  amyotrophic lateral sclerosis ( als ) is a neurodegenerative disorder primarily affecting motor neurons in the spinal cord , brain stem , and cerebral cortex
. five to ten percent of als cases are familial ; the others are believed to be sporadic .
mutations in the cu / zn superoxide dismutase gene ( sod1 ; omim 147450 ) are the most frequent genetic defects known to underlie als , accounting for 20% of familial cases ( fals ) and one to seven percent of apparently sporadic cases ( sals ) [ 17 ] .
recently , other mutations like the tardbp gene ( tdp-43 ) [ 8 , 9 ] , ang gene , fus / tls gene , and optn gene   were identified as causative of als . despite this genetic heterogeneity ,
, we report the results of screening for sod1 mutations in the 184 sals cases in our hospital and 265 als cases all over japan by high - resolution melting ( hrm ) analysis .  
hrm analysis is a mutation scanning technique that monitors the progressive change in fluorescence caused by the release of an intercalating dna dye from a dna duplex as it is denatured with marginal increases in temperature .
the shifts and shapes of melting curves , there are obtained as fluorescence difference plots , are used to distinguish between mutations and controls .
hrm analysis of pcr products amplified in the presence of lc green plus can detect all heterozygous and most homozygous sequence variations through differences in shape and position of a melting curve compared with a wild - type melting profile .
although single - strand conformation polymorphism ( sscp ) [ 2 , 3 , 1420 ] and denaturing high - performance liquid chromatography ( dhplc ) [ 5 , 6 ] seem to be the main screening strategies for sod1 mutations , hrm analysis has its own advantages .
this is the first report of hrm analysis being applied to the sod1 screening . in this paper , we report the high sensitivity of hrm analysis for known sod1 mutations , and the prevalence and clinical features of sod1 mutations in japanese sals cases .
  a total of consecutive 184 sals cases ( 109 males and 75 females ) visited our neurology division at the jichi medical university hospital in tochigi , japan .
ethical approval was granted by the bioethics committee for human gene analysis of our university and informed consent was obtained from all subjects according to the declaration of helsinki .
every patient fulfilled the diagnostic criteria for als as outlined by the el escorial revisited   classification ; 177 definite , probable or possible als and 7 suspected als .
there was no significant difference in onset age between 109 males and 75 females ( males : 60.4 years on average with a range of 2780 ; females : 64.3 years with a range of 3483 ) .   in 2006 , the japanese consortium for amyotrophic lateral sclerosis research ( jacals ) was organized with the aim of investigating the relationships of clinical and genetic aspects of als in japan .
the inclusion criteria for registration with the jacals are : ( 1 ) adult onset , steady progressive course , ( 2 ) definite , probable or possible als based on the el escorial revisited   criteria for diagnosis of als , and ( 3 ) informed consent for the genetic study and clinical checking every three months . from 2006 to 2008 , 265 patients ( 10 fals and 255 sals ) were registered , and blood samples and clinical data having been obtained by neurologists .
we used 19 reported sod1 mutations in all five exons ( table 1 ) to determine the sensitivity of the hrm analysis .
watanabe y. ( tottori university , japan ) , and they were already direct sequenced and confirmed they had the mutations .  
dna samples were amplified with double - stranded dna - binding dye lc green plus ( idaho technology ) .
pcr was performed with a veriti 96-well thermal cycler ( applied biosystems ) in 10  l reaction mixtures comprising 10  ng dna , 1xpcr buffer , lc green plus ( idaho technology ) , and 1  u taq polymerase , with 0.25  m each forward and reverse primers .
initial denaturation was performed at 95c for 2  min , followed by 45 cycles of 94c for 30  sec and 6268c for 30  sec , with a final cycle of 94c for 30  sec and 25c for 30  sec .  
the plate was heated from 80 to 98c at 0.1c / sec with a 300  ms frame interval , 15  ms exposure , and 100% led power .
sequencing of samples indicated to include mutations was then carried out using a bigdye terminator v1.1 cycle sequencing kit ( applied biosystems ) and an abi 310 automated sequencer ( pe applied biosystems ) .  
next , we applied this method to japanese als patients for mutation screening of sod1 .
  hrm analysis clearly distinguished 18 of 19 previously identified sod1 mutations from normal controls .
the melting curves of control samples ( wild - type ) were tightly grouped for all fragments , and altered difference curves were easily identified for the 18 mutations ( figure 1 ) .
we found sod1 mutations in three out of the 184 sals cases ( 1.63% ) in the group 1 .
the mutations identified were all novel : cys 6 tyr ( c6y ) and gln 22 his ( q22h ) in exon 1 , and ser 134 thr ( s134 t ) in exon 5 ( figure 2 ) .   in case 1 , a 34-year - old woman , there was a single - base pair substitution in exon 1 at codon 6 ( tgc to tac ) .
she awoke with painful cramping and weakness in the right leg almost every morning at the age of 33 years .
the cramping resolved , but her right leg weakness progressed and become accompanied by fasciculation .
one year after the onset , neurological examination showed marked muscle atrophy and prominent fasciculation in her right leg .
the muscle weakness and atrophy progressed , and spread to the other parts of her body despite treatment with intravenous gamma globulin , cyclophosphamide , and plasmapheresis .
she expired 3 years after disease onset .   in case 2 , a 48-year - old man , there was a single - base pair substitution in exon 1 at codon 22 ( cag to cac ) .
two years after the onset , neurological examination showed muscle weakness , atrophy and fasciculation were observed in the left leg .
atrophy spread to the right leg , confining him to a wheelchair at 51 years old and to bed at 52 years old .
he underwent tracheotomy because of progressive respiratory failure , and artificial ventilation support was started eight years after disease onset .
five years after artificial ventilation support was started , he moved to another hospital and thus we could not follow him further .   in case 3 , a 69-year - old man , there was a single - base pair substitution in exon 5 at codon 134 ( agt to act ) .
he noticed gait disturbance due to muscle weakness of the lower limbs at the age of 62 years .
the weakness progressively worsened , and he could not walk by himself at 67 years old .
neurological examination showed muscle weakness , and fasciculation were evident in the upper and lower limbs .
although upper motor neuron impairment was not confirmed , als was considered as the most probable diagnosis .
the weakness progressed very slowly , and he died of respiratory insufficiency seven years after disease onset .  
of these , four had family histories , mutations being leu 38 val ( l38v ) and his 46 arg ( h46r ) in exon 2 , gly 93 ser ( g93s ) in exon 3 and gly 141 ala ( g141a ) in exon 5 .
the g141a found in a woman whose brother probably died of als was a novel mutation . in this case , left hand weakness occurred at 57 years old .
the clinical course was rapid that she died at 3 years and 11 months after the onset .
the remaining four sod1 mutations were found in sporadic cases , mutations being lys 3 glu ( k3e ) in exon 1 and gly 93 ser ( g93s ) in exon 3 .
k3e was a novel mutation found in a woman who noticed right leg weakness at 52 years old , and artificial ventilation support was started 6 years after the onset .
the prevalence of sod1 mutations in the sals cases was 1.56% ( 4 of 255 sals cases ) in the group 2 .
we have demonstrated that hrm analysis is a rapid and   sensitive ( 94.7% sensitivity ) method for mutation scanning of sod1 .
sscp is a method that most laboratories use for the screening of gene mutations , but the sensitivity is not high ( 80% to 90% ) .
dhplc using wave system is also a screening method , but it can not detect the d90a mutation , which is one of the worldwide detected sod1 mutations , and the most appropriate condition for analysis is difficult to determine . using hrm analysis , we can analyze within 5 to 10 minutes on 96 samples and the running cost is not expensive .   the one mutation that hrm analysis could not detect was guanine to cytosine at nucleotide 341 substituting glycine ( ggc ) to alanine ( gcc ) at codon 114 . on the other hand , guanine ( ttg ) to cytosine ( ttc ) mutations ( l144f ) , and alanine ( gct ) to alanine ( gca ) mutations ( a140a ) in other samples
were detected with this method , indicating the possibility that the g to c mutation detection failure may be a sequence - specific phenomenon .  
we applied this method to our own 184 ( group 1 ) and 255 ( group 2 ) japanese cases of sals , finding three different novel sod1 mutations in three cases in the former ( mutation prevalence , 1.63% ) , and one novel and three known mutations in four cases in the latter ( mutation prevalence , 1.57% ) .
we listed the prevalence and identified mutations of sod1 in sals cases in other countries ( table 2 ) .
the prevalence was high in the scottish population ( 7% ) and widely ranged in italy ( 0%6% ) , but in   other countries , it was 2 to 4% , similar to our data .
this time we found four novel mutations in sals cases , and these mutations were not found in the japanese control group .   in a sporadic als patient carrying an sod1 mutation , it is also difficult to ascertain whether it is a genuine sporadic case , a case due to a mutation , or a familial case with incomplete penetrance . to date
, an sals case with h80a is the only one with a proven de novo mutation . in our analysis ,
the g93s mutation was found in three unrelated patients from the tokai district of japan ( personal communication ) .
there are at least 6 japanese families with g93s , 4 of the 6 families being reported to be residents of the tokai district [ 2426 ] .
the accumulation of g93s in japanese sals cases suggests the possibility of decreased penetrance or an incomplete family history rather than a de novo mutation .
clinical characteristics such as onset age , onset symptoms , and clinical course of so far reported sals patients having sod1 mutations are summarized in table 3 . since a4v , d90a , and i113 t
have been observed worldwide and are considered to be the most common mutations in both familial and sporadic als cases [ 4 , 7 ] . because of the difficulty to define true sporadic , we did not include these three mutations in the table .
based on the results of analysis of these 20 sod1 mutations in 27 sporadic als patients ( 13 men , 10 women , and 4 unknown ) , the average age at onset was 43.8 ( range 1877 ) years , which is about 10 years younger that the mean age at onset reported for the sporadic als population .
the onset symptom was limb weakness in 21 cases and bulbar dysfunction only in one case .
the clinical courses were under three years ( rapid ) in seven cases , over six years ( slow ) in nine cases , and three to six years ( moderate ) in five cases .
the clinical characteristics of sals involving sod1 mutations indicate a relatively young onset age and a high percentage of limb involvement at onset .
these characteristics are similar to the features of alsod ( als patients having sod1 mutations ) , not those of sporadic als .  
the c6y mutation in our case was difficult to diagnose because the main symptom was lower motor neuron dysfunction and the onset age was young ( midthirties ) .
but this clinical course was similar to that in the case of de novo mutation h80a .
there were nine ( bold ) patients whose onset ages were under forty , and eight of them had rapid or moderate clinical course ( table 3 ) .
on the other hand , there are four ( underlined ) patients whose onset ages were over 55 , and three of them had slow clinical course ( table 3 ) .
gamez and his colleagues reported   there were three types of sporadic als patients who were particular candidates for genetic testing for sod1 : ( a ) those with the typical scandinavian phenotype , ( b ) those with clinical onset before 55 years of age , and ( c ) patients with slow progression / long survival . compare with this theory ( b ) and ( c ) , only one patient ( n19s ) is an exception for sod1 screening .
  we have demonstrated that hrm analysis is a rapid and sensitive method for the mutation scanning of sod1 . with this method ,
four novel sod1 mutations were found in sals cases , the prevalence of sod1 mutations in japanese sals cases being 1.6% .
the clinical characteristics of sals involving sod1 mutations are a young onset age and a high percentage of limb involvement at onset .
we will screen other causative genes for als ( tdp-43 , ang , fus / tls , optn and others ) by hrm analysis and determine the cause of disease appearance .
 amyotrophic lateral sclerosis ( als ) is a progressive neurodegenerative disorder , and the majority of als are sporadic ( sals ) . 
 recently , several causative genes for familial als ( fals ) were identified , but the cause of the sals is still unknown . 
 this time , we aimed to identify the genetic background of sals . 
 first , we applied the new sensitive screening methods : high - resolution melting ( hrm ) analysis . 
 hrm analysis detected 18 out of 19 known sod1 gene mutations ( 94.7% sensitivity ) . 
 next , we screened sod1 , three novel mutations ( c6y , q22h , and s134 t ) were identified in our own 184 sals cases ( 1.63% prevalence ) , and four mutations in another 255 sals cases ( 1.56% prevalence ) registered from all over japan . 
 the patients with sod1 mutations suggested a relatively young onset and limb involvement at onset . 
 the hrm analysis is a sensitive and easy screening method ; we will use this method for screening other als causative genes and revealing the genetic background of sals . 
1. Introduction
2. Patients and Methods
3. Results
4. Discussion
5. Conclusion