BACKGROUND
intellectual disability  affects 1–3% of the global population.
a significant proportion of id is caused by genetic defects, yet despite extensive testing including by clinical chromosomal microarray , ~30% of cases remain idiopathic  <cit> .
genome-wide sequencing can identify previously unknown genes causative for id.
whole exome sequencing  is limited by poor ability or inability to detect non-coding and structural variation, and capturing less than 100% of the exome  <cit> .
in contrast, whole genome sequencing  offers a comprehensive screen of a variety of dna variation types.
current evidence suggests wgs is able to detect coding variants in 42% of cases missed by wes  <cit> .
we report comprehensive wgs analyses for eight patients with id and brain malformations, whose family history suggested a de novo mutation.
despite a diagnostic odyssey, including genome-wide clinical and research cma, they were idiopathic.
wgs was conducted on trios composed of the affected child and both unaffected parents , and data was analyzed using both alignment and assembly approaches to detect all possible causative genetic changes- single nucleotide variants , copy number variants  and structural variants  .
we validated our findings using wes data from an independent positive control cohort of  <dig> patients with id and other neurocognitive phenotypes, and a negative control wgs cohort of  <dig> normal subjects.
in addition we probed molecular themes indicated by our discovery cohort findings in the positive control cohort, leveraging its large size.
we also conducted a screen for de novo variants in possible regulatory sequences of known and hypothesized pathogenic genes.fig.
<dig> schematic of complete study design.
abbreviations: cnv = copy number variant; sv = structural variant; snv = single nucleotide variant; ddd = deciphering developmental disabilities study; upp = ubiquitin proteolysis pathway; igv = integrated genome viewer; dgv = database of genome variation
methods
subjects
patients were enrolled from the british columbia children’s and women’s hospital provincial medical genetics program after obtaining informed consent.
this study is approved by the british columbia children’s and women’s hospital research ethics boards.
all patients presented with id  and brain morphological defects detected by mri or ct scan.
patients had no family history of id, and all were products of normal pregnancies with no reported teratogenic exposures as ascertained by clinical assessment by board certified medical genetics specialists at the recruiting facility.
saliva samples were collected and dna extracted using dna genotek® collection kits, reagents and protocols from child, father and mother.
methods
wgs methods, variant calling protocols, verification methods, and secondary control study methods including bootstrap analysis, are summarized below and detailed in additional file  <dig>  briefly; dna was extracted using dnagenotek® extraction kits.
paired-end wgs libraries were prepared using illumina’s pcr-free protocol .
sequencing was by illuminahiseq  <dig> platform  generating 100 bp paired-end reads, using three lanes per sample .
alignment and variant calling was by canada’s michael smith genome science center standard pipelines .
variants were identified and filtered as follows, briefly; putative snvs were identified using samtools mpileup version  <dig> .
<dig> run on each sample separately.
relatedness was tested for each trio by comparing snp concordance between child, mother and father using vcftools- <dig> .
<dig>  <cit>  .
de novo variants were selected by intersecting the child’s snvs with that of each parent, and selecting variants only present in the child and not in either parent.
for variants in the coding region, we selected de novo missense, nonsense and splicing variants, i.e., functional variants.
we next selected rare variants by excluding alleles with minor allele frequency >1% in dbsnpv <dig> , exome variant server, exome aggregation consortium , and in-house databases of > <dig> exomes, and > <dig> genomes .
we then used pathway enrichment analyses to prioritize de novo rare variants; selecting snvs in genes enriched in pathways involved in brain development and function conducted using qiagens ingenuity® pathway analysis , david  and panther .
for those variants passing the pathway enrichment screen, pathogenicity predictions and conservation scores were annotated using sift  <cit> , phylop  <cit> , polyphen  <cit> , mutationtaster  <cit>  and cadd  <cit>  scores.
these steps yielded de novo, functional, rare variants, that are highly conserved and predicted to be damaging and in biologically relevant pathways.
in addition to the above prioritization, the rare functional variants were subsequently also screened under a series of additional genetic models , and manually checked for alignment quality with integrated genomic viewer .
snvs that were highly conserved and were predicted to be damaging by at least one pathogenicity prediction software, were selected for verification by sanger sequencing in the child, mother and father.
cnv analyses was conducted using freec  <cit> , cnaseq  <cit> , delly  <cit>  and abyss  <cit> .
the first three algorithms align reads to the reference genome while abyss uses de novo assembly to reconstruct the patient’s genome.
sv analyses was conducted using only delly and abyss.
first, de novo cnvs/svs were identified by comparing the child’s data to that of either parent .
de novo cnvs from each algorithm were filtered by manual assessment of local read configuration on igv, and genuine ones were prioritized based on functional relevance of the included/cnv-affected genes.
svs, i.e., translocations and inversions, were filtered by either igv read visualization and then by using qc metrics specific to each algorithm; qc metrics generated by the program were used for delly, and checking of blat scores for breakpoint-junction contigs and number of supporting reads were used for abyss.
candidate cnvs/svs that were selected from the above filtering were verified using an independent method as detailed below.
all de novo variants, i.e., snvs, cnvs and svs, were verified by sanger sequencing of pcr-captured amplicons of the affected sequence, either bearing the snv or spanning the breakpoint junction  in the trio, with forward and reverse primers .
all verified candidate snvs were subjected to genotype-phenotype correlations assessment as per the guidelines of the american college of medical genetics   <cit> .
secondary control study - wes data from the uk10k project  <cit>  for  <dig> patients with neurofunctional phenotypes , and wgs data from  <dig> normal individuals from the  <dig> genomes project  <cit> ; a publicly available repository of variation in healthy individuals, was obtained.
‘possibly damaging snvs’ , were extracted from these datasets , and a gene-wise pds burden for all genes in the human genome was determined in both the positive and negative control cohorts.
subsequently the gene-wise pds burden only in our candidate genes was compared between the positive and negative control cohorts.
we further bootstrapped the positive control cohort to determine if the pds burden in our six candidate genes could be due to random sampling.
finally, we tested to see what functional pathways genes with pds in the positive control cohort were involved in, and conducted a kyoto encyclopedia of genes and genomes  pathway enrichment analyses, testing which of the total  <dig> functional pathways from kegg were most enriched for genes bearing pds in this large dataset.
regulatory region variation – for our regulatory region analysis we selected ‘high confidence’ de novo snvs defined as having a mapping quality >  <dig> and read depth ≥  <dig> and ≤  <dig>  and ‘high confidence’ de novo cnvs defined as those that were detected by two or more cnv detection algorithms.
we then intersected both the de novo high confidence snvs and cnvs with six non-coding sequence annotation datasets.
results from the above, i.e., de novo high confidence snvs and cnvs with involvement in putative regulatory regions, were then intersected with candidate gene lists and appropriate flanking sequence  to determine their possible association to a candidate known or hypothesized id gene.
RESULTS
de novo snvs identified by objective molecular pathway-based filtration
genes with functional rare de novo snvs were screened using three pathway analyses programs  in order to refine candidates involved in brain development and function; ipa returned  <dig> candidate genes, david returned  <dig>  and panther returned  <dig>  a total of  <dig> unique genes involved in brain development and function were yielded by the combined analyses .
from these, highly conserved and predicted damaging snvs  were sanger tested, and six snvs in six genes in five children were confirmed as heterozygous de novo ; arid1b  nm_017519:c.1595delg , phf <dig> nm_001015877:c.820c > t , spry <dig>  nm_001127496:c.508 t > a , cacnb <dig> nm_0012069:c.1289g > a , sqstm <dig>  nm_03900: c.115_116delinsta  and upf <dig>  nm_002911:c.1576_1577delinsaa .
the latter two were found in a single patient while the rest each appeared in a separate patient.
as best practice, we also screened our de novo rare functional variants for location within published known  <cit>  and candidate id genes  <cit> , however no new findings were yielded.
except arid1b and phf <dig>  the other genes are novel for id.
table  <dig> provides variant classification as per the acmg variant interpretation guidelines  <cit>   and our interpretation of their causative effect.
brief genotype-phenotype correlations are given below;table  <dig> patient phenotype and variant summary
arid1b
phf6
spry4
ap4e1
ap4e1
cacnb3
scn3a
51b
sqstm1
upf1
lrp2
lrp2
abbreviations: id intellectual disability, ofc occipito-frontal circumference, ct computerized tomography scan, mri magnetic resonance imaging scan, pvs <dig> null variant in a gene where lof is a known mechanism of disease, ps2
de novo in a patient with the disease and no family history, pm <dig> absent from controls in exome sequencing project,  <dig> genomes project or exome aggregation consortium, pp <dig> multiple lines of computational evidence support a deleterious effect on the gene or gene product
aage at examination is given in 5 year intervals in order to protect patient anonymity
bpatient  <dig> also bears a de novo likely contributory cnv as detailed in the text, in addition to the snvs given here
arid1b c.1595delg  in patient 43
this single base deletion in exon  <dig> of the known id gene arid1b causes a frame-shift leading to predicted loss of function .
our patient presents with id, autism, absence of corpus callosum, absence of speech, feeding difficulties and failure to thrive .
haploinsufficiency of arid1b was reported to cause corpus callosum abnormalities, id, speech impairment and autism  <cit> , suggesting the arid1b lof is causative and sufficient in this case.
phf <dig> c.820c > t  in patient 58
phf <dig> encodes the plant homeodomain finger protein  <dig>  the nonsense variant in phf <dig> is located in the ephd <dig> domain in which causative de novo truncating and missense variants for börjeson-forssman-lehmann syndrome    <cit> , and coffin-siris syndrome    <cit>  are known.
de novo truncating and other mutations in phf <dig> are reported to cause a distinct syndrome in girls  <cit>  and reported for a female specific form of bfls  <cit> .
roles for phf <dig> are reported in the chromatin remodeling swi/snf complex  <cit> , and in the nurd epigenetic regulatory complex where it acts as a possible regulator for the latter in neurogenesis  <cit> .
rnai knock down of phf <dig> profoundly impairs neuronal migration in vivo  <cit> , thus leading to formation of white matter heterotopias.
in keeping with this, this patient reports pachygyria, which results from abnormal migration of neurons in the developing brain.
she also presents with an unusual asymmetrical growth phenotype that was reported in the one patient with the female specific bfls  <cit> .
these data indicate the variant is a good candidate in this case.
spry <dig> c.508 t > a  in patient 59
spry <dig> encodes a specific inhibitor of the mitogen-activated protein kinase family.
spry <dig> is expressed in the mouse developing brain  <cit> , and is essential for the normal morphogenesis and cytoarchitecture of the cerebellum  <cit> .
morphogenic changes in axon growth have been shown when the protein is down regulated both in vivo and in vitro  <cit> .
in zebrafish, spry <dig> is a principal regulator of mid-brain development  <cit> , and mediates hindbrain patterning  <cit> .
these data support the notion that the spry <dig> missense variant may contribute to the brain morphological phenotype in this patient.
spry <dig> expression plays a role in xenopus limb bud development  <cit> , of note as our patient reports short and crowded toes.
cacnb <dig> c.1289g > a  in patient 45
cacnb <dig> encodes a regulatory subunit of a voltage-dependent calcium channel .
mice lacking cacnb <dig> presented visual impairment  <cit> , high pain threshold  <cit> , and behavioral phenotypes  <cit> , all of which features are seen in this patient.
mutations in other members of vdcc subunit encoding genes are known to cause neurological disease, including epilepsy  <cit>  present in our patient.
this variant is found in eight of  <dig>  individuals in the exac database, where its non-absence disqualifies likely pathogenicity as per acmg criteria, despite being de novo and deleterious by multiple lines of computational evidence.
neither does it meet criteria to be a benign variant, and therefore is of uncertain significance.
sqstm1c.115_116delinsta , upf <dig> c.1576_1577delinsaa  and a 1q <dig> deletion cnv in patient 51
the patient is severely affected, with significant id and several major congenital anomalies .
the heterozygous indel formed by two adjacent snvs in sqstm <dig> causes a stop-gain.
sqstm <dig> encodes p <dig>  a regulatory factor in nuclear factor kappa-b  signaling, nf-e2-related factor  <dig>  activation, ubiquitin-mediated authophagy, and transcription  <cit> .
the snv is located in the pb1domain, mutations of which cause paget disease of bone  and frontotemporal dementia and/or amyotrophic lateral sclerosis    <cit> ; both neurodegenerative conditions that include morphological brain changes.
the adjacent snvs in upf <dig>  together cause a likely pathogenic missense amino acid change .
upf <dig> has an essential role in nonsense-mediated mrna decay  <cit> .
interestingly, upf <dig> has been shown to remarkably reduce als-associated neuronal toxicity in vitro  <cit>  and to protect against motor dysfunction and forelimb paralysis in a rat model for als  <cit> .
it is plausible haploinsufficiency of sqstm <dig> may have caused neurofunctional defects, which the haploinsufficiency of upf <dig> may have exacerbated.
in this regard, it is notable that at 19 years of age, patient  <dig> presents significant motor deficits, being wheelchair bound, indicative of a possible early onset of als.
while scoliosis and hearing loss, both among the presentation for pds is already seen in her.
these data support the notion that the snvs in both genes maybe contributory toward her presentation.
we further verified a de novo ~165 kb heterozygous deletion that spans cep <dig>  in whole and sdccag <dig>  in part  in this patient.
cep <dig> encodes a component of the centrosome  <cit> .
sdccag <dig> is also involved in centrosome function  <cit> , dna damage response signaling  <cit>  and neuronal migration  <cit> .
both genes are suggested as candidates for corpus callosum abnormalities via 1q <dig> microdeletion  <cit> , however this has been contested  <cit>  .
our patient presents partial phenotypic overlap with microdeletion 1q <dig> index cases.
the demonstrated roles for sdccag <dig> in dna-mismatch repair, and for both genes in cell cycle progression, supports the notion this cnv may be contributory.
notably, the haploinsufficiency of a dna-mismatch repair gene could lead to the high mutation burden detected in this child .
we also confirmed at least one maternally inherited balanced translocation , which is unlikely to be contributory.fig.
<dig> details of cnv analyses.
a igv images for heterozygous deletion cnv in patient  <dig>  showing proximal and distal breakpoint.
the cnv involves whole of cep <dig> and part of sdcaag <dig> genes.
top, middle and bottom panels are child’s .bam file, mother’s .bam file and father .bam file respectively.
read-depth coverage shows cnv is de novo .
b cartoon of breakpoint junction seuqence showing a 24 bp chromosome  <dig>  and 107 bp chromosome  <dig> sequence  inserted between the proximal and distal breakpoints on chromosome 1q <dig>  yellow shaded segment shows sequnce microhomology- this 14 bp seuqence  is found at chromosome 5: <dig> ,598- <dig> , <dig> and at chromosome 1: <dig> ,747- <dig> , <dig>  hg19).
sanger sequence trace images are overlaid confirming the cnv breakpoint.
grey arrows denote pcr forward and reverse primers.
n denotes dna repeat sequence.
c genomic interval involved in the de novo cnv detected in patient 51- ucsc genome browser .
red highlighted box shows region involved in the deletion in our patient.
yellow boxes show critical region for 1q43- <dig> sydrome defined by nagamani et al.
green box shows critical region as defined by perlman et al.
n.b, nagamani et al.
also highlight zbtb <dig>  in their critical region
mutation burden assessment in large secondary positive and negative control cohorts support candidacy of novel genes
we investigated the candidacy of the above verified genes by assessment for incidence of damaging mutation in large positive and negative cohorts with comparable ngs data.
we looked for ‘potentially damaging snvs’   in our candidate genes, from wes of  <dig> patients with neurodevelopmental and neurocognitive phenotypes from the uk10k cohort  <cit>   and compared that to incidence in wgs from  <dig> healthy people from the  <dig> genomes project  <cit> .
we first screened for the exact variant detected in our discovery cohort, and did not find any case of an exact match.
we then conducted a gene-wise pds screen and observed that incidence for pds in arid1b, spry <dig>  cacnb <dig>  sqstm <dig> and upf <dig> were significantly enriched in the positive versus negative control cohorts .
there was no significance for phf6; however, the two pds found in  <dig> people was insufficient for meaningful statistical assessment.
the extremely high pds burden in the positive control cohort for sqstm <dig> and upf <dig> is noteworthy, as these genes have previously not been reported for id to our knowledge, and further, the indels in both are found in the same patient in our cohort.fig.
<dig> validation study.
a showing incidence for potentially damaging snvs  in both the positive control  and negative control  control cohorts.
* denotes statistical signficance  b and c results of bootstrap analyses for pdss in  <dig> randomly selected genes.
red vertical bar shows the mean and median result for pdss in our  <dig> candidate genes
while we do not have access to clinical data to conduct a classical genotype-phenotype correlation between cases in the positive control cohort and our patients who have the same gene affected, the large number of such cases in the positive control cohort also impedes such a study within the scope of this work.
we therefore assessed if our findings could be due to random chance effect, by bootstrapping the uk10k cohort for pds in six randomly selected genes each, a thousand times.
we found from the bootstrap analysis that the mean and median gene-wise variant frequency for our six candidate genes was greater than that of the corresponding distribution, indicating that our findings were not likely due to chance .
these data are consistent with an association of at least five of our candidate genes with neurodevelopmental abnormalities.
novel candidate genes converge unto the ubiquitin proteasome pathway, which also bears significant mutation burden in  <dig> positive control wes samples
we investigated molecular links between our pathogenic and candidate genes; focused ipa and string pathway analyses revealed that all six connected to the ubiquitin proteasome degradation pathway   which has important roles in the structural development and function of the brain  <cit> .
we assessed the relative importance of this pathway and found the upp was among significantly enriched pathways for pds when compared with all kegg pathway categories  , in the uk10k patient cohort , substantiating the importance of the upp pathway in brain development.
mendelian inheritance and n of  <dig> analyses provides additional candidate variants
in addition to our in-silico refinement and test for candidate de novo snvs above, we also conducted a classical series of n of  <dig> studies for these eight patients; manually assessing the possible candidacy of variants selected by all possible mendelian inheritance patterns .
compound heterozygous missense mutations were identified in lrp <dig> , causative of the autosomal recessive donnai barrow syndrome  in patient  <dig>  absence of the corpus callosum, reported in our patient, presents in donnai barrow syndrome.
compound heterozygous mutations were identified in ap4e <dig>  causative of autosomal recessive spastic paraplegia type  <dig>  in patient  <dig>  this patient reported a seizure at 12 years of age, however does not exhibit the severe neurological phenotypes nor the shy demeanour reported for a possible syndromic form of id  <cit>  caused by defects in adaptor protein complex- <dig>  of which ap4e <dig> encodes one of the four subunits.
a missense scn3a  snv  in patient  <dig> was selected due to scn3a association to epilepsy  <cit>  , and the predicted deleterious effect of the variant, and was sanger verified as de novo.
functional studies are underway to further investigate the role of scn3a in epilepsy.
extensive copy number variant  and structural variant  analyses identifies likely causative cnv missed by clinical cma, and balanced benign translocation
we conducted both alignment-based  and de novo assembly-based  cnv/sv analyses.
cnvs, i.e., duplications  and deletions  were identified by all four platforms, while svs, i.e.
translocations and inversions, were identified by delly and abyss .
an average of  <dig> de novo gain cnvs and  <dig> de novo loss cnvs across all eight patients were detected.
however, only  <dig> cnvs were called by over one platform, and none were called by more than two , with the majority of each algorithm’s findings being unique.
we carried out extensive visual in silico curation for all cnvs, and selected three to verify of which, only the previously discussed 1q <dig> loss cnv, sanger verified as de novo- it was detected by freec and cnaseq, and is clearly visible on igv .
breakpoint junction sequence reveals a complex architecture .table  <dig> number of copy number variants and structural variants identified
fig.
<dig> venn driagram showing cnvs found by each algorithm
similar to our cnv results, sv results from delly and abyss were divergent .
only one translocation  in patient  <dig>  was called by both, and there was no concordance among inversions.
upon extensive manual in silico curation we selected  <dig> translocations and  <dig> inversion to verify , but none verified as de novo.
sanger verification for these lesions was challenging as breakpoints mapped to repeat-masked regions, nevertheless one translocation verified as maternally inherited; a chromosome x- <dig>  translocation not causing any gene-disruption, in patient  <dig>  the breakpoint junction shows a single base addition .
genome assembly yields small insertions/deletions  missed by genome re-alignment
in contrast to the re-alignment based algorithms, abyss  <cit>  identified over  <dig> potential de novo indels , via genome assembly.
forty three indels were refined as likely true positives with a functional importance, due to having at least seven spanning reads, and producing a protein coding change; the majority being in patient  <dig>  for consistency, we conducted a pathway analyses for the indel-bearing genes, and a manual curation, in order to select candidates for verification as we had done for our snvs.
this resulted in  <dig> indels that were sanger tested ; however one was false positive, five were inherited, and eight did not pass pcr quality checks , indicating location to repeated dna sequence, thus hampering any ability to amplify the region for sanger sequencing.
gene regulatory region variation identified in known and hypothesized id genes
we investigated gene regulatory sequence variation which we term ‘de novo variants in possible regulatory regions’ .
we filtered the dvprr for potentially pathogenic changes using two approaches: by screening for involvement in known id genes, and on the basis of our hypothesized involvement of the upp.
an average ~ <dig>  de novo snvs were found across our eight patients in the non-coding genome .
of these, an average  <dig> located to transcription factor binding sites, an average  <dig> to putative gene promoters, an average  <dig> of those located to promoters were also located to transcription factor binding site regions, an average  <dig> located to regions annotated as enhancers by the fantom consortium  <cit> , an average  <dig> belonged to 5′ or 3′ utr regions and an average  <dig> located to highly conserved ultra-sensitive regions  <cit>  – we considered these to be dvprr and therefore there were an average  <dig> dvprr across all eight patients .
we then intersected dvprrs with  <dig> genes known to cause developmental delay   <cit>  in a disease gene screen approach, and with the total  <dig> genes of the upp , − as our candidate genes converged upon the upp - in a hypothesis-driven approach.
as a final step for enhancers and ultra-sensitive regions, we further selected dvprr where it, and the candidate gene , were located within the same topological domain  <cit> , postulating that their physical proximity would imply that the regulatory region in question did in fact impact the targeted gene.
in summary we found an average of  <dig> and  <dig> dvprr per patient in our gene-screen and hypothesis driven approach respectively, by these filtrations combined  .
we also interrogated high-confidence cnvs in the same manner, but only found association to sdccag <dig>  a known id gene present in the previously discussed 1q <dig> microdeletion .fig.
<dig> schematic of filtration pipeline for variants in non-coding regions.
a schematic for snvs.
b schematic for cnvs.
abbeviations; snv- single nucleotide variant, tfbs – transcription factor binding site, fantom-enhancer sequence as annotated by the fantom consortium.
utr – untranslated regions.
ddd- deciphering development disabilities.
upp – ubiquitin proteosome degredation pathway.
cn- copy number.
patient  <dig> had dvprr in the utrs of two genes; cbl and ube3b.
patient  <dig> had a dvprr in the promoter of ube3a, patient  <dig> had a dvprr in the promoter of cul4b, and patient  <dig> had dvprrs in the promoters of ube3a, cul4b and cul <dig>
occurrence of de novo snvs in non-coding rnas
we found an average of  <dig> high confidence de novo snvs that located to sequence annotated as ncrna across all eight patients.
a majority of these  fall within introns while an average  <dig> are exonic, an average  <dig>  are predicted in splice junction sequence and average  <dig> and  <dig> are located to 3′ and 5′ utr respectively.
discussion
selection of candidate snvs: comparison of strategies
an effective strategy is essential to select causative snvs from ngs data.
standard filtration approaches  yield potential de novo variants that then must be careful sifted for likely true candidates.
in keeping with others  <cit> , we found an average of  <dig> +/−  <dig> candidate unverified de novo snvs , and it was necessary to implement an effective prioritization approach for verification.
discovery wgs and wes studies published to date have used a large sample size  <cit> , detailed pedigree information  <cit> , or well characterized rare syndromes  <cit>  as study cohorts, leveraging the power of numbers, inheritance pattern, and phenotypic commonality, respectively, as filtration strategies.
in as much as we did not have a large cohort, all of our cases were sporadic, and none had a recognized dysmorphic syndrome, we refined snvs objectively, by selecting genes known to be involved in brain development pathways.
we reasoned that this systematic approach would reduce subjective bias inherent in an n of  <dig> genotype-phenotype correlation, and thereby identified potential candidates.
however a subjective screen for snvs yielded the likely damaging variant in scn3a, which was not stratified by our objective approach –highlighting the limitation of pathway analyses programs that depend on available gene-functional annotations.
notably scn3a was selected by a team of biochemical geneticists specifically with respect to the epilepsy presented by the child.
thus a subjective approach may also miss results detected from objective screening, as exemplified in this case, where the two analyses were done by independent members and each did not report the result of the other.
interpreting detected variants; discovery study findings further inform genetic complexity for id
variable expressivity and reduced penetrance are well known in the pathogenicity of id, and it is increasingly recognized that a single mutation in a single gene may only rarely explain the full phenotypic spectrum  <cit> .
our results provide further indications of such complex heritability; in patient  <dig>  the 1q <dig> deletion, and snvs in sqstm <dig> and upf <dig> may act in concert to produce the complex and severe phenotype in this patient.
while in patient  <dig>  we have identified both compound heterozygous variants in the known id gene ap4e <dig> that act in a recessive model, as well as de novo variant in a novel gene spry <dig>  which has important functions in brain development.
de novo mutation is recognized to play an important role particularly in the pathogenicity of id  <cit> , and it is difficult to determine to what extent each of these variants, if at all, contributes to disease burden in this patient.
the same is true for patient  <dig> in whom de novo variants for two novel genes, cacnb <dig> and scn3a were identified.
we note that patient  <dig> who bears the most complex genotype, is the most severely affected in our cohort, and in this case, clinical severity does co-relate with number and complexity of genomic alterations, suggesting that gradation of clinical severity may provide useful toward assessing the contribution of genomic alterations.
it is recognized that genes responsible for id converge onto common networks  <cit> .
the candidate genes we identified converge onto the upp, which is critically involved in neurodegenerative disease  <cit>  and has important roles in neurodevelopmental disorders  <cit> .
this observation is consistent with the notion that they may be good candidates, and exemplifies the usefulness of probing molecular links among novel findings.
large secondary positive wes cohort analysis supports novel findings
novel snv findings from ngs studies require rigorous additional studies to support proof of pathogenicity  <cit> .
in our case, several of our novel candidates cause missense variation, whose effect is difficult to model, as opposed to clear loss of function mutations which are amenable to functional studies in model organisms.
conversely we were unable to conduct traditional genotype-phenotype correlations studies as none of our patients had a recognizable syndrome to match with other patients.
therefore, our approach of using a large secondary positive control cohort, despite the phenotypic spectrums not matching our cases precisely, gave us sufficient ability to test the predicted causality of our candidate genes and was the best strategy available.
we were hampered by the lack of an optimal comparison negative control cohort.
we used wgs data from the  <dig> genomes project, which we recognize is primarily comprised of low coverage samples whose phenotypic spectrum is poorly characterized , yet the similarity of sample size between the two groups allowed us to explore the pds distribution for these genes reasonably, providing a useful contributory analysis toward assessing their likely pathogenicity.
finally this large cohort enabled us to further probe the convergence of our candidate genes upon upp, by assessing its contribution versus other biological pathways.
wgs is able to detect structural variants below the threshold of clinical cma, and enables mechanistic insights into cnv formation
by using wgs instead of wes, we were able to detect a cnv below clinical cma resolution, isolate it’s breakpoints, and uncover a possible complex genomic landscape in one patient.
we wanted to conduct a comprehensive screen for cnvs and other structural variants to maximize sensitivity.
therefore we used four approaches that are fundamentally different; cnaseq and freec are sequence based copy-number estimators that use categorically different algorithmic approaches for background correction.
delly is an alignment based assembler, whilst abyss is a de-novo genome assembler.
since each algorithm was optimized differently, it therefore yielded different results.
for example, cnaseq executes read-depth based binning, and hence aggregates results at telomeres and centromeres where a larger number of reads re-align due to pervasive repeat sequence .
the verified cnv we detected was only identified by delly and freec, but missed by the other algorithms.
therefore, we caution against using only one cnv detection algorithm as this would reduce sensitivity.
the breakpoint junction sequence in the case of the confirmed 1q <dig> microdeletion is consistent with the notion that it could be caused by chromotripsis, a mechanism only recently reported in the constitutional genome  <cit> , further demonstrating the utility of wgs data.
wgs enabled a de novo genome assembly that unmasked hidden genome complexity
abyss de novo assembly identified a translocation missed by delly, and also detected a higher than usual number of putative indels in patient  <dig>  who was found to have a remarkably unstable genome masked by standard genome re-alignment based analysis .
however, we experienced difficulty confirming these events via sanger sequencing, which was due, in part, to the high degree of repeated sequence at breakpoint junctions.
genome assembly is able to call events in repetitive sequence better than alignment based algorithms  <cit> , though conversely such events are harder to independently verify.
we are among the first to use de novo assembly to interrogate patients with id, and our findings suggest variation located to repeat enriched sequence is currently under-ascertained in the constitutional genome.fig.
<dig>
a sanger sequencing verification of translocation in patient  <dig>  with karyotype cartoon of balanced translocation.
pcr amplicon trace file shows sequence mapping across the chromosome x - <dig> translocation boundary.
zoomed-in view shows single base addition at breakpoint jucntion.
b circos plot showing mutation burden for patient  <dig> called by abyss genome assembly
wgs is able to interrogate regulatory genomic sequence
meaningful interpretation of snvs within regulatory sequence is hampered by the sparsity of annotations for the non-coding genome.
we implemented two different filtering strategies in order to identify non-coding snvs that could have a functional impact, and also used topological domain data to further refine good candidates.
though we were able to reduce the number of candidate dvprr from an average > <dig> to dozens in the case of our gene-screen approach and a handful in the case of our hypothesis driven approach, nonetheless without further focused studies, meaningful interpretations are precluded.
in contrast, assessing the impact of cnv-based dvprr is theoretically less challenging, as it is more straightforward to predict functional outcome for a complete loss or gain of a possible regulatory sequence.
in summary, though clinically relevant conclusions for dvprr will require a case-by-case analysis and extensive follow-up functional studies, nevertheless we note it is possible to stratify dvprr in the context of known causative genes for id using wgs.
wgs versus wes
wgs yields a comprehensive screen of the genome as, in addition to coding variation, it includes ability to investigate structural variation at a fine scale as discussed above, and also variation in possible gene regulatory sequence as well as ‘non-coding genes’ such as ncrnas for which there is a paucity of information in the context of neurodevelopmental disease.
while we show strategic stratification for dvprr can yield results potentially relevant to id causation, much less is possible for annotation of snvs within ncrna sequence, of which we detect an average  <dig> across our samples.
nevertheless, initial screens such as ours, importantly generate exploratory information for non-coding sequence variation possible only by wgs.
we note that all the snvs we identified as involved in disease would have been possible to detect by wes.
however, wgs yields a more complete view of possible pathogenic variation in each child.
this is exemplified in the case of patient  <dig>  for whom had only wes been performed, while the sqstm <dig> and upf <dig> snvs would likely have been detected, the 1q <dig> microdeletion would not have been identified.
in the case of this patient, it is unclear what the gene-effect size for each variant is.
conversely, in the case of patient  <dig>  for whom we detected the snv in arid1b, we are more certain of the penetrance of this variant due to the normal results for other causative variation in their genome  from our wgs data analyses.
these data argue in favor of wgs over wes for clinical use.
CONCLUSIONS
this is the first study to present extensive analyses of wgs data in the context of id, for causative snv and cnv/sv in both coding and non-coding sequence, and the first to present results from de novo assembly of the genome.
in a heterogeneous group of eight children with id and morphological brain defects, we were able to identify candidate causative variants, highlight neurodevelopmental pathways, and unearth hidden genome instability, demonstrating the efficacy of a discovery approach to wgs analyses in the context of id.
additional files
additional file 1: supplementary methods - additional details on methods presented succinctly in main text.
additional file 2: table s <dig>  test of relatedness - table showing relatedness for each trio by comparing snp concordance between child, mother and father.
additional file 3: table s <dig>  primers for verification - all primer sequences used for snv, cnv and sv verification.
additional file 4: table s <dig>  uk10k study cohorts that comprise the positive control cohort – table giving descriptions of the study cohorts from the uk10k project that comprised the positive control cohorts and conditions for use.
additional file 5: supplementary figures.
figure s <dig>  igv image and sanger verification trace files for indel in arid1b and missense variation in upf <dig>  figure s <dig>  uk10k mutation load – counts as per variant annotation type on one patient.
figure s <dig>  histogram of mutation burden per patient in the uk10k cohort.
figure s <dig>  pathway interactions showing convergence onto upp pathway.
figure s <dig>  plots for cnv distribution for two chromosomes as called by cnaseq.
additional file 6: table s <dig>  detailed variant information for all verified variants, including acmg classification – table showing details for variant classification such as pathogenicity prediction algorithms results and details of acmg criteria application.
additional file 7: table s <dig>  significantly enriched kegg pathways for pds burden in positive control cohort – details for pathway enrichment analysis showing all kegg pathways and burden of enrichment for each in the positive control cohort.
additional file 8: table s <dig>  raw data for snvs detected across all patients by mendelian inheritance pattern filtering – all snvs detected per trio, organized as one trio per sheet, using the mendelian inheritance filtering.
sheet  <dig> gives the legend.
all snvs are annotated, including cadd and rvis scores.
additional file 9: table s <dig> cnv/sv/indel verification summary – table giving a list of all indels, cnvs and svs , and details on their verification.
additional file 10: table s <dig>  summary of snvs and cnvs across  <dig> id patients in dvprr – details on all snvs and cnvs detected in dvprr for all patients, organized by those intersecting with ddd genes and those with upp genes.
abbreviations
1000g project
http://www.internationalgenome.org/
cmaclinical microarray
cnvcopy number variant
dbsnp135
https://www.ncbi.nlm.nih.gov/snp
dgvdatabase of genomic variants
dgv
http://dgv.tcag.ca/dgv/app/home
dvprrde novo variants in possible regulatory regions
fantom consortium
http://fantom.gsc.riken.jp/
grch37-lite/hg19a
http://www.bcgsc.ca/downloads/genomes/9606/hg19/1000genomes/bwa_ind/genome/readme.grch37-lite
kegg
www.genome.jp/kegg/pathway.html
ngsnext generation sequencing
nhlbi-esp
http://evs.gs.washington.edu/evs/
omim
www.omim.org
pdspotentially damaging snvs
picard
https://broadinstitute.github.io/picard/
snvsingle nucleotide variant
string
www.string-db.org
svstructural variant
uk10k project
www.uk10k.org
uppubiquitin proteasome pathway
weswhole exome sequencing
wgswhole genome sequencing
electronic supplementary material
the online version of this article  contains supplementary material, which is available to authorized users.

 intellectual disability affects 1–3% of the global population. a significant proportion of id is caused by genetic defects, yet despite extensive testing including by clinical chromosomal microarray, ~30% of cases remain idiopathic. genome-wide sequencing can identify previously unknown genes causative for id. whole exome sequencing is limited by poor ability or inability to detect non-coding and structural variation, and capturing less than 100% of the exome. in contrast, whole genome sequencing offers a comprehensive screen of a variety of dna variation types. current evidence suggests wgs is able to detect coding variants in 42% of cases missed by wes. we report comprehensive wgs analyses for eight patients with id and brain malformations, whose family history suggested a de novo mutation. despite a diagnostic odyssey, including genome-wide clinical and research cma, they were idiopathic. wgs was conducted on trios composed of the affected child and both unaffected parents, and data was analyzed using both alignment and assembly approaches to detect all possible causative genetic changes- single nucleotide variants, copy number variants and structural variants. we validated our findings using wes data from an independent positive control cohort of patients with id and other neurocognitive phenotypes, and a negative control wgs cohort of normal subjects. in addition we probed molecular themes indicated by our discovery cohort findings in the positive control cohort, leveraging its large size. we also conducted a screen for de novo variants in possible regulatory sequences of known and hypothesized pathogenic genes.fig. schematic of complete study design. abbreviations: cnv = copy number variant; sv = structural variant; snv = single nucleotide variant; ddd = deciphering developmental disabilities study; upp = ubiquitin proteolysis pathway; igv = integrated genome viewer; dgv = database of genome variation methods subjects patients were enrolled from the british columbia children’s and women’s hospital provincial medical genetics program after obtaining informed consent. this study is approved by the british columbia children’s and women’s hospital research ethics boards. all patients presented with id and brain morphological defects detected by mri or ct scan. patients had no family history of id, and all were products of normal pregnancies with no reported teratogenic exposures as ascertained by clinical assessment by board certified medical genetics specialists at the recruiting facility. saliva samples were collected and dna extracted using dna genotek® collection kits, reagents and protocols from child, father and mother. methods wgs methods, variant calling protocols, verification methods, and secondary control study methods including bootstrap analysis, are summarized below and detailed in additional file briefly; dna was extracted using dnagenotek® extraction kits. paired-end wgs libraries were prepared using illumina’s pcr-free protocol. sequencing was by illuminahiseq platform generating 100 bp paired-end reads, using three lanes per sample. alignment and variant calling was by canada’s michael smith genome science center standard pipelines. variants were identified and filtered as follows, briefly; putative snvs were identified using samtools mpileup version. run on each sample separately. relatedness was tested for each trio by comparing snp concordance between child, mother and father using vcftools-.. de novo variants were selected by intersecting the child’s snvs with that of each parent, and selecting variants only present in the child and not in either parent. for variants in the coding region, we selected de novo missense, nonsense and splicing variants, i.e., functional variants. we next selected rare variants by excluding alleles with minor allele frequency >1% in dbsnpv, exome variant server, exome aggregation consortium, and in-house databases of > exomes, and > genomes. we then used pathway enrichment analyses to prioritize de novo rare variants; selecting snvs in genes enriched in pathways involved in brain development and function conducted using qiagens ingenuity® pathway analysis, david and panther. for those variants passing the pathway enrichment screen, pathogenicity predictions and conservation scores were annotated using sift, phylop, polyphen, mutationtaster and cadd scores. these steps yielded de novo, functional, rare variants, that are highly conserved and predicted to be damaging and in biologically relevant pathways. in addition to the above prioritization, the rare functional variants were subsequently also screened under a series of additional genetic models, and manually checked for alignment quality with integrated genomic viewer. snvs that were highly conserved and were predicted to be damaging by at least one pathogenicity prediction software, were selected for verification by sanger sequencing in the child, mother and father. cnv analyses was conducted using freec, cnaseq, delly and abyss. the first three algorithms align reads to the reference genome while abyss uses de novo assembly to reconstruct the patient’s genome. sv analyses was conducted using only delly and abyss. first, de novo cnvs/svs were identified by comparing the child’s data to that of either parent. de novo cnvs from each algorithm were filtered by manual assessment of local read configuration on igv, and genuine ones were prioritized based on functional relevance of the included/cnv-affected genes. svs, i.e., translocations and inversions, were filtered by either igv read visualization and then by using qc metrics specific to each algorithm; qc metrics generated by the program were used for delly, and checking of blat scores for breakpoint-junction contigs and number of supporting reads were used for abyss. candidate cnvs/svs that were selected from the above filtering were verified using an independent method as detailed below. all de novo variants, i.e., snvs, cnvs and svs, were verified by sanger sequencing of pcr-captured amplicons of the affected sequence, either bearing the snv or spanning the breakpoint junction in the trio, with forward and reverse primers. all verified candidate snvs were subjected to genotype-phenotype correlations assessment as per the guidelines of the american college of medical genetics. secondary control study - wes data from the uk10k project for patients with neurofunctional phenotypes, and wgs data from normal individuals from the genomes project; a publicly available repository of variation in healthy individuals, was obtained. ‘possibly damaging snvs’, were extracted from these datasets, and a gene-wise pds burden for all genes in the human genome was determined in both the positive and negative control cohorts. subsequently the gene-wise pds burden only in our candidate genes was compared between the positive and negative control cohorts. we further bootstrapped the positive control cohort to determine if the pds burden in our six candidate genes could be due to random sampling. finally, we tested to see what functional pathways genes with pds in the positive control cohort were involved in, and conducted a kyoto encyclopedia of genes and genomes pathway enrichment analyses, testing which of the total functional pathways from kegg were most enriched for genes bearing pds in this large dataset. regulatory region variation – for our regulatory region analysis we selected ‘high confidence’ de novo snvs defined as having a mapping quality >  and read depth ≥  and ≤  and ‘high confidence’ de novo cnvs defined as those that were detected by two or more cnv detection algorithms. we then intersected both the de novo high confidence snvs and cnvs with six non-coding sequence annotation datasets. results from the above, i.e., de novo high confidence snvs and cnvs with involvement in putative regulatory regions, were then intersected with candidate gene lists and appropriate flanking sequence to determine their possible association to a candidate known or hypothesized id gene.  de novo snvs identified by objective molecular pathway-based filtration genes with functional rare de novo snvs were screened using three pathway analyses programs in order to refine candidates involved in brain development and function; ipa returned candidate genes, david returned and panther returned a total of unique genes involved in brain development and function were yielded by the combined analyses. from these, highly conserved and predicted damaging snvs were sanger tested, and six snvs in six genes in five children were confirmed as heterozygous de novo; arid1b nm_017519:c.1595delg, phf nm_001015877:c.820c > t, spry nm_001127496:c.508 t > a, cacnb nm_0012069:c.1289g > a, sqstm nm_03900: c.115_116delinsta and upf nm_002911:c.1576_1577delinsaa. the latter two were found in a single patient while the rest each appeared in a separate patient. as best practice, we also screened our de novo rare functional variants for location within published known and candidate id genes, however no new findings were yielded. except arid1b and phf the other genes are novel for id. table  provides variant classification as per the acmg variant interpretation guidelines and our interpretation of their causative effect. brief genotype-phenotype correlations are given below;table patient phenotype and variant summary arid1b phf6 spry4 ap4e1 ap4e1 cacnb3 scn3a 51b sqstm1 upf1 lrp2 lrp2 abbreviations: id intellectual disability, ofc occipito-frontal circumference, ct computerized tomography scan, mri magnetic resonance imaging scan, pvs null variant in a gene where lof is a known mechanism of disease, ps2 de novo in a patient with the disease and no family history, pm absent from controls in exome sequencing project, genomes project or exome aggregation consortium, pp multiple lines of computational evidence support a deleterious effect on the gene or gene product aage at examination is given in 5 year intervals in order to protect patient anonymity bpatient also bears a de novo likely contributory cnv as detailed in the text, in addition to the snvs given here arid1b c.1595delg in patient 43 this single base deletion in exon of the known id gene arid1b causes a frame-shift leading to predicted loss of function. our patient presents with id, autism, absence of corpus callosum, absence of speech, feeding difficulties and failure to thrive. haploinsufficiency of arid1b was reported to cause corpus callosum abnormalities, id, speech impairment and autism, suggesting the arid1b lof is causative and sufficient in this case. phf c.820c > t in patient 58 phf encodes the plant homeodomain finger protein the nonsense variant in phf is located in the ephd domain in which causative de novo truncating and missense variants for börjeson-forssman-lehmann syndrome, and coffin-siris syndrome are known. de novo truncating and other mutations in phf are reported to cause a distinct syndrome in girls and reported for a female specific form of bfls. roles for phf are reported in the chromatin remodeling swi/snf complex, and in the nurd epigenetic regulatory complex where it acts as a possible regulator for the latter in neurogenesis. rnai knock down of phf profoundly impairs neuronal migration in vivo, thus leading to formation of white matter heterotopias. in keeping with this, this patient reports pachygyria, which results from abnormal migration of neurons in the developing brain. she also presents with an unusual asymmetrical growth phenotype that was reported in the one patient with the female specific bfls. these data indicate the variant is a good candidate in this case. spry c.508 t > a in patient 59 spry encodes a specific inhibitor of the mitogen-activated protein kinase family. spry is expressed in the mouse developing brain, and is essential for the normal morphogenesis and cytoarchitecture of the cerebellum. morphogenic changes in axon growth have been shown when the protein is down regulated both in vivo and in vitro. in zebrafish, spry is a principal regulator of mid-brain development, and mediates hindbrain patterning. these data support the notion that the spry missense variant may contribute to the brain morphological phenotype in this patient. spry expression plays a role in xenopus limb bud development, of note as our patient reports short and crowded toes. cacnb c.1289g > a in patient 45 cacnb encodes a regulatory subunit of a voltage-dependent calcium channel. mice lacking cacnb presented visual impairment, high pain threshold, and behavioral phenotypes, all of which features are seen in this patient. mutations in other members of vdcc subunit encoding genes are known to cause neurological disease, including epilepsy present in our patient. this variant is found in eight of individuals in the exac database, where its non-absence disqualifies likely pathogenicity as per acmg criteria, despite being de novo and deleterious by multiple lines of computational evidence. neither does it meet criteria to be a benign variant, and therefore is of uncertain significance. sqstm1c.115_116delinsta, upf c.1576_1577delinsaa and a 1q deletion cnv in patient 51 the patient is severely affected, with significant id and several major congenital anomalies. the heterozygous indel formed by two adjacent snvs in sqstm causes a stop-gain. sqstm encodes p a regulatory factor in nuclear factor kappa-b signaling, nf-e2-related factor activation, ubiquitin-mediated authophagy, and transcription. the snv is located in the pb1domain, mutations of which cause paget disease of bone and frontotemporal dementia and/or amyotrophic lateral sclerosis; both neurodegenerative conditions that include morphological brain changes. the adjacent snvs in upf together cause a likely pathogenic missense amino acid change. upf has an essential role in nonsense-mediated mrna decay. interestingly, upf has been shown to remarkably reduce als-associated neuronal toxicity in vitro and to protect against motor dysfunction and forelimb paralysis in a rat model for als. it is plausible haploinsufficiency of sqstm may have caused neurofunctional defects, which the haploinsufficiency of upf may have exacerbated. in this regard, it is notable that at 19 years of age, patient presents significant motor deficits, being wheelchair bound, indicative of a possible early onset of als. while scoliosis and hearing loss, both among the presentation for pds is already seen in her. these data support the notion that the snvs in both genes maybe contributory toward her presentation. we further verified a de novo ~165 kb heterozygous deletion that spans cep in whole and sdccag in part in this patient. cep encodes a component of the centrosome. sdccag is also involved in centrosome function, dna damage response signaling and neuronal migration. both genes are suggested as candidates for corpus callosum abnormalities via 1q microdeletion, however this has been contested. our patient presents partial phenotypic overlap with microdeletion 1q index cases. the demonstrated roles for sdccag in dna-mismatch repair, and for both genes in cell cycle progression, supports the notion this cnv may be contributory. notably, the haploinsufficiency of a dna-mismatch repair gene could lead to the high mutation burden detected in this child. we also confirmed at least one maternally inherited balanced translocation, which is unlikely to be contributory.fig. details of cnv analyses. a igv images for heterozygous deletion cnv in patient showing proximal and distal breakpoint. the cnv involves whole of cep and part of sdcaag genes. top, middle and bottom panels are child’s.bam file, mother’s.bam file and father.bam file respectively. read-depth coverage shows cnv is de novo. b cartoon of breakpoint junction seuqence showing a 24 bp chromosome and 107 bp chromosome sequence inserted between the proximal and distal breakpoints on chromosome 1q yellow shaded segment shows sequnce microhomology- this 14 bp seuqence is found at chromosome 5:,598-, and at chromosome 1:,747-, hg19). sanger sequence trace images are overlaid confirming the cnv breakpoint. grey arrows denote pcr forward and reverse primers. n denotes dna repeat sequence. c genomic interval involved in the de novo cnv detected in patient 51- ucsc genome browser. red highlighted box shows region involved in the deletion in our patient. yellow boxes show critical region for 1q43- sydrome defined by nagamani et al. green box shows critical region as defined by perlman et al. n.b, nagamani et al. also highlight zbtb in their critical region mutation burden assessment in large secondary positive and negative control cohorts support candidacy of novel genes we investigated the candidacy of the above verified genes by assessment for incidence of damaging mutation in large positive and negative cohorts with comparable ngs data. we looked for ‘potentially damaging snvs’ in our candidate genes, from wes of patients with neurodevelopmental and neurocognitive phenotypes from the uk10k cohort and compared that to incidence in wgs from healthy people from the genomes project. we first screened for the exact variant detected in our discovery cohort, and did not find any case of an exact match. we then conducted a gene-wise pds screen and observed that incidence for pds in arid1b, spry cacnb sqstm and upf were significantly enriched in the positive versus negative control cohorts. there was no significance for phf6; however, the two pds found in people was insufficient for meaningful statistical assessment. the extremely high pds burden in the positive control cohort for sqstm and upf is noteworthy, as these genes have previously not been reported for id to our knowledge, and further, the indels in both are found in the same patient in our cohort.fig. validation study. a showing incidence for potentially damaging snvs in both the positive control and negative control control cohorts. * denotes statistical signficance b and c results of bootstrap analyses for pdss in randomly selected genes. red vertical bar shows the mean and median result for pdss in our candidate genes while we do not have access to clinical data to conduct a classical genotype-phenotype correlation between cases in the positive control cohort and our patients who have the same gene affected, the large number of such cases in the positive control cohort also impedes such a study within the scope of this work. we therefore assessed if our findings could be due to random chance effect, by bootstrapping the uk10k cohort for pds in six randomly selected genes each, a thousand times. we found from the bootstrap analysis that the mean and median gene-wise variant frequency for our six candidate genes was greater than that of the corresponding distribution, indicating that our findings were not likely due to chance. these data are consistent with an association of at least five of our candidate genes with neurodevelopmental abnormalities. novel candidate genes converge unto the ubiquitin proteasome pathway, which also bears significant mutation burden in positive control wes samples we investigated molecular links between our pathogenic and candidate genes; focused ipa and string pathway analyses revealed that all six connected to the ubiquitin proteasome degradation pathway which has important roles in the structural development and function of the brain. we assessed the relative importance of this pathway and found the upp was among significantly enriched pathways for pds when compared with all kegg pathway categories, in the uk10k patient cohort, substantiating the importance of the upp pathway in brain development. mendelian inheritance and n of analyses provides additional candidate variants in addition to our in-silico refinement and test for candidate de novo snvs above, we also conducted a classical series of n of studies for these eight patients; manually assessing the possible candidacy of variants selected by all possible mendelian inheritance patterns. compound heterozygous missense mutations were identified in lrp, causative of the autosomal recessive donnai barrow syndrome in patient absence of the corpus callosum, reported in our patient, presents in donnai barrow syndrome. compound heterozygous mutations were identified in ap4e causative of autosomal recessive spastic paraplegia type in patient this patient reported a seizure at 12 years of age, however does not exhibit the severe neurological phenotypes nor the shy demeanour reported for a possible syndromic form of id caused by defects in adaptor protein complex- of which ap4e encodes one of the four subunits. a missense scn3a snv in patient was selected due to scn3a association to epilepsy, and the predicted deleterious effect of the variant, and was sanger verified as de novo. functional studies are underway to further investigate the role of scn3a in epilepsy. extensive copy number variant and structural variant analyses identifies likely causative cnv missed by clinical cma, and balanced benign translocation we conducted both alignment-based and de novo assembly-based cnv/sv analyses. cnvs, i.e., duplications and deletions were identified by all four platforms, while svs, i.e. translocations and inversions, were identified by delly and abyss. an average of de novo gain cnvs and de novo loss cnvs across all eight patients were detected. however, only cnvs were called by over one platform, and none were called by more than two, with the majority of each algorithm’s findings being unique. we carried out extensive visual in silico curation for all cnvs, and selected three to verify of which, only the previously discussed 1q loss cnv, sanger verified as de novo- it was detected by freec and cnaseq, and is clearly visible on igv. breakpoint junction sequence reveals a complex architecture.table number of copy number variants and structural variants identified fig. venn driagram showing cnvs found by each algorithm similar to our cnv results, sv results from delly and abyss were divergent. only one translocation in patient was called by both, and there was no concordance among inversions. upon extensive manual in silico curation we selected translocations and inversion to verify, but none verified as de novo. sanger verification for these lesions was challenging as breakpoints mapped to repeat-masked regions, nevertheless one translocation verified as maternally inherited; a chromosome x- translocation not causing any gene-disruption, in patient the breakpoint junction shows a single base addition. genome assembly yields small insertions/deletions missed by genome re-alignment in contrast to the re-alignment based algorithms, abyss identified over potential de novo indels, via genome assembly. forty three indels were refined as likely true positives with a functional importance, due to having at least seven spanning reads, and producing a protein coding change; the majority being in patient for consistency, we conducted a pathway analyses for the indel-bearing genes, and a manual curation, in order to select candidates for verification as we had done for our snvs. this resulted in indels that were sanger tested; however one was false positive, five were inherited, and eight did not pass pcr quality checks, indicating location to repeated dna sequence, thus hampering any ability to amplify the region for sanger sequencing. gene regulatory region variation identified in known and hypothesized id genes we investigated gene regulatory sequence variation which we term ‘de novo variants in possible regulatory regions’. we filtered the dvprr for potentially pathogenic changes using two approaches: by screening for involvement in known id genes, and on the basis of our hypothesized involvement of the upp. an average ~ de novo snvs were found across our eight patients in the non-coding genome. of these, an average located to transcription factor binding sites, an average to putative gene promoters, an average of those located to promoters were also located to transcription factor binding site regions, an average located to regions annotated as enhancers by the fantom consortium, an average belonged to 5′ or 3′ utr regions and an average located to highly conserved ultra-sensitive regions – we considered these to be dvprr and therefore there were an average dvprr across all eight patients. we then intersected dvprrs with genes known to cause developmental delay in a disease gene screen approach, and with the total genes of the upp, − as our candidate genes converged upon the upp - in a hypothesis-driven approach. as a final step for enhancers and ultra-sensitive regions, we further selected dvprr where it, and the candidate gene, were located within the same topological domain, postulating that their physical proximity would imply that the regulatory region in question did in fact impact the targeted gene. in summary we found an average of and dvprr per patient in our gene-screen and hypothesis driven approach respectively, by these filtrations combined. we also interrogated high-confidence cnvs in the same manner, but only found association to sdccag a known id gene present in the previously discussed 1q microdeletion.fig. schematic of filtration pipeline for variants in non-coding regions. a schematic for snvs. b schematic for cnvs. abbeviations; snv- single nucleotide variant, tfbs – transcription factor binding site, fantom-enhancer sequence as annotated by the fantom consortium. utr – untranslated regions. ddd- deciphering development disabilities. upp – ubiquitin proteosome degredation pathway. cn- copy number. patient had dvprr in the utrs of two genes; cbl and ube3b. patient had a dvprr in the promoter of ube3a, patient had a dvprr in the promoter of cul4b, and patient had dvprrs in the promoters of ube3a, cul4b and cul occurrence of de novo snvs in non-coding rnas we found an average of high confidence de novo snvs that located to sequence annotated as ncrna across all eight patients. a majority of these fall within introns while an average are exonic, an average are predicted in splice junction sequence and average and are located to 3′ and 5′ utr respectively. discussion selection of candidate snvs: comparison of strategies an effective strategy is essential to select causative snvs from ngs data. standard filtration approaches yield potential de novo variants that then must be careful sifted for likely true candidates. in keeping with others, we found an average of +/− candidate unverified de novo snvs, and it was necessary to implement an effective prioritization approach for verification. discovery wgs and wes studies published to date have used a large sample size, detailed pedigree information, or well characterized rare syndromes as study cohorts, leveraging the power of numbers, inheritance pattern, and phenotypic commonality, respectively, as filtration strategies. in as much as we did not have a large cohort, all of our cases were sporadic, and none had a recognized dysmorphic syndrome, we refined snvs objectively, by selecting genes known to be involved in brain development pathways. we reasoned that this systematic approach would reduce subjective bias inherent in an n of genotype-phenotype correlation, and thereby identified potential candidates. however a subjective screen for snvs yielded the likely damaging variant in scn3a, which was not stratified by our objective approach –highlighting the limitation of pathway analyses programs that depend on available gene-functional annotations. notably scn3a was selected by a team of biochemical geneticists specifically with respect to the epilepsy presented by the child. thus a subjective approach may also miss results detected from objective screening, as exemplified in this case, where the two analyses were done by independent members and each did not report the result of the other. interpreting detected variants; discovery study findings further inform genetic complexity for id variable expressivity and reduced penetrance are well known in the pathogenicity of id, and it is increasingly recognized that a single mutation in a single gene may only rarely explain the full phenotypic spectrum. our results provide further indications of such complex heritability; in patient the 1q deletion, and snvs in sqstm and upf may act in concert to produce the complex and severe phenotype in this patient. while in patient we have identified both compound heterozygous variants in the known id gene ap4e that act in a recessive model, as well as de novo variant in a novel gene spry which has important functions in brain development. de novo mutation is recognized to play an important role particularly in the pathogenicity of id, and it is difficult to determine to what extent each of these variants, if at all, contributes to disease burden in this patient. the same is true for patient in whom de novo variants for two novel genes, cacnb and scn3a were identified. we note that patient who bears the most complex genotype, is the most severely affected in our cohort, and in this case, clinical severity does co-relate with number and complexity of genomic alterations, suggesting that gradation of clinical severity may provide useful toward assessing the contribution of genomic alterations. it is recognized that genes responsible for id converge onto common networks. the candidate genes we identified converge onto the upp, which is critically involved in neurodegenerative disease and has important roles in neurodevelopmental disorders. this observation is consistent with the notion that they may be good candidates, and exemplifies the usefulness of probing molecular links among novel findings. large secondary positive wes cohort analysis supports novel findings novel snv findings from ngs studies require rigorous additional studies to support proof of pathogenicity. in our case, several of our novel candidates cause missense variation, whose effect is difficult to model, as opposed to clear loss of function mutations which are amenable to functional studies in model organisms. conversely we were unable to conduct traditional genotype-phenotype correlations studies as none of our patients had a recognizable syndrome to match with other patients. therefore, our approach of using a large secondary positive control cohort, despite the phenotypic spectrums not matching our cases precisely, gave us sufficient ability to test the predicted causality of our candidate genes and was the best strategy available. we were hampered by the lack of an optimal comparison negative control cohort. we used wgs data from the genomes project, which we recognize is primarily comprised of low coverage samples whose phenotypic spectrum is poorly characterized, yet the similarity of sample size between the two groups allowed us to explore the pds distribution for these genes reasonably, providing a useful contributory analysis toward assessing their likely pathogenicity. finally this large cohort enabled us to further probe the convergence of our candidate genes upon upp, by assessing its contribution versus other biological pathways. wgs is able to detect structural variants below the threshold of clinical cma, and enables mechanistic insights into cnv formation by using wgs instead of wes, we were able to detect a cnv below clinical cma resolution, isolate it’s breakpoints, and uncover a possible complex genomic landscape in one patient. we wanted to conduct a comprehensive screen for cnvs and other structural variants to maximize sensitivity. therefore we used four approaches that are fundamentally different; cnaseq and freec are sequence based copy-number estimators that use categorically different algorithmic approaches for background correction. delly is an alignment based assembler, whilst abyss is a de-novo genome assembler. since each algorithm was optimized differently, it therefore yielded different results. for example, cnaseq executes read-depth based binning, and hence aggregates results at telomeres and centromeres where a larger number of reads re-align due to pervasive repeat sequence. the verified cnv we detected was only identified by delly and freec, but missed by the other algorithms. therefore, we caution against using only one cnv detection algorithm as this would reduce sensitivity. the breakpoint junction sequence in the case of the confirmed 1q microdeletion is consistent with the notion that it could be caused by chromotripsis, a mechanism only recently reported in the constitutional genome, further demonstrating the utility of wgs data. wgs enabled a de novo genome assembly that unmasked hidden genome complexity abyss de novo assembly identified a translocation missed by delly, and also detected a higher than usual number of putative indels in patient who was found to have a remarkably unstable genome masked by standard genome re-alignment based analysis. however, we experienced difficulty confirming these events via sanger sequencing, which was due, in part, to the high degree of repeated sequence at breakpoint junctions. genome assembly is able to call events in repetitive sequence better than alignment based algorithms, though conversely such events are harder to independently verify. we are among the first to use de novo assembly to interrogate patients with id, and our findings suggest variation located to repeat enriched sequence is currently under-ascertained in the constitutional genome.fig. a sanger sequencing verification of translocation in patient with karyotype cartoon of balanced translocation. pcr amplicon trace file shows sequence mapping across the chromosome x - translocation boundary. zoomed-in view shows single base addition at breakpoint jucntion. b circos plot showing mutation burden for patient called by abyss genome assembly wgs is able to interrogate regulatory genomic sequence meaningful interpretation of snvs within regulatory sequence is hampered by the sparsity of annotations for the non-coding genome. we implemented two different filtering strategies in order to identify non-coding snvs that could have a functional impact, and also used topological domain data to further refine good candidates. though we were able to reduce the number of candidate dvprr from an average > to dozens in the case of our gene-screen approach and a handful in the case of our hypothesis driven approach, nonetheless without further focused studies, meaningful interpretations are precluded. in contrast, assessing the impact of cnv-based dvprr is theoretically less challenging, as it is more straightforward to predict functional outcome for a complete loss or gain of a possible regulatory sequence. in summary, though clinically relevant conclusions for dvprr will require a case-by-case analysis and extensive follow-up functional studies, nevertheless we note it is possible to stratify dvprr in the context of known causative genes for id using wgs. wgs versus wes wgs yields a comprehensive screen of the genome as, in addition to coding variation, it includes ability to investigate structural variation at a fine scale as discussed above, and also variation in possible gene regulatory sequence as well as ‘non-coding genes’ such as ncrnas for which there is a paucity of information in the context of neurodevelopmental disease. while we show strategic stratification for dvprr can yield results potentially relevant to id causation, much less is possible for annotation of snvs within ncrna sequence, of which we detect an average across our samples. nevertheless, initial screens such as ours, importantly generate exploratory information for non-coding sequence variation possible only by wgs. we note that all the snvs we identified as involved in disease would have been possible to detect by wes. however, wgs yields a more complete view of possible pathogenic variation in each child. this is exemplified in the case of patient for whom had only wes been performed, while the sqstm and upf snvs would likely have been detected, the 1q microdeletion would not have been identified. in the case of this patient, it is unclear what the gene-effect size for each variant is. conversely, in the case of patient for whom we detected the snv in arid1b, we are more certain of the penetrance of this variant due to the normal results for other causative variation in their genome from our wgs data analyses. these data argue in favor of wgs over wes for clinical use.  this is the first study to present extensive analyses of wgs data in the context of id, for causative snv and cnv/sv in both coding and non-coding sequence, and the first to present results from de novo assembly of the genome. in a heterogeneous group of eight children with id and morphological brain defects, we were able to identify candidate causative variants, highlight neurodevelopmental pathways, and unearth hidden genome instability, demonstrating the efficacy of a discovery approach to wgs analyses in the context of id. additional files additional file 1: supplementary methods - additional details on methods presented succinctly in main text. additional file 2: table s test of relatedness - table showing relatedness for each trio by comparing snp concordance between child, mother and father. additional file 3: table s primers for verification - all primer sequences used for snv, cnv and sv verification. additional file 4: table s uk10k study cohorts that comprise the positive control cohort – table giving descriptions of the study cohorts from the uk10k project that comprised the positive control cohorts and conditions for use. additional file 5: supplementary figures. figure s igv image and sanger verification trace files for indel in arid1b and missense variation in upf figure s uk10k mutation load – counts as per variant annotation type on one patient. figure s histogram of mutation burden per patient in the uk10k cohort. figure s pathway interactions showing convergence onto upp pathway. figure s plots for cnv distribution for two chromosomes as called by cnaseq. additional file 6: table s detailed variant information for all verified variants, including acmg classification – table showing details for variant classification such as pathogenicity prediction algorithms results and details of acmg criteria application. additional file 7: table s significantly enriched kegg pathways for pds burden in positive control cohort – details for pathway enrichment analysis showing all kegg pathways and burden of enrichment for each in the positive control cohort. additional file 8: table s raw data for snvs detected across all patients by mendelian inheritance pattern filtering – all snvs detected per trio, organized as one trio per sheet, using the mendelian inheritance filtering. sheet gives the legend. all snvs are annotated, including cadd and rvis scores. additional file 9: table s cnv/sv/indel verification summary – table giving a list of all indels, cnvs and svs, and details on their verification. additional file 10: table s summary of snvs and cnvs across id patients in dvprr – details on all snvs and cnvs detected in dvprr for all patients, organized by those intersecting with ddd genes and those with upp genes. abbreviations 1000g project http://www.internationalgenome.org/ cmaclinical microarray cnvcopy number variant dbsnp135 https://www.ncbi.nlm.nih.gov/snp dgvdatabase of genomic variants dgv http://dgv.tcag.ca/dgv/app/home dvprrde novo variants in possible regulatory regions fantom consortium http://fantom.gsc.riken.jp/ grch37-lite/hg19a http://www.bcgsc.ca/downloads/genomes/9606/hg19/1000genomes/bwa_ind/genome/readme.grch37-lite kegg www.genome.jp/kegg/pathway.html ngsnext generation sequencing nhlbi-esp http://evs.gs.washington.edu/evs/ omim www.omim.org pdspotentially damaging snvs picard https://broadinstitute.github.io/picard/ snvsingle nucleotide variant string www.string-db.org svstructural variant uk10k project www.uk10k.org uppubiquitin proteasome pathway weswhole exome sequencing wgswhole genome sequencing electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. 
we report results from a large secondary control study of exomes probing the pathogenicity of the above genes. the online version of this article contains supplementary material, which is available to authorized users. we verified de novo pathogenic single nucleotide variants in arid1b c.1595delg and phf c.820c > t, potentially causative de novo two base indels in sqstm c.115_116delinsta and upf c.1576_1577delinsa, and de novo snvs in cacnb c.1289g > a, and spry c.508 t > a, of uncertain significance. we also interrogated regulatory sequence variation for known and hypothesized id genes and present useful strategies for wgs data analyses for non-coding variation. intellectual disability is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. intellectual disabilitywhole genome sequencingarid1bphf6spry4cacnb3sqstm1upf11q microdeletiongenome assemblyhttp://dx.doi.org/http://dx.doi.org/ /501100000024canadian institutes of health researchmop-102600issue-copyright-statement© the author 2017 we analyzed structural variation by four different algorithms including de novo genome assembly. whole genome sequencing is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. the de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms.
 intellectual disability is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. whole genome sequencing is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. methods we conducted wgs on eight children with idiopathic id and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches.  we verified de novo pathogenic single nucleotide variants in arid1b c.1595delg and phf c.820c > t, potentially causative de novo two base indels in sqstm c.115_116delinsta and upf c.1576_1577delinsa, and de novo snvs in cacnb c.1289g > a, and spry c.508 t > a, of uncertain significance. we report results from a large secondary control study of exomes probing the pathogenicity of the above genes. we analyzed structural variation by four different algorithms including de novo genome assembly. we confirmed a likely contributory 165 kb de novo heterozygous 1q microdeletion missed by clinical microarray. the de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. we also interrogated regulatory sequence variation for known and hypothesized id genes and present useful strategies for wgs data analyses for non-coding variation.  this study provides an extensive analysis of wgs in the context of id, providing genetic and structural insights into id and yielding diagnoses. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords intellectual disabilitywhole genome sequencingarid1bphf6spry4cacnb3sqstm1upf11q microdeletiongenome assemblyhttp://dx.doi.org/http://dx.doi.org/ /501100000024canadian institutes of health researchmop-102600issue-copyright-statement© the author 2017 