNP p.Q206H-SNP 0 0 0 0 1 1 0.8%

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Birmingham(31.0%)casesincluding nine from Liverpool [C117-(BLi0224), C130-( BLi0105),C148-(BLi0259),C160-(BLi0032),C162-(BLi0198), C175-(BLi0021),C18 5-(BLi0240), C188-(BLi0074) and C195-(BLi0204)], one from Oxford [C110-(BOx0 008)], onefromBelfastCity Hospital [C153-(BBe0002)] and one from Bristol Fren- chay Hospital [C150-(BBr0020)]; 28 London (28.0%) cases including two from Po- ole General Hospital [C119-(LPo0059) and C122-(LPo0087)], two from Cambridge [C189-(LCa0093) and C190-(LCa0134)] and one from Plymouth Derriford Hospital [C125-(LPy0011)] and 41 Sheffield (41.1%) cases including six from Newcastle [C1 02-(SNc0201), C109-(SNc0158), C135-(SNc0125), C144-(SNc0078), C146-(SNc016 7)andC157-(SNc0015)],tenfromManchester[C111-(SMa0096),C124-(SMa0227), C133-(SMa0009), C139-(SMa0188), C151-(SMa0050), C163-(SMa0054), C167-(SM a0203), C170-(SMa0232), C171-(SMa0133) and C194-(SMa0013)], one from Prest- on [C104-(SPr0016)], one from Nottingham [C177-(SNt0075)] and two from Durh- am[C197-(SDu0009)andC120-(SDu0007)].Ageatconsultationrangedbetween35 and83yearswithameanof63.4±10yrs. The M:F ratio was 1.78:1 (Figure 4.4) [APP ENDIX TABLE B5].

SALS Control

M:F 2.17:1 1.78:1

Age 59.7±11yrs 63.4±10yrs

Total 250 100

Figure 4.4 Age Frequency Distribution of Patient and Control LCL’s in the ECACC Replication Cohort (n=350)

Abbreviations: ALS - Amyotrophic Lateral Sclerosis, Ctrl - control, ECACC - European Collection of Cell Cultures, F - female, LCL - lymphoblastoid cell line, M - male and S - sporadic

Fr eq ue nc y 60 50 40 30 20 10 0 30 35 40 45 50 55 60 65 70 75 80 85 90 Age (years) SALS Ctrl

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4.1.2.1.2 Sporadic Cases

SALS patient derived LCL’s (n=250) [171-M, 79-F] recruited to the ECACC replicati- oncohortwerecomprisedof107Birmingham(42.8%)casesincludingonefromBe- lfastCityHospital[S432-(BBe0015)];96 London (38.4%) cases including four from Southampton General Hospital [S322-(LSh0002), S326-(LSh0016), S405-(LSh0028 )andS473-(LSh0004)]andninefromPooleGeneralHospital [S289-(LPo0003), S29 8-(LPo0001),S349-(LPo0016),S362-(LPo0019),S383-(LPo0018),S394-(LPo0009), S429-(LPo0039), S485-(LPo0005) and S489-(LPo0017)] and 47 Sheffield (18.8%) cases including two from Preston [S446-(SPr0005)andS456-(SPr0001)], two from Durham [S327-(SDu0001) and S476-(SDu0005)], seven from Nottingham [S274-(S Nt0008), S359-(SNt0012), S407-(SNt0042), S427-(SNt0021), S463-(SNt0013), S46 4-(SNt0040)andS492-(SNt0019)]and five from Newcastle [S299-(SNc0028), S305 -(SNc0043), S380-(SNc0049), S420-(SNc0057)andS466-(SNc0020)]. The M:F ratio was 2.17:1 (Figure 4.4) [APPENDIX TABLE B6].

The majority of SALS patients, 97.2% (n=243/250) were diagnosed (Section 1.1.3) (Figure 1.1) with either definite (n=81/243) or probable ALS (n=162/243) includ- ing one incidence of concomitant PD (Section 1.2.1) [S455-(BP6152)] (Table 4.1). Of the remaining 2.8% (n=7/250) of the cohort, two patients were diagnosed with PLS (Section 1.2.2.2) [S301-(SP3402) and S497-(SP3234)] and five were diagnosed with PBP (Section 1.2.2.1) [S272-(SP3253, S299-(SNc0028), S308-(BP6328), S349- (BP6100) and S359-(SNt0012)]. Age at symptom onset ranged between 31 and 87 years with a mean of 59.7±11yrs. Survival ranged from 5 months to 10 years and 3 months in 80.0% (n=200/250) of cases with a mean of 3.72±2.0yrs. For 14.8% (n= 37/250)ofSALSthisinformationwas not available and in the remaining 5.2% (n=1 3/250)ofcasestheindividualwasstillreported to be alive as of June 2013 with mi- nimum disease duration of either 7 [S281-(LP0482) and S304-(LP0400)], 8 [S298- (LPo0001), S326-(LSh0016), S351-(LP0451), S452-(LP0195), S453-(LP0380) and S454-(LP0364)], 9 [S311-(LP0086), S334-(LP0442) and S395-(LP0109)], 10 [S321 -(LP0003)]or12years[S300-(LNh0020)],respectively.Onsetoccurred focally in at least 90.0% (n=225/250) of cases. Of these, 71.5% (n=161/225) [150-SALS and 2- PLS(sporadic)]presented with asymmetric weakness in the upper and/or lower li- mbs,26.7%(n=60/225)[55-SALSand 5-PBP (sporadic)] experienced bulbar symp-

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tomsandafurther1.8%(n=4/225)[4-SALS]hadimpairedrespiratoryfunction.The remaining10.0%(n=25/250)ofthecohortcomprised20 SALS cases of mixed pres- entationandfiveincidenceswherethesiteofonsetwasnotknown.ALSFRS-Rscores rangedbetween0 (severe disability) and 48 (normal function) with an average of 3 4.2±10 for 55.6% of SALS (n=139/250) (Figure 4.5).

Figure 4.5 ALSFRS-R Frequency Distribution of SALS (n=250) Recruited to the ECACC Replication Cohort Revised ALS Functional Rating Scale scores on a

scale of 0 to 48 where zero represents severe disability and a score of forty-eight signifies that normal function is preserved. Mean ALSFRS-R plotted along the x- axis is computed based on measurements that are incorporated from a series of twelve questions relating to the patient’s ability to perform common daily tasks including but not limited to: speech, salivation, swallowing, dressing and hygiene, walking, climbing stairs, handwriting and shortness of breath (SOB) (Cedarbaum et al 1999). Relative counts or frequencies along the y-axis were calculated as a percentage of the total number of SALS (n=139/250) in the ECACC replication cohort for which this information was available.

Abbreviations: ALS - Amyotrophic Lateral Sclerosis, ECACC - European Collection of Cell Cultures, FRS-R - Revised Functional Rating Scale and S - sporadic.

FortheSheffieldcasessome additional clinical information could be extracted from the local database. Of these 4.3% (n=2/47) [2-SALS] were prone to EL, 10.6% (n=5 /47) [5-SALS] suffered pronounced dyspnoea with a fifth in need of NIPPV support (Section 1.1.4) and one individual presented with the rarer FA variant (Section 1.1. 5.1)[S467-(SP3198)].Dysarthriaanddysphagiawerealsoapparentin23.4%(n=11 /47)[10-SALSand1-PBP(sporadic)]ofthecohortwithPEG feeding becoming a ne- cessaryinterventioninthecase the PBP patient. Three individuals were non-ambu- lantwithafurther 12.8% (n=6/47) [6-SALS] unable to walk without assistance. Cu-

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rrent smokers comprised 10.6% (n=5/47) [5-SALS] of the cohort, ex-smokers who hadnotsmokedacigaretteinthelast 8 years a further 4.3% (n=2/47) [2-SALS] and non-smokers 42.6% (n=20/47) [19-SALS and 1-PBP (sporadic)]. For the remaining 42.5% (n=20/47) [13-SALS, 1-SALS+PD, 4-PBP (sporadic) and 2-PLS (sporadic)] of cases this information was not disclosed.

There were reports of at least one other neurological condition occurring in a first degreerelative of 8.5% (n=4/47) of Sheffield pedigrees which received a diagnosis of SALS in the ECACC replication cohort including PD (Section 1.2.1) [S306-(SP343 9)andS482-(SP3367)],HD (Section 1.4.1.2.1) [S284-(SP3069)], tremor [S461-(SP3 107)] and ALZ (Section 1.3.1.3.1) [S272-(SP3253)]. Incidences of cancer affecting 6 .4%(n=3/47)[1-bowel,1-breastand1-other],strokes2.1%(n=1/47)orpost-viral motor/immune disorder 2.1% (n=1/47) were also reported.

4.1.2.2 Therapeutics Used to Treat the Cohort

Riluzole,administered at a dose of 50mg twice daily, was being used to treat 88.4% ofSALS cases(n=221/250)[213-SALS,1-SALS+PD, 4-PBP and 2-PLS (sporadic)]. In asmallnumberofinstances,approximately3.6%(n=8/221)[8-SALS]theindividual experiencedadversesideeffectsandthemedicationwaswithdrawn.VitaminC/Eor multivitaminsupplementswerecollectively being taken by 26.4% (n=66/250) [63- SALS,2-PBPand1-PLS(sporadic)]of the cohort. As well as the Riluzole therapy, se- veral patients (n=4/250) [4-SALS] were also participants of the Novartis [S450-(LP 0014)], ONO-2506 (Arundic acid) [S413-(LP0019)], minocycline [S253-(BP6180)] or glatiramer acetate (Copaxone®_{) [S333-(LP0432)] drug trials [APPENDIX TABLE}

B4].Incontrast,14.0%(n=35/250)[34-SALSand1-PBP(sporadic)]chosenotto re- ceive any kind of disease modifying therapy and for the remaining 0.8% (n=2/250) [2-SALS] of the cohort this information was not available.

4.1.2.3 Genetic Status of the Cohort

TARDBPmutationswere reported in the ECACC replication cohort at a frequency of

1.2%(n=3/250).Theyincludedac.269C>T(p.A90V)SNPinexon3[S412-(SP3147)] andtwosinglebasesubstitutions,namelyc.859G>A(p.G287S)[S461-(SP3107)]and

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c.962C>T(p.A321V)[S352-(SP3484)]inexon6.Inaddition, 5.6% (n=6/107) of Bir- mingham, 9.4% (n=9/104) of London and 2.1% (n=1/48) of Sheffield SALS carried aminimumofthirtycopiesoftheG4C2repeatexpansioninC9ORF72. There was also

oneexampleofoligogenicinheritance(Section5.3.2.2)involving patient sample [S3 52-(SP3484)] which was C9ORF72+ and also found to harbour a heterozygous mis- sense c.962C>T (p.A321V) mutation in exon 6 of TARDBP (Kirby et al 2010) (Table 4.3).

Birmingham London Sheffield ALL

TARDBP exon 3 exon 6 p.A90V-SNP p.G287S 0 0 0 0 1 1 0.8%

C9ORF72 non-coding G4C2 repeat 6 9 1 6.4%

C9ORF72+TARDBP exon 6 p.A321V 0 0 1 0.4%

Total 6 9 3 7.2%

Table 4.3 Summary of Genetic Variants Reported in the ECACC Replication Cohort

Abbreviations: C9ORF72 - chromosome 9 open reading frame 72, ECACC - European Collection of

Cell Cultures, SNP - single nucleotidepolymorphism andTARDBP-transactiveresponse(TAR) DNA

binding protein.