prior to this study , the affected index patient in family 1 was prescreened for smn1 mutations .
whole exome sequencing ( wes ) was performed by axeq technologies ( seoul , south korea ) using the illumina ( san diego , ca ) trueseq kit to generate sequencing data .
annotated wes data were examined for variants in genes selected for relevance to the phenotype .
preceding sanger sequencing analysis of setx , als2 , sod1 , lmna , mfn2 , and trpv4 did not identify any pathogenic mutations in the affected individual of family 2 ; subsequently wes was carried out at the university of washington genome center .
data from vcf files were analyzed using the in - house seave analysis pipeline ( kinghorn centre for clinical genomics , sydney , australia ) under homozygous and potentially compound heterozygote models .
the effect of amino acid substitutions for sequence variants was assessed using the software sift and polyphen2 .
sanger sequencing was performed to confirm the mutations in the probands and for segregation analysis in family members .
two affected siblings were from a nonconsanguineous family of ashkenazi jewish origin ( figure 1 ) .
in retrospect , she had difficulties performing sporting activities from the age of 15 years .
she presented to medical attention at the age of 35 years with progressive lower limb weakness requiring the use of a walking stick .
the impairment of motor function was progressive and at age 56 years she required a scooter and long arm crutch to mobilize .
there were no significant ocular , bulbar , or sensory symptoms seen over the course of the illness and she had normal intellectual function .
( b ) schematic graph of the vrk1 coding region and the corresponding vrk1 protein shows the position of mutations identified in family 1 ( blue ) and 2 ( green ) .
clinical examination was consistent with a profound symmetric distal muscle wasting affecting the upper and lower limbs symmetrically ( figure 2 ) .
there was a predominantly distal pattern of weakness of greater severity in the lower than upper limbs , with relative preservation of proximal strength .
neurophysiology showed preservation of sensory responses but severe and progressive reduction in compound motor action potential amplitudes with relative preservation of conduction velocities .
motor evoked potentials from the upper limbs were prolonged with a central component suggesting upper motor neuron involvement in the disease , which is consistent with mri findings of atrophy of the spinal cord ( figure 3a ) .
pontocerebellar hypoplasia or other developmental anomalies as reported in other patients previously described with vrk1 mutations were absent ( figure 3 ) .
respiratory muscle weakness resulted in reduced mean inspiratory and expiratory pressures and a nonprogressive elevation of blood gas co2 .
other respiratory parameters had been stable for 5 years with no requirement for assisted ventilation .
serum creatine kinase was not elevated and an mri of the lower limbs showed profound proximal and distal muscle atrophy with fat replacement .
there was relative preservation of the iliopsoas and adductor compartments bilaterally ( figure 3 ) .
( a ) mri of proband in family 1 ( sma ) . ( a.aa.c ) t2 images and ( a.d ) sagittal t1 image demonstrate mild nonspecific generalized atrophy and absence of pontocerebellar hypoplasia .
( a.e ) t2 image sagittal and ( a.f ) transverse spinal image demonstrate cord atrophy .
( a.ga.i ) t2 images of upper thigh , calf , and stir sequence of thigh demonstrate diffuse fatty replacement of muscle and relative sparing of adductor compartments .
( b.ab.c ) t2 images and ( b.d ) sagittal t1 image demonstrate absence of pontocerebellar hypoplasia . the proband has a similarly affected older brother with a slightly later onset of progressive motor weakness in the late teenage years , progression to requiring a walking stick in his early 30s , and the requirement for an articulated walking device and wheelchair in his late 40s .
the patient was a 3-year - old girl with short stature and microcephaly who presented with initial distal lower limb muscle weakness and amyotrophy combined with pathologically brisk deep tendon reflexes and normal sensation .
weakness progressed in a caudalocephalic direction , with loss of ambulation by 10 years of age and complete dependence for activities of daily living at 18 years .
respiratory dysfunction developed with dyspnea and weak cough accompanied by reduction of forced vital capacity to below 40% predicted .
neurophysiology studies at age 11 years demonstrated severe reduction in compound muscle action potential amplitudes combined with chronic and active denervation , consistent with motor neuropathy / neuronopathy .
sensory responses were initially preserved but subsequently were reduced on repeat testing at age 19 years .
the clinical possibility of juvenile als , with the presence of upper and lower motor neuron signs in more than 2 spinal regions and clinical progression , prompted sanger sequencing as described above .
participating individuals were enrolled through the neurogenetics clinic concord hospital and the paediatric neuromuscular clinic at sydney children 's hospital randwick .
these procedures were performed with informed consent according to protocols approved by the human ethics committees of sydney local health district , concord hospital ( hrec/11/crgh/105 ) , and south eastern sydney local health district ( hrec/13/powh/203 ) , australia .
prior to this study , the affected index patient in family 1 was prescreened for smn1 mutations .
whole exome sequencing ( wes ) was performed by axeq technologies ( seoul , south korea ) using the illumina ( san diego , ca ) trueseq kit to generate sequencing data .
annotated wes data were examined for variants in genes selected for relevance to the phenotype .
preceding sanger sequencing analysis of setx , als2 , sod1 , lmna , mfn2 , and trpv4 did not identify any pathogenic mutations in the affected individual of family 2 ; subsequently wes was carried out at the university of washington genome center .
data from vcf files were analyzed using the in - house seave analysis pipeline ( kinghorn centre for clinical genomics , sydney , australia ) under homozygous and potentially compound heterozygote models .
the effect of amino acid substitutions for sequence variants was assessed using the software sift and polyphen2 .
sanger sequencing was performed to confirm the mutations in the probands and for segregation analysis in family members .
two affected siblings were from a nonconsanguineous family of ashkenazi jewish origin ( figure 1 ) .
in retrospect , she had difficulties performing sporting activities from the age of 15 years .
she presented to medical attention at the age of 35 years with progressive lower limb weakness requiring the use of a walking stick .
the impairment of motor function was progressive and at age 56 years she required a scooter and long arm crutch to mobilize .
there were no significant ocular , bulbar , or sensory symptoms seen over the course of the illness and she had normal intellectual function .
( b ) schematic graph of the vrk1 coding region and the corresponding vrk1 protein shows the position of mutations identified in family 1 ( blue ) and 2 ( green ) .
clinical examination was consistent with a profound symmetric distal muscle wasting affecting the upper and lower limbs symmetrically ( figure 2 ) .
there was a predominantly distal pattern of weakness of greater severity in the lower than upper limbs , with relative preservation of proximal strength .
neurophysiology showed preservation of sensory responses but severe and progressive reduction in compound motor action potential amplitudes with relative preservation of conduction velocities .
motor evoked potentials from the upper limbs were prolonged with a central component suggesting upper motor neuron involvement in the disease , which is consistent with mri findings of atrophy of the spinal cord ( figure 3a ) .
pontocerebellar hypoplasia or other developmental anomalies as reported in other patients previously described with vrk1 mutations were absent ( figure 3 ) .
respiratory muscle weakness resulted in reduced mean inspiratory and expiratory pressures and a nonprogressive elevation of blood gas co2 .
other respiratory parameters had been stable for 5 years with no requirement for assisted ventilation .
serum creatine kinase was not elevated and an mri of the lower limbs showed profound proximal and distal muscle atrophy with fat replacement . there was relative preservation of the iliopsoas and adductor compartments bilaterally ( figure 3 ) .
( a.aa.c ) t2 images and ( a.d ) sagittal t1 image demonstrate mild nonspecific generalized atrophy and absence of pontocerebellar hypoplasia .
( a.e ) t2 image sagittal and ( a.f ) transverse spinal image demonstrate cord atrophy .
( a.ga.i ) t2 images of upper thigh , calf , and stir sequence of thigh demonstrate diffuse fatty replacement of muscle and relative sparing of adductor compartments .
( b.ab.c ) t2 images and ( b.d ) sagittal t1 image demonstrate absence of pontocerebellar hypoplasia .
the proband has a similarly affected older brother with a slightly later onset of progressive motor weakness in the late teenage years , progression to requiring a walking stick in his early 30s , and the requirement for an articulated walking device and wheelchair in his late 40s .
the patient was a 3-year - old girl with short stature and microcephaly who presented with initial distal lower limb muscle weakness and amyotrophy combined with pathologically brisk deep tendon reflexes and normal sensation .
weakness progressed in a caudalocephalic direction , with loss of ambulation by 10 years of age and complete dependence for activities of daily living at 18 years .
severe thoracolumbar scoliosis required surgical rod insertion at age 14 years . respiratory dysfunction developed with dyspnea and weak
neurophysiology studies at age 11 years demonstrated severe reduction in compound muscle action potential amplitudes combined with chronic and active denervation , consistent with motor neuropathy / neuronopathy .
sensory responses were initially preserved but subsequently were reduced on repeat testing at age 19 years .
the clinical possibility of juvenile als , with the presence of upper and lower motor neuron signs in more than 2 spinal regions and clinical progression , prompted sanger sequencing as described above .
participating individuals were enrolled through the neurogenetics clinic concord hospital and the paediatric neuromuscular clinic at sydney children 's hospital randwick .
these procedures were performed with informed consent according to protocols approved by the human ethics committees of sydney local health district , concord hospital ( hrec/11/crgh/105 ) , and south eastern sydney local health district ( hrec/13/powh/203 ) , australia .
testing identified compound heterozygous vrk1 mutations nm_003384.2 ( vrk1):c.(356a > g ) ; ( 1072c > t ) ( p.[h119r];[r358 * ] ) in the individuals with sma in family 1 and nm_003384.2 ( vrk1):c.(403 g > a ) ; ( 583t > g ) ( p.[g135r];[l195v ] ) in the patient with juvenile als in family 2 .
no pathogenic variants were identified in other genes associated with sma or als in either patient or genes associated with short stature .
the present cases with adult - onset distal sma and childhood motor neuron disease broaden the clinical spectrum of patients with vrk1 mutations .
the identification of vrk1 mutations among these various motor phenotypes , with clinical overlap among juvenile als , dhmn + ps , and sma , may serve to further unite concepts of pathogenesis among motor neuron disorders .
the results for family 1 are consistent with an autosomal recessive mode of inheritance with segregation of both vrk1 variants with the disease ( figure 1a ) .
the c.1072c > t mutation is well - characterized and described as pathogenic in the ashkenazi jewish population when in a homozygous state .
the c.356a > g variant ( esp frequency < 0.01% ) encodes a histidine residue that is conserved across multiple vertebrate species and is in the protein kinase domain of the vrk1 gene ( figure 1b ) .
it was assessed with in silico tools ( polyphen2 score 1 and 0.999 , provean score 3.804 ) and is classified as damaging . the c.356a > g missense substitution was reported in one recent case of a 32-year - old man with a 5-year history of als without cerebellar hypoplasia .
this sequence variant is most likely to be pathogenic as it is present in both the affected members of family 1 and a reported sporadic als case . in family 2 ,
both substituted amino acids are highly conserved across multiple species and the frequency of the novel c.583t > g mutation in public databases was 2 in 121382 .
online database tools supported pathogenicity for c.403 g > a and c.583t > g mutations ( polyphen2 scores 1 and 0.997 , probably damaging ; sift score 0 and 0 , deleterious ; and cadd score 22.6 and 19.05 , respectively ) .
the mutations were sanger sequenced for confirmation and each parent was found to carry one of the variants , supporting autosomal recessive inheritance ( figure 1 ) .
three - dimensional structural protein analysis of the p.gly135arg vrk1 variant with the hope protein structure analysis suite database suggested a difference in charge , size , and hydrophobicity resulting in disturbances in local structure and protein misfolding .
similarly , the p.leu195val vrk1 variant altered the amino acid size , causing an empty space in the core of the protein .
gonzaga - jauregui et al . described childhood - onset distal sensory motor axonal neuropathy with microcephaly with a simplified gyral pattern and in a second case there was a similar motor and sensory axonal neuropathy with an underdeveloped cerebellar vermis .
the latter case and this report suggest that vrk1 is not only important in development of motor system architecture , but are also important in the longer term in maintenance of motor neurons .
vrk1 mutations should therefore be considered in the differential diagnosis of patients presenting with adult - onset sma and childhood motor neuron disease .
marion stoll : organized and performed genetic testing on proband and family members ( family 1 ) , performed bioinformatics analysis and interpretation of wes data , writing of the manuscript .
hooi ling teoh : collected data of the proband ( family 2 ) , writing of the manuscript .
lee : clinically examined and interviewed the proband ( family 1 ) , writing of the manuscript .
stephen reddel : clinically examined and performed neurophysiology on proband ( family 1 ) , critical revision of manuscript .
ying zhu : supervised the creation of the bioinformatic pipeline for genomic analysis , assisted with bioinformatics analysis and interpretation of whole exome data , critical revision of manuscript .
michael buckley : organized and performed genetic testing on proband and family members ( family 2 ) , interpretation of variant results , critical revision of manuscript .
hugo sampaio : clinically examined and interviewed the proband ( family 2 ) , critical revision of manuscript .
tony roscioli : obtained research funding for genomic sequencing , developed genomic consent forms for patient enrolment , supervised the creation of the bioinformatic pipeline for genomic analysis , performed bioinformatics analysis and interpretation of wes data , critical revision of manuscript .
michelle farrar : provided clinical and scientific direction for the manuscript , drafted , reviewed and approved the final manuscript .
garth nicholson : provided clinical and scientific direction for the manuscript , planned , drafted , reviewed , and finalized the manuscript , clinically examined , characterized , and interviewed the proband ( family 1 ) .
supported by motor neurone disease research institute of australia . no industry , governmental , or institutional funding
j. lee , s. reddel , y. zhu , m. buckley , h. sampaio , t. roscioli , m. farrar , and g. nicholson report no disclosures relevant to the manuscript .
 objective : to describe the phenotypes in 2 families with vaccinia - related kinase 1 ( vrk1 ) mutations including one novel vrk1 mutation.methods:vrk1 mutations were found by whole exome sequencing in patients presenting with motor neuron disorders.results:we identified pathogenic mutations in the vrk1 gene in the affected members of 2 families . in family 1 , compound heterozygous mutations were identified in vrk1 , c.356a > g ; p.h119r , and c.1072c > t ; p.r358 * , in 2 siblings with adult onset distal spinal muscular atrophy ( sma ) . in family 2 , a novel vrk1 mutation , c.403g > a ; p.g135r and 
 c.583t > g ; p.l195v , were identified in a child with motor neuron disease.conclusions:vrk1 mutations can produce adult - onset sma and motor neuron disease in children without pontocerebellar hypoplasia . 
METHODS
Genetic analysis.
Clinical phenotype: Family 1.
Clinical phenotype: Family 2.
Standard protocol approvals, registrations, and patient consents.
RESULTS
DISCUSSION
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE