patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally . informed consent was obtained from the patient or his or her parent or legal guardian . the study was approved by the institutional review boards of austin health and the university of washington . we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology . known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) . we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members . we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic . maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) . we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) . patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally . informed consent was obtained from the patient or his or her parent or legal guardian . the study was approved by the institutional review boards of austin health and the university of washington . we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology . known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) . we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members . we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic . maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) . we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) . we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples . we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) . a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo . patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy . patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) . this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) . clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms . variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) . upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) . lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies . many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene . it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder . we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) . family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) . this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent . family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family . spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 . two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features . eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) . three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory . one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional . brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) . d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) . after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures . we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples . we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) . a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo . patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy . patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) . this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) . clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms . variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) . upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) . lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies . many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene . it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder . we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) . family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) . this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent . family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family . spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 . two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features . eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) . three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory . one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional . brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) . d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) . after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures . we first identified depdc5 in familial focal epilepsy , and here we show its relevance to epileptic spasms , illustrating the convergence of phenotypes in genetic mutations of the mtor pathway . our findings suggest that greater significance should be attributed to a family history of focal seizures in patients with epileptic spasms . affected family members had focal epilepsies emanating from different cortical regions , consistent with the pattern of ffevf . infantile spasms have an identifiable etiology in 60% cases and may include hypoxic - ischemic or metabolic encephalopathies , malformations , infection , and chromosomal anomalies . a family history of spasms is rare but has been described in conditions such as tsc and specific genetic diseases such as arx . the importance of de novo mutations has recently been emphasized , with a pathogenic mutation attributed to a single gene identified in 5%16% of cases ( n = 268 from 3 studies using next - generation sequencing technologies ) ( table e-1 ) . the most frequently mutated genes were stxbp1 ( n = 6 ) , cdkl5 ( n = 2 ) , kcnq2 ( n = 2 ) , and alg13 ( n = 2 ) . our finding of a depdc5 variant in up to 2.3% of patients in our cohort suggests that this gene may be one of the more frequent genes associated with epileptic spasms , taking into account that this cohort had been previously screened for many of the known genes . as only 1 patient showed classic hypsarrhythmia , the cohort may have some fundamental differences from other studies of infantile spasms in which hypsarrhythmia is essential for inclusion . we identified 3 truncating mutations : 1 occurred de novo and the remaining 2 were inherited . this is in keeping with previous reports in which the overwhelming majority of pathogenic depdc5 mutations resulted in premature truncation of the protein ( figure 2 ) , suggesting that depdc5 mutations cause disease by haploinsufficiency of the protein . this is further supported by the presence of only 15 truncating variants in the 61,000 exomes in exac . however , we report 2 missense variants , pro1031his ( maf 0.7% ) and tyr281phe ( maf 0.007% ) , and there are more than 400 missense variants in exac . tyr281phe is exceedingly rare , and incomplete penetrance of depdc5 mutations may explain the presence of these variants in the population . however , the 0.7% maf of pro1031his ( arose de novo in family c ) seems too high in this population to be explained solely by incomplete penetrance , and this result needs to be interpreted with caution . this may also be true for several reported missense mutations also present in controls at low frequencies ( figure 2 ) . it will be important to develop robust functional experiments to assess the pathogenicity of these missense variants in the future . we have combined a targeted resequencing approach in 130 patients and results from clinical diagnostics and extended phenotyping in a known mutation - positive family to determine several notable features that highlight the variability of onset and outcome of depdc5-associated spasms . our findings expand the depdc5 phenotypic spectrum to include more severe epilepsies presenting with spasms . first , the outcome may be excellent with normal intellect , although most of our patients had intellectual disability with or without autistic features . third , 1 patient had later onset of spasms in the third year with further cognitive decline in adolescence . fourth , 2 patients had malformations with fcd ; in 1 , surgery resulted in seizure improvement . surprisingly , only 1 patient showed hypsarrhythmia on eeg ; however , all had highly abnormal eegs with abundant epileptiform activity , which can be associated with epileptic spasms . the absence of classic hypsarrhythmia means that these patients differ from those with west syndrome , which has been the focus of many recent genomic studies . of note , spasms were present in patients with fcd ( 2 patients ) and those without fcd ( 3 patients ) after careful scrutiny of the mri . because many patients with focal epilepsies and depdc5 mutations have normal mri , the presence of detectable lesions is not necessary for the development of seizures . rather , loss - of - function mutations in depdc5 , an inhibitor of the mtor pathway , presumably lead to excessive signaling of this pathway , which has many functions that could conceivably contribute to hyperexcitability . this scenario would be analogous to tsc , in which a second mutation in the mtor regulators tsc1 and tsc2 is reported in some tumors of patients with tsc . in patients with depdc5 mutations , second hit  could occur either on the other allele or on another gene involved in the mtor pathway . alternatively , an acquired cause , such as a human papillomavirus , has been conjectured to be a deep targeted or even whole - exome sequencing should be performed on resected tissue to explore this hypothesis . given the incomplete penetrance of depdc5 mutations and the discovery of both inherited and de novo mutations , molecular approaches for epileptic spasms should interrogate both inheritance patterns . the detection of inherited mutations has important reproductive counseling implications for families of children with spasms , which needs to incorporate increased risk for comorbidities such as intellectual disability and autism spectrum disorders . the recognition of spasms in depdc5 epilepsies parallels other mtoropathies such as tsc and ste20-related kinase adaptor alpha syndrome . although many depdc5 epilepsies are milder , the convergence of the phenotypic spectrum and molecular pathways suggests that targeted mtor therapies may benefit patients with the more severe depdc5 disorders . g.l.c . : drafted / revised the manuscript , study design , acquisition of data , and data analysis . d.e.c . : drafted / revised the manuscript , acquisition of data , and data analysis . b.m.r . : , s.f.b . , and j. sullivan : acquisition of data and data analysis . i.e.s . and h.c.m . : drafted / revised the manuscript , study design , acquisition of data , and data analysis . this work was supported by funding from the nih ( ninds 5r01ns069605 ) to h.c.m . is supported by a postdoctoral fellowship from the epilepsy foundation and the lennox and lombroso fund and the nih ( ninds 1k99ns089858 ) . i.e.s . , s.f.b . , and l.d . are supported by a national health and medical research council of australia ( nhmrc ) program grant . is supported by an nhmrc practitioner fellowship and l.d . is supported by an nhmrc career development fellowship ( 1032603 ) . the work was also supported by the university of washington intellectual and developmental disabilities research center genetics core ( nih u54hd083091 ) . dr . carvill is a member of the scientific advisory board of ambry genetics and has received grants from the nih / national institute of neurological disorders and stroke , the epilepsy foundation and lennox and lombroso trust , and citizens united for research in epilepsy . crompton has received research support from the national health and medical research council of australia . mcmahon has received research support from the national health and medical research council of australia . howell has received research support from the national health and medical research council gustav nossal postgraduate scholarship and her husband holds stock options for general electric ( medical imaging equipment ) . mandelstam has been an employee of the royal children 's hospital , the melbourne brain centre , and the university of melbourne . dr . leventer has received honoraria for travel from the asia oceania congress of child neurology and has received research support from the national health and medical research council project grant 1068278 , the national health and medical research council project grant 1059666 , and campbell edwards trust research support . mullen has been an employee of the florey institute of neuroscience and mental health , the university of melbourne , australia austin health , and australia eastern health ; has received travel funding from the american epilepsy society young investigator award ; and has received research support from the nhmrc early career fellowship and nhmrc project grant . berkovic has served on the scientific advisory board of ucb pharma ; has served on the editorial boards of brain , epileptic disorders , and lancet neurology ; has received research support from the national health and medical research council of australia ; has received honoraria from ucb ; has a patent for pcdh19 testing planned and is one of the inventors listed on a patent held by bionomics inc . on diagnostic testing of using the scn1a gene ; has received payment for development of educational presentations from ucb pharma , novartis pharmaceuticals , sanofi - aventis , and janssen - cilag ; and has been a consultant to bionomics and athena diagnostics . dr . sullivan has been the chair of sab for nonprofit entity pcdh19 alliance ; has received honoraria from zogenix ; and has received research support from marinus pharmaceuticals . scheffer has served on scientific advisory boards for ucb and janssen - cilag emea ; has served on the editorial boards of the annals of neurology , neurology , epilepsy currents , progress in epileptic disorders series , virtual neuro centre , and epileptic disorders ; holds patents for methods of treatment and diagnosis of epilepsy by detecting mutations in the scn1a gene , a diagnostic method for epilepsy , mutations in ion channels , diagnostic and treatment methods relating to autosomal dominant nocturnal frontal lobe epilepsy ( pending ) , gene and mutations thereof associated with seizure disorders , a gene and mutations thereof associated with seizure and movement disorders , and diagnostic and therapeutic methods for efmr ; may accrue future revenue on pending patent wo61/010176 ( filed : 2008 ) : therapeutic compound ; has received speaker honoraria from glaxosmithkline , athena diagnostics , ucb , biocodex , and janssen - cilag emea ; has received funding for travel from athena diagnostics , ucb , biocodex , glaxosmithkline , janssen - cilag emea , aoccn taiwan , weizmann institute , the american academy of neurology , irccs oasi maria ss , sanofi china , qbi university of queensland , international league against epilepsy , australian academy of science , commonwealth department of industry , westmead hospital , perpetual , university of california , matthew 's friends , sbs , pts for nfle conference , turkish child neurology association , european congress on epileptology , international child neurology association , ucb , movement disorder society , international epilepsy congress , university of auckland , world congress of neurology , epileptic disorders , eisai , aoccn , sanofi , and transgenomic ; and has received research support from the national health and medical research council of australia , nih , australian research council , health research council of new zealand , cure , american epilepsy society , us department of defense autism spectrum disorder research program , the jack brockhoff foundation , the shepherd foundation , perpetual charitable trustees , the university of melbourne , the epilepsy association of tasmania , melbourne neurosciences institute , weizmann institute , cure sudep award , and perpetual philanthropic services . dr . mefford has received research support from the nih / national institute of neurological disorders and stroke , the burroughs wellcome fund , the american thoracic society foundation partner award , and simon 's foundation for autism research ; has served on the editorial board of science translational medicine , is a consultant for sera prognostics and the simons foundation ( sfari gene advisory board ) ; and has served on the scientific advisory boards of the lennox gastaut foundation and supporting families with koolen - de vries syndrome .
objective : to assess the presence of depdc5 mutations in a cohort of patients with epileptic spasms.methods:we performed depdc5 resequencing in 130 patients with spasms , segregation analysis of variants of interest , and detailed clinical assessment of patients with possibly and likely pathogenic variants.results:we identified 3 patients with variants in depdc5 in the cohort of 130 patients with spasms . 
 we also describe 3 additional patients with depdc5 alterations and epileptic spasms : 2 from a previously described family and a third ascertained by clinical testing . 
 overall , we describe 6 patients from 5 families with spasms and depdc5 variants ; 2 arose de novo and 3 were familial . 
 two individuals had focal cortical dysplasia . 
 clinical outcome was highly variable.conclusions:while recent molecular findings in epileptic spasms emphasize the contribution of de novo mutations , we highlight the relevance of inherited mutations in the setting of a family history of focal epilepsies . 
 we also illustrate the utility of clinical diagnostic testing and detailed phenotypic evaluation in characterizing the constellation of phenotypes associated with depdc5 alterations . 
 we expand this phenotypic spectrum to include epileptic spasms , aligning depdc5 epilepsies more with the recognized features of other mtoropathies .