amyotrophic lateral sclerosis ( als ) is a progressive neurodegenerative condition affecting upper ( umn ) and lower motor neurons ( lmn ) ( kiernan et al . , 2011 ; turner et al . , understanding the pathophysiology of als is challenging , due to significant variability of clinical phenotype , patient characteristics and disease progression ( chi et al . 2013 ) . despite this variability , common patterns have been observed across a wide range of als subjects . a well - studied example is the phenomenon , in which there is disproportionate weakness of the thenar / first dorsal interosseous muscle group ( eisen et al . , 2014 ; 2014 ) and speech ( devine et al . , 2013 ) has also been observed . these findings have prompted suggestions that functions which humans have evolved more recently , such as the pincer grip and upright stance , are more susceptible to als ( eisen et al . however , the factors determining the side of onset and direction of spread remain unclear . since humans have evolved strong population - wide upper limb dominance ( fitch and braccini , 2013 ) , it is important to explore this as another potential source of vulnerability in als . it has been shown that the dominant upper limb , but not lower limb , is more susceptible to onset of weakness ( turner et al . , 2011 ) . we have also described that spread of weakness and umn signs are affected by dominance , suggesting importance of central factors ( devine et al . , 2014 ) . the aim of this study was to investigate gray matter ( gm ) asymmetry in als , and thus identify regions asymmetrically affected by the disease . applying voxel - based morphometry ( vbm ) analysis of structural mri , we performed direct comparisons between als subjects and controls , as well as using a novel asymmetry protocol to assess interhemispheric differences ( rose et al . , 2012 ) . our hypothesis was that this asymmetry protocol would detect patterns of disproportionate atrophy in als , which would be affected by whether weakness first occurred in a dominant or non - dominant limb . ethical approval was obtained from the royal brisbane and women 's hospital ( rbwh ) human research ethics committee . all subjects provided written informed consent , and all research was conducted in accordance with the declaration of helsinki . thirty right - handed subjects were recruited from als outpatient clinics at the rbwh ( 20082013 ) . all had diagnoses of clinically probable or definite als , according to revised el escorial criteria ( brooks et al . , 2000 ) . we chose to study only right - handed subjects due to their predominance in the population ( meguerditchian et al . , 2013 ) , as well as greater uniformity of motor and language lateralisation ( adamo and taufiq , 2011 ) . subjects were grouped according to the index limb , defined as the first limb affected by weakness ( either the limb of onset , or the first limb affected after bulbar onset ) ( devine et al . , 2014 ) . fifteen subjects had a right - sided ( dominant ) index limb and 15 had a left - sided ( non - dominant ) index limb . each subject was administered the als functional rating scale  revised ( alsfrs - r ) as a measure of disability . to adjust the degree of disability for the disease duration , we calculated  disease progression  as : ( 48    alsfrs - r score )  /  ( disease duration ) . seventeen right - handed healthy controls were closely age and sex - matched with each group of 15 als subjects . none of the control or als subjects had a history of cerebrovascular events , intracranial pathology , or other neurological diseases . each subject underwent an mri scan acquired with a 3  t siemens timtrio ( siemens , erlangen , germany ) , using sequences from vb17 neuro applications and a 12-channel head coil . a high - resolution structural image was acquired for each subject using a 1  mm isotropic 3d t1 mprage ( fov 24    25.6    17.6  cm , tr / te / ti 2300/2.26/900  ms , flip angle 9 ) . structural images were processed according to the protocol previously reported ( rose et al . , 2012 ) . the software package fsl - vbm ( version 4.1 ) , an optimised vbm protocol ( good et al . , 2001a ) carried out with fsl tools ( smith et al . , 2004 ) , gm segmentation was performed using fast ( zhang et al . , 2001 ) , with the segmentations then aligned to mni152 standard space using affine registration , flirt ( jenkinson and smith , 2001 ) , followed by non - linear registration using fnirt ( andersson , 2007 ) . each image was non - linearly re - registered to that template , before being modulated by dividing by the jacobian determinant of the warp field and smoothed with an isotropic gaussian kernel ( sigma  =  4  mm ) . mirror images were then generated for each of the smoothed , modulated gm images in standard space , for each of the 47 subjects . all statistical comparisons were performed using randomise ( nichols and holmes , 2002 ) , and adjusted for multiple comparisons using threshold - free cluster enhancement ( tfce ) ( smith and nichols , 2009 ) . firstly , a voxelwise unpaired t - test was performed , comparing the gm density of all als subjects ( n  =  30 ) with all controls ( n  =  17 ) . als subjects were then subdivided into two groups ( 15 with a right - sided index limb , and 15 with a left - sided index limb ) , and each group was compared with 15 age and sex - matched controls . finally , the als subjects with a right - sided index limb were compared directly with those having a left - sided index limb . for each test , age and disease progression were introduced as nuisance covariates . in order to identify areas of hemispheric asymmetry , a voxelwise paired t - test was performed between the native and mirror images . this was performed separately for each of the three groups of subjects ( 17 controls , 15 als subjects with a right - sided index limb and 15 with a left - sided index limb ) . the limb subscore ( questions 49 ) of the alsfrs - r was introduced as a covariate . the anatomical location of each cluster of gm asymmetry was determined using the talairach daemon . the threshold for statistical significance was set at p    0.01 ( tfce - corrected ) . specific subject characteristics are presented in table 1 . compared with controls ( n  =  17 ) , subjects with als ( n  =  30 ) showed a multifocal cluster of reduced gm density , involving the left precentral gyrus and adjacent regions of the left middle frontal gyrus and bilateral medial frontal gyri ( 2087  voxels ; centre - of - gravity : 22 , 11 , 52 ; p    0.05 ) . there was a separate cluster of reduced gm density involving bilateral anterior cingulate gyri ( 425  voxels ; centre - of - gravity : 1 , 39 , 6 ; p    0.05 ) . , there was a negative correlation ( p    0.05 ) between age and gm density in widespread regions of the frontal , parietal , temporal and occipital lobes , representative of age - related atrophy . however , there was no confounding effect of age or disease progression on the patterns of atrophy in als . 1b , als subjects with a right ( dominant ) index limb ( n  =  15 ) showed patchy reductions in gm density affecting the left precentral gyrus , at a threshold of p    0.05 . these changes were not significant at a higher threshold of p    0.01 . subjects with a left ( non - dominant ) index limb ( n  =  15 ) did not demonstrate any significant reductions in gm density at either the left or right precentral gyri at a threshold of p    0.05 ( fig . direct voxelwise comparison between als subjects with either a right or left index limb also did not reveal any significant differences in gm density . in the 17 right - handed control subjects , multiple statistical clusters of both rightward and leftward asymmetries were identified ( table 2 , fig . 2a ) . of particular note was an area of leftward asymmetry ( p    0.01 ) encompassing a dorsolateral region of the precentral and postcentral gyri . this area corresponded closely with the centre - of - gravity of the dominant thenar representation area , previously defined using transcranial magnetic stimulation ( tms ) ( niskanen et al . , 2010 ) . control subjects also demonstrated significant leftward asymmetry of a region of the superior and transverse temporal gyri , adjacent to the sylvian fissure . there were no significant asymmetries of lower limb or bulbar representation areas , indicating that these regions were of a similar density in the right and left hemispheres . in the 15 als subjects with a right - sided ( dominant ) index limb , there was complete absence of leftward asymmetry at the precentral gyrus hand representation area , at the threshold of p    0.01 ( table 2 , fig . this indicated disproportionate loss of gm supplying the dominant hand , relative to the remainder of the bilateral motor strip ( including lower limb and bulbar representation areas ) . leftward asymmetry was also lost at the adjacent region of the postcentral gyrus , as well as the superior and transverse temporal gyri and anterior insula . in contrast , leftward asymmetry of the dorsolateral postcentral gyrus , superior and transverse temporal gyri and anterior insula was preserved in the 15 als subjects with a left - sided ( non - dominant ) index limb ( table 2 , fig . 2c ) . however , these subjects still showed complete absence of leftward asymmetry at the precentral gyrus hand representation area . this disproportionate loss of gm density in the left motor cortex occurred despite first weakness occurring in a limb controlled by the right hemisphere . these subjects also demonstrated a new cluster of leftward asymmetry involving the middle frontal gyrus ( mfg ) , which was not present in controls ( table 2 ) . this may indicate either relative gain of gm density at the left mfg or gm loss at the right mfg , with the latter favoured . these included rightward asymmetry of the inferior frontal , rectal and orbital gyri , posterior thalamus , posterior cingulate gyrus and cuneus , and leftward asymmetry of the posterolateral cerebellum and occipital lobes . several of these regions , including rightward asymmetry of the antero - inferior frontal lobes and leftward asymmetry of the occipital lobes , have been previously reported in larger populations of healthy subjects ( toga and thompson , 2003 ; watkins et al . , 2001 ) . the objective of this study was to identify als - related changes to the normal patterns of gm asymmetry , and interpret these in the context of limb dominance . we have established that a vbm asymmetry protocol applied to structural t1 mri ( rose et al . , 2012 ) is a useful tool for assessing gm changes in both healthy and disease states , especially in smaller cohorts in which standard voxelwise comparisons are less sensitive . using this protocol , we found that right - handed als subjects with dominant limb onset disproportionately lost gm in left hemispheric sensorimotor ( upper limb representation ) and language regions . however , unlike other regions , disproportionate atrophy of the left motor cortical hand area occurred independent of whether onset of weakness was in a dominant or non - dominant limb . these findings support previous clinical studies of als laterality , and have implications for cortical organisation , its evolution , and selective vulnerability . multiple large studies have demonstrated motor cortical atrophy in als , which may initially be left - hemisphere predominant ( menke et al . , 2014 ) . furthermore , motor impairment has been shown to be focal , using both clinical examination ( ravits et al . , 2007 ) and neuroimaging ( bede et al . , 2013 ) . however , in the current study , major differences between als and controls were only apparent when analysing all 30 subjects together , and at a more relaxed statistical threshold ( p    0.05 ) . comparisons involving 15 als subjects only detected minor , patchy reductions in motor cortical gm density , and were unable to differentiate subjects with right or left - sided disease onset . in contrast , the vbm asymmetry protocol was sufficiently powered to identify differences in gm between the two clinical groups of als subjects , at a stricter threshold ( p    0.01 ) . therefore , this method is ideal for exposing subtle asymmetric changes in less common diseases such as als . despite previous authors correlating the size or density of motor areas with handedness ( herv et al . , 2006 ; rose et al . , 2012 ; takao et al . , 2011 ) , some have not demonstrated this ( good et al . , 2001b ) . in the current study , we defined a significant cluster of leftward gm asymmetry in right - handed controls , incorporating the cortical hand representation area . in contrast , the absence of this asymmetry across all right - handed als subjects suggests that the left hemisphere hand area disproportionately loses gm density relative to the remainder of the bilateral motor cortex . our results also suggest that this region of motor cortex is particularly vulnerable to atrophy , regardless of whether the patient or clinician had noticed first weakness in a right or left - sided limb . vulnerability of the dominant upper limb in als has been previously described using history and clinical examination ( devine et al . , 2014 ; turner et al . , our study provides neuroimaging evidence to support this phenomenon , and suggests an important role for central factors in driving this effect . our findings also confirm the lack of preferential involvement of either lower limb , which has been observed clinically ( turner et al . , 2011 ) . it has been proposed that certain functions , such as the  split hand  , are more vulnerable to the pathology of als due to more recent development in human evolution ( eisen et al . , 2014 ) . another key feature of human development has been a population - wide bias toward right - handedness ( fitch and braccini , 2013 ) , which is likely to have been facilitated by the onset of upright gait ( meguerditchian et al . , the current results suggest that the increased complexity of left hemispheric motor networks in right - handers may lead to greater susceptibility to als . the cause of neuronal vulnerability in als remains unknown , although potential contributory factors include cellular density , excitability , and hormonal influences ( amunts et al . , 1996 ; bumer et al . , 2007 ; vivekananda et al . , 2011 ) . in our study , subjects with dominant limb onset of weakness also showed disproportionate atrophy of areas involved in language and communication ( left superior and transverse temporal gyri ) . it is known that 98% of right - handers have left hemispheric language lateralisation ( adamo and taufiq , 2011 ) . although these subjects did not undergo formal cognitive testing , this finding may reflect the occurrence of language dysfunction as part of the als  ftd spectrum , including milder language deficits in subjects without frank dementia ( taylor et al . , 2013 ) . alternatively , some authors have proposed that the dominant upper limb and speech form a single network for communication , with gestures and vocalisations being linked ( gentilucci et al . , 2008 ; meguerditchian et al . , 2013 due to the current cohort being restricted to right - handers , we were unable to assess changes to gm asymmetry in left - handed or ambidextrous subjects . however , this would require recruitment of a larger cohort , since hemispheric lateralisation of motor and language is less predictable in non - right - handed individuals ( adamo and taufiq , 2011 ) . the size of our cohort also limited the ability to separately analyse smaller subgroups , for example only those subjects with upper limb onset of weakness . formal neuropsychological testing was also not performed , therefore the cognitive correlates of some changes ( such as those affecting the left frontal and bilateral cingulate gyri ) remain unclear . finally , it remains to be seen whether the current results can be translated to individual subjects , for example in a diagnostic setting . overall , this study provides evidence that certain areas of gm are disproportionately and asymmetrically vulnerable to the pathology of als , and that these regions can be identified using a sensitive vbm protocol . in particular , we have identified the dominant hand area as being particularly susceptible to atrophy , supporting previous history and examination - based studies . this finding warrants further investigation , including assessment of associated white matter asymmetry using diffusion tensor imaging .
limb weakness in amyotrophic lateral sclerosis ( als ) is typically asymmetric . 
 previous studies have identified an effect of limb dominance on onset and spread of weakness , however relative atrophy of dominant and non - dominant brain regions has not been investigated . 
 our objective was to use voxel - based morphometry ( vbm ) to explore gray matter ( gm ) asymmetry in als , in the context of limb dominance . 
 30 als subjects were matched with 17 healthy controls . 
 all subjects were right - handed . 
 each underwent a structural mri sequence , from which gm segmentations were generated . 
 patterns of gm atrophy were assessed in als subjects with first weakness in a right - sided limb ( n  =  15 ) or left - sided limb ( n  =  15 ) . within each group 
 , a voxelwise comparison was also performed between native and mirror gm images , to identify regions of hemispheric gm asymmetry . 
 subjects with als showed disproportionate atrophy of the dominant ( left ) motor cortex hand area , irrespective of the side of first limb weakness ( p  <  0.01 ) . 
 asymmetric atrophy of the left somatosensory cortex and temporal gyri was only observed in als subjects with right - sided onset of limb weakness . 
 our vbm protocol , contrasting native and mirror images , was able to more sensitively detect asymmetric gm pathology in a small cohort , compared with standard methods . 
 these findings indicate particular vulnerability of dominant upper limb representation in als , supporting previous clinical studies , and with implications for cortical organisation and selective vulnerability .