the proband ( ii-1 ) was a 58-year - old man who developed progressive gait dysfunction at age 35 .
when seen at age 45 , he showed spastic gait , baclofen - responsive spasticity in the lower extremities , mild spastic dysarthria , minimal upper extremity dysfunction , and normal extraocular movements .
imaging studies of his brain and complete spine were normal . over the next 13 years
, he developed progressive spasticity and inability to move his legs , with increasing dysfunction in his hands and a brisk jaw jerk . by age 53 , he had developed progressive loss of weight and muscle bulk , with a decrease in deep tendon reflexes .
a younger brother and 2 younger sisters developed a similar syndrome ( figure 1a ) . a third sister developed more progressive spasticity .
the family has been followed over the years , and no family members have developed any sensory involvement or bladder problems . both parents and the other 2 siblings are free of neurologic symptoms .
. dna samples from 3 affected individuals ( ii-1 , ii-4 , and ii-7 ) were used for whole - exome sequencing .
candidate variants were validated , and cosegregation analysis was performed using the dna samples from all 9 members of this family .
the compound missense mutations , l695p and i743 t , cosegregated with pls in this family .
dna and messenger rna samples from lymphoblastoid cells from patients with pls were sanger - sequenced ; heterozygous mutations are indicated by arrows . ( + ) = positive ; (  ) = absence ; cf = cerebellar feature ; lmn = lower motor neuron signs ; nn = neurologically normal ; wcb = wheelchair bound .
the family history , clinical data , consent forms , and blood samples from 5 affected and 4 unaffected family members were collected , and permanent lymphoblast cell lines were established .
the illumina hq - seq 2500 ngs platform was used to sequence the exomes of 3 patients from the family .
sanger sequencing and western blot were performed . to determine whether the p695 and t743 compound mutations in spg7
are associated with altered mitochondrial activity , we performed metabolic measurements on lymphoblasts from both patients and unaffected heterozygous individuals using a seahorse xf24 instrument . the seahorse metabolic analyzer measures oxygen consumption rates under 4 conditions : basal , complex v inhibited ( oligomycin ) , uncoupler stimulated ( carbonyl cyanide-4-(trifluoromethoxy ) phenylhydrazone [ fccp ] ) , and in the presence of complex i and iii inhibitors ( rotenone , antimycin a ) .
we followed the manufacturer 's protocols , with steps and conditions optimized for lymphoblasts . in a typical experiment ,
2 patient samples and 2 samples from unaffected heterozygous individuals were analyzed in a 5-well per sample setting .
the proband ( ii-1 ) was a 58-year - old man who developed progressive gait dysfunction at age 35 .
when seen at age 45 , he showed spastic gait , baclofen - responsive spasticity in the lower extremities , mild spastic dysarthria , minimal upper extremity dysfunction , and normal extraocular movements .
imaging studies of his brain and complete spine were normal . over the next 13 years
, he developed progressive spasticity and inability to move his legs , with increasing dysfunction in his hands and a brisk jaw jerk . by age 53 , he had developed progressive loss of weight and muscle bulk , with a decrease in deep tendon reflexes .
a younger brother and 2 younger sisters developed a similar syndrome ( figure 1a ) . a third sister developed more progressive spasticity .
the family has been followed over the years , and no family members have developed any sensory involvement or bladder problems . both parents and the other 2 siblings are free of neurologic symptoms .
. dna samples from 3 affected individuals ( ii-1 , ii-4 , and ii-7 ) were used for whole - exome sequencing .
candidate variants were validated , and cosegregation analysis was performed using the dna samples from all 9 members of this family .
the compound missense mutations , l695p and i743 t , cosegregated with pls in this family .
dna and messenger rna samples from lymphoblastoid cells from patients with pls were sanger - sequenced ; heterozygous mutations are indicated by arrows . ( + ) = positive ; (  ) = absence ; cf = cerebellar feature ; lmn = lower motor neuron signs ; nn = neurologically normal ; wcb = wheelchair bound .
the family history , clinical data , consent forms , and blood samples from 5 affected and 4 unaffected family members were collected , and permanent lymphoblast cell lines were established .
the illumina hq - seq 2500 ngs platform was used to sequence the exomes of 3 patients from the family .
to determine whether the p695 and t743 compound mutations in spg7 are associated with altered mitochondrial activity , we performed metabolic measurements on lymphoblasts from both patients and unaffected heterozygous individuals using a seahorse xf24 instrument . the seahorse metabolic analyzer measures oxygen consumption rates under 4 conditions : basal , complex v inhibited ( oligomycin ) , uncoupler stimulated ( carbonyl cyanide-4-(trifluoromethoxy ) phenylhydrazone [ fccp ] ) , and in the presence of complex i and iii inhibitors ( rotenone , antimycin a ) .
we followed the manufacturer 's protocols , with steps and conditions optimized for lymphoblasts . in a typical experiment ,
2 patient samples and 2 samples from unaffected heterozygous individuals were analyzed in a 5-well per sample setting .
the exome sequencing data from 3 affected individuals showed that 21 variants in 20 genes were shared by these 3 patients .
subsequent sanger sequencing and cosegregation analysis demonstrated that 5 variants in 4 genes were present in all 5 patients .
variants in 3 genes , including sptn2 , tpcn2 , and orc74 , were also present in the unaffected individuals ( table 1 ) .
uniquely , 2 missense variants in spg7 , c.2084t > c / p.l695p and c.2228t >
the l695p was present in the unaffected father and 2 unaffected siblings , and the i743 t was present in the unaffected mother ( figure 1a ) .
we performed sanger sequencing of a cohort of 480 caucasian controls and did not find these variants .
the l695p variant was not present in the exome aggregation consortium ( exac ) , which includes 60,706 unrelated individuals sequenced as part of various disease - specific and population genetic studies .
the i743 t was observed in 6 individuals in a heterozygous condition but in none in a homozygous condition in the exac .
these data indicate that the compound heterozygous mutations l695p and i743 t in spg7 are linked to pls in this family .
novel variation segregation analyses for family with primary lateral sclerosis to test whether the pls - linked spg7 mutations affect its messenger rna ( mrna ) and protein stability , we performed mrna analysis using the lymphoblastoid cells and found that both alleles are expressed at a similar level ( figure 1b ) . both p695 and i743
are conserved in several vertebrate species ( figure 2a ) and are located within the spg7 c - terminal domain that is required for interaction with binding partner afg3l2 .
western blots using cell lysates from lymphoblastoid cells suggested an increased amount of spg7 protein in the compound heterozygous patients compared with the unaffected single heterozygous siblings ( figure 2b ) .
( a ) the mutated amino acids l695 and i743 are highly conserved in vertebrate species .
( b ) western blot was performed using the lysates of the lymphoblastoid cells from the unaffected heterozygous ( lanes 1 and 2 ) and the affected compound heterozygous ( lanes 3 and 4 ) individuals in this family .
the relative ratios of band density for spg7 over gapdh are shown in the right panel .
the ocr was measured under 4 conditions : basal , complex v inhibited , uncoupler stimulated , and in the presence of complex i and iii inhibitors .
the lymphoblastoid cells from 3 different patients and 2 unaffected heterozygous individuals were used in normal ( c ) or stress ( d ) conditions using the standard seahorse protocols . in all assays ,
the cells were allowed to attach for 1 hour before beginning the assay . * p < 0.05 .
the seahorse experiment 's data showed that the patient cells exhibit a higher level ( p = 0.037 ) of oxygen consumption in the presence of the uncoupler fccp than do the unaffected cells ( figure 2c ) . in a second experiment ,
this stress condition partially inhibits glycolysis and glucose uptake . as shown in figure 2d , the basal level and the fccp - induced oxygen consumption are greatly reduced ( p = 0.003 ) in the patient - derived cells but do not change significantly in the unaffected individuals , which suggests that mitochondria with the compound heterozygous mutants were more sensitive to glucose reduction
mutations in spg7 , which encodes a mitochondrial protein paraplegin , typically cause a recessive form of spastic paraplegia . recently
, it has been shown that homozygous or compound heterozygous mutations in spg7 also lead to chronic progressive external ophthalmoplegia ( cpeo ) and are a major cause of previously unexplained adult - onset ataxia .
several mutations were found in patients with sporadic adult - onset umn syndrome , none of which produced symptoms in the bulbar region .
we report compound heterozygous missense mutations that lead to pls in an autosomal recessive fashion in a large family with 5 affected individuals , therefore expanding the spectrum of spg7-linked diseases .
pls typically occurs sporadically , and its etiology and pathogenic mechanisms are not well understood .
we previously found that loss - of - function mutations in alsin cause recessive amyotrophic lateral sclerosis ( als ) type 2 and juvenile primary lateral sclerosis ( jpls ) .
we observed that the mutations affecting both isoforms led to als , whereas the mutations affecting only the long form , but leaving the short form intact , resulted in jpls .
this observation led us to propose that the short isoform of alsin may retain some functions that protect lower motor neurons , therefore resulting in the phenotype differences . in mice , there are 2 paraplegia isoforms .
the long isoform is targeted to the mitochondria , and the short isoform is targeted to the endoplasmic reticulum .
knockout of the long isoform causes mitochondrial abnormalities . in the knockout model , the short isoform
is retained and is not affected . of note , spg7 has 6 isoforms due to alternative splicing ; the expression profile and functional importance of the 6 isoforms are not clear .
it is interesting that in all the patients reported with cpeo or adult - onset ataxia , 1 spg7 allele was a missense or a truncation mutation affecting the 2 longest isoforms and not affecting the remaining 4 short isoforms , whereas the mutations on the other spg7 allele varied greatly .
these observations raise the possibility that full - length paraplegin with these missense mutations and the 4 short paraplegin isoforms may retain partial functions . the most prevalent missense mutation in spg7 identified in cpeo or adult - onset ataxia is a510v , which accounts for more than half of the mutant alleles in these patients .
although the initial symptoms vary , patients with these mutations may eventually develop spasticity after a long duration of disease , resulting in an overlap of symptoms with complicated or syndromic spastic paraplegia .
the mutations identified in the pls family in our report include l695p and i743 t .
the l695p mutation does not affect the 4 short isoforms , whereas the i743 t affects only the longest isoform , leaving the 5 short isoforms intact .
therefore , it is plausible that the differential expression profile and relative functional importance of the paraplegin isoforms , together with age and other genetic and environmental factors , may determine the clinical variance of the spg7-linked symptoms , especially in the early stages of the diseases .
yi yang : study concept and design , involved in all of the experiments and analyses related to this manuscript , and wrote the draft of this manuscript .
david lynch : the clinical and diagnostic workup of the patients and collection of the blood samples from family .
han - xiang deng : analyzed sequencing data and helped write the draft of this manuscript .
teepu siddique : study concept and design , analysis of data , and manuscript finalization .
the experiments were carried out in teepu siddique 's laboratory and in other laboratories at northwestern university feinberg school of medicine .
yi yang , thomas lukas , han - xiang deng , and teepu siddique wrote the manuscript .
les turner als foundation , les turner als foundation / herbert c. wenske foundation professorship , vena e. schaff als research fund , foglia family fund for als research , sp foundation , and nih grants list : ro1 es022310 .
david r. lynch has served on the editorial board of journal of neurogenetics ; holds a patent for the creation of a test for anti - nmda receptor encephalitis ; has consulted for bracket inc ; and has received research support from edison pharmaceutical , shire pharmaceutical , reata pharmaceutical , horizon pharma , nih ( r21 ) , and fara .
thomas lukas holds a patent for anti - inflammatory and protein kinase inhibitor compositions and related methods for downregulation of detrimental cellular responses and inhibition of cell death ( us8088774-b2 ) .
sleiman , eanna ryan , kimberly a. schadt , jordan h. newman , and han - xiang deng report no disclosures .
nailah siddique has received research support from ninds ( ns050641 ) and les turner als foundation .
teepu siddique has served on the editorial board of neurogenetics and amyotrophic lateral sclerosis & frontotemporal degeneration and has received funding from niehs ( ro1 ns078504 ) , ninds ( ro1 ns078504 ) , les turner als foundation , the foglia family fund for als research , les turner als foundation / herbert c. wenske foundation professorship , vena e. schaff als research fund , and sp foundation .
 objective : to identify the genetic defect for adult - onset primary lateral sclerosis ( pls ) in a family with 5 patients.methods:whole-exome sequencing was performed to identify the shared genetic variants in 3 affected members in a pls family with 5 affected individuals . 
 sanger sequencing was used for validation of the variants and for cosegregation analysis . 
 mitochondrial activity for both patients and unaffected siblings was measured using a seahorse metabolic analyzer.results:whole-exome sequencing and subsequent cosegregation analysis demonstrated that compound heterozygous missense variants l695p and i743 t in spg7 were the only mutations cosegregating with the disease in an autosomal recessive fashion in this family . 
 the parents and siblings are genetically heterozygous and clinically unaffected . 
 functional studies suggested that the pls - associated spg7 mutants affect mitochondrial function when glucose is reduced.conclusions:compound heterozygote mutations in spg7 are associated with adult - onset pls , extending the spectrum of spg7-linked neurologic diseases . 
 patients with the pls phenotype should have genetic testing for paraplegin , especially when the condition is familial . 
METHODS
Clinical presentation.
Standard protocol approvals, registrations, and patient consents.
Genetic mutation identification.
Mitochondrial activity.
RESULTS
DISCUSSION
Supplementary Material
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE