Elouan Chérot
email: elouan.cherot@chu-rennes.fr

Boris Keren
email: boris.keren@aphp.fr

Christèle Dubourg
email: christele.dubourg@chu-rennes.fr

Wilfrid Carré
email: wilfrid.carre@chu-rennes.fr

Mélanie Fradin
email: melanie.fradin@chu-rennes.fr

Alinoë Lavillaureix
email: alinoe.lavillaureix@aphp.fr

Alexandra Afenjar
email: alexandra.afenjar@aphp.fr

Lydie Burglen
email: lydie.burglen@aphp.fr

Sandra Whalen
email: sandra.whalen@aphp.fr

Perrine Charles
email: perrine.charles@aphp.fr

Isabelle Marey
email: isabelle.marey@aphp.fr

Solveig Heide
email: solveig.heide@aphp.fr

Aurélia Jacquette
email: aurelia.jacquette@aphp.fr

Delphine Heron
email: delphine.heron@aphp.fr

Diane Doummar
email: diane.doummar@aphp.fr

Diana Rodriguez
email: diana.rodriguez@aphp.fr

Thierry Billette De Villemeur

Marie-Laure Moutard
email: marielaure.moutard@aphp.fr

Agnès Guët
email: agnes.guet@aphp.fr

Jean Xavier
email: jean.xavier@aphp.fr

Didier Périsse
email: didier.perisse@aphp.fr

David Cohen
email: david.cohen@aphp.fr

Florence Demurger
email: florence.demurger@chu-rennes.fr

Chloé Quélin
email: chloe.quelin@chu-rennes.fr

Christel Depienne
email: depiennc@igbmc.fr

Sylvie Odent
email: sylvie.odent@chu-rennes.fr

Caroline Nava
email: caroline.nava@aphp.fr

Véronique David
email: veronique.david@chu-rennes.fr

Laurent Pasquier
email: laurent.pasquier@chu-rennes.fr

Cyril Mignot
email: cyril.mignot@aphp.fr

Using medical exome sequencing to identify the causes of neurodevelopmental disorders: experience of two clinical units and 216 patients Running title: Medical exome and NDD

Keywords: medical exome, molecular strategy, intellectual disability, autism

Using medical exome sequencing to identify the causes of neurodevelopmental disorders: Experience of 2 clinical units and 216 patients

INTRODUCTION

Intellectual disability (ID) is the most frequent neurodevelopmental disorder (NDD) affecting about 1-3% of the population worldwide. More than 700 genes are known to cause ID [START_REF] Vissers | Genetic studies in intellectual disability and related disorders[END_REF] and this large genetic heterogeneity is challenging for diagnosis because the phenotype in many patients is either non-syndromic or corresponds to syndromes that are either not recognized or for which the molecular cause is still unknown. To decipher the genetic etiologies of NDD, chromosomal microarray is a first tier diagnosis tool [START_REF] Miller | Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies[END_REF] , together with the search for a FMR1 gene 5'UTR triplet amplification. When these investigations are negative and if no targeted genetic testing is considered, clinical geneticists have to choose between gene panels [START_REF] Redin | Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing[END_REF] and whole exome sequencing (WES).

Because of the extreme genetic heterogeneity of NDD and because each genetic cause is very rare, the probability to find molecular causes of NDD tends to increase with the number of analyzed genes, which implies that WES is a more appropriate tool than gene panels [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][START_REF] Yang | Molecular findings among patients referred for clinical whole-exome sequencing[END_REF][START_REF] Thevenon | Diagnostic odyssey in severe neurodevelopmental disorders: Towards clinical whole-exome sequencing as a first-line diagnostic test[END_REF][7] . However, the cost of WES is still high and unaffordable in daily practice for many genetic centers that have to choose between locally designed gene panels and commercialized gene panels comprising all known disease-related genes, called "medical exome" (ME). A few articles reported the use of ME for the diagnosis of genetic disorders [START_REF] Pajusalu | Large Gene Panel Sequencing in Clinical Diagnostics -Results from 501 Consecutive Cases[END_REF][START_REF] Okazaki | Clinical Diagnosis of Mendelian Disorders Using a Comprehensive Gene-Targeted Panel Test for Next-Generation Sequencing[END_REF] . We report here the experience of two French genetic centers using the TruSight One sequencing panel, which targets genes associated with known phenotypes, for the diagnosis of NDD in 216 patients.

Accepted Article

MATERIAL AND METHODS

Patients

We studied 216 index patients with a presumed genetic NDD but without molecular etiology recruited consecutively in the daily practice of two clinical genetic centers (Groupe Hospitalier Pitié-Salpêtrière and Centre Hospitalier Universitaire of Rennes). Inclusion criteria were: 1) the family asks for the establishment of a definite risk for a first degree relative of having a child with the disease of the index case, 2) negative previous genetic testing including chromosomal microarray analysis, fragile X testing, as well as normal metabolic screening and/or targeted genetic studies varying from one patient to another.

The overall series included 133 males and 83 females. Ages at disease onset ranged from 1 to 56 years. Consanguinity was reported in 17 families (7.8%).

Three patients only had a definite clinical diagnosis (Nicolaides-Baraitser syndrome (MIM 601358), Coffin-Siris syndrome (MIM 135900) and microcephaly, lymphoedema, retinal dysplasia syndrome (MIM 152950) with unavailable or expensive molecular testing.

We classified the neurodevelopmental phenotypes of the patients into four categories: 1) patients with non-syndromic ID (NSID), includes those with normal growth parameters and without dysmorphic features/malformations, neuromotor involvement (pyramidal, extrapyramidal, cerebellar syndrome) and sensory organ involvement, 2) patients with syndromic ID (SID), 3) patients with pediatric neurodegenerative disorders (NDEG), 4) patients with autism spectrum disorder (ASD). Patients with early developmental delay and ID

Accepted Article

meeting the criteria of the Autism Diagnosis Interview (ADI) for ASD were classified as having ID. Patients meeting the ADI criteria for ASD with normal early development during the first 12-18 months of life followed by autistic regression and those with ASD and preserved intelligence were classified in the ASD category. We considered macrocephaly and microcephaly in patients with a head circumference above or below two standard deviations (SD) to the mean, respectively. We considered epilepsy as a non-specific feature.

Informed consent was obtained from all individual participants, parents or legal representatives, included in the study. Samples from each center were sent to local laboratories.

The following patients have been previously reported in articles: #3 and #4 10 , #8 [START_REF] Ruaud | DYRK1A mutations in two unrelated patients[END_REF] , #13 [START_REF] Platzer | GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects[END_REF] , #14 [START_REF] Depienne | Genetic and phenotypic dissection of 1q43q44 microdeletion syndrome and neurodevelopmental phenotypes associated with mutations in ZBTB18 and HNRNPU[END_REF] , #23 [START_REF] Saunier | Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency[END_REF] , #26 [START_REF] Stessman | Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders[END_REF] , #51 [START_REF] Wolff | Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders[END_REF] .

Sequencing technologies

All samples were prepared with the Illumina TruSight One preparation kit (which covers 4,813 genes associated with known phenotypes and 11,884,205 base pairs) and sequenced on either an Illumina MiSeq or NextSeq 500 sequencer using 2x150 bp sequencing kits. We performed ME sequencing in 196 parentoffspring trios (90.7%), 10 duos (proband + one parent; 4.6%) and 10 singletons (4.6%), including the three patients with a definite clinical diagnosis.

Bioinformatics pipeline

Alignment on the reference genome was made with BWA-mem and variant calling algorithms were Freebayes and the GATK Unified Genotyper and Haplotype Caller in Rennes. Annotations from ANNOVAR were added. The

Accepted Article

Basespace cloud computing platform (with BWA 2.1 and GATK Unified Genotyper 1.6) and the Variant Studio software provided by Illumina were used in La Pitié-Salpêtrière. More than 95% of targets were covered with a 20x depth of sequencing in both centers. Only point variants and small indels were investigated with this pipeline, as usually done in WES series [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][7][START_REF] Pajusalu | Large Gene Panel Sequencing in Clinical Diagnostics -Results from 501 Consecutive Cases[END_REF][START_REF] Okazaki | Clinical Diagnosis of Mendelian Disorders Using a Comprehensive Gene-Targeted Panel Test for Next-Generation Sequencing[END_REF] .

Variant validation and interpretation

All variants with a potential deleterious effect were confirmed by Sanger

Accepted Article

RESULTS

Detailed results are available in Tables 1 and2. ME sequencing revealed one or two pathogenic (class 5) variants in 39/216 patients (17.9%), one or two likely pathogenic (class 4) variants in 17/216 (7.8%) and one or two variants partially explaining the phenotype in 2/216 others (0.9%). Excluding the latter category, the overall diagnostic yield was 25.9%.

Mode of inheritance and types of variants

Of the 58 full or partial diagnoses, 40 (69%) were dominant disorders due to i) heterozygous de novo variants (n=35 including two in the same gene for patient #53), ii) variants inherited from an unaffected parent with proven or suspected somatic mosaicism (n=5) and iii) variant inherited from an affected father (n=1).

Nine patients (15.5%) had recessively inherited disorders related to i) compound heterozygous variants (n=6), ii) homozygous variants (n=2) or iii) paternally inherited variant combined with a maternally inherited gene deletion (n=1). Nine patients (15.5%) had an X-linked disorder due to i) de novo variants (n=2), ii) maternally inherited variants (n=5), iii) one variant inherited from a mosaic father.

We identified the molecular causes of NDD in 5/20 patients studied as index cases or in duos, including the three patients with a definite clinical diagnosis.

Thus, we identified a pathogenic variant for 2/17 (11%) patients without definite clinical diagnosis studied as index cases or in duos, and 27% in parent-offspring trios (all without definite clinical diagnosis).

Accepted Article

Altogether, we identified 64 variants including 47 novel mutations (Table S1).

Variant types include 33 variants leading to a premature termination codon (nonsense, frameshift, canonic splice site variants) and 31 missense variants.

Truncating/missense variants were distributed as follows according to the mode of inheritance: autosomal dominant disorders 22/19 (including the two variants of patient #53), autosomal recessive disorders 7/7 (including two homozygous variants), X-linked disorders 4/5.

Intriguingly, for patient #53, we identified 2 heterozygous de novo variants in the same gene SMARCA2 responsible for Nicolaides-Baraister syndrome. We were not able to answer about the cis/trans position; Indeed, we confirmed that both variations were not present in parent's DNA within Sanger sequencing method and were not located on the same read, looking at our NGS data.

Rate of diagnoses and phenotypes

Diagnostic yields according to the phenotypic categories defined above (Table S1) were as follows: NSID 30% (10/33), SID 32% (40/122), NDEG 57% (4/7), ASD 3.7% (2/54). The number of diagnoses relative to the number of tested patients with SID was not significantly different from the number of diagnoses made in those with NSID. On the contrary, the number of diagnoses made in all patients with ID (50/155, 32%) was significantly higher than the number of diagnoses made in those with ASD (p= 0.0002, Fisher's exact test).

Pathogenic variants identified in KAL1 in patient #57 and PRODH in patient #58 partly explained their SID phenotypes (hypogonadotrophic hypogonadism and ID with schizophrenia, respectively).

Accepted Article

DISCUSSION

Many previous studies have shown that WES is an excellent option for genetic testing in patients with NDD when fragile X syndrome, chromosomal imbalances and other hypotheses with available targeted genetic studies have been ruled out [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][START_REF] Yang | Molecular findings among patients referred for clinical whole-exome sequencing[END_REF][START_REF] Thevenon | Diagnostic odyssey in severe neurodevelopmental disorders: Towards clinical whole-exome sequencing as a first-line diagnostic test[END_REF][7] . The huge genetic heterogeneity of NDD and the rarity of each cause imply that the likelihood of finding pathogenic variants increases with the number of the studied genes. ME, i.e. panels including all disease-associated genes, is an alternative strategy when WES is not available or too expensive. In the only article reporting of the use of the TruSight One gene panel in the daily practice, this panel was applied to the diagnosis of all kinds of genetic diseases [START_REF] Pajusalu | Large Gene Panel Sequencing in Clinical Diagnostics -Results from 501 Consecutive Cases[END_REF] . Our study on patients with NDD only provides more insight into the use of ME in a context of extreme genetic heterogeneity.

Mutated genes and associated phenotypes

As in series of patients with NDD studied by WES [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][START_REF] Yang | Molecular findings among patients referred for clinical whole-exome sequencing[END_REF][START_REF] Thevenon | Diagnostic odyssey in severe neurodevelopmental disorders: Towards clinical whole-exome sequencing as a first-line diagnostic test[END_REF][7] , genetic heterogeneity was the rule in our series since we found pathogenic variants in 48 different genes involved in NDD (excluding KAL1). Eight genes were found mutated twice

Accepted Article

are responsible for epileptic encephalopathies [START_REF] Wolff | Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders[END_REF][START_REF] Stamberger | STXBP1 encephalopathy: A neurodevelopmental disorder including epilepsy[END_REF] . Patient #30 with a de novo SCN2A variant had an unusual phenotype associating ASD and mild ID without seizures. We considered this variant as the cause of the NDD because rare patients with ASD had been previously reported [START_REF] Tammimies | Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder[END_REF] . The largest series of patients with SCN2A variants published thereafter (including ours) demonstrated that 15% of them had no epilepsy but ID with or without ASD. The de novo missense variant in STXBP1 found in patient #55 is of interest because: i) it affects only one of the two isoforms of the protein, while patients with STXBP1related phenotypes usually have variants affecting both isoforms, ii) patient #55 had no epilepsy, ataxia or tremor, which are usually observed in patients with STXBP1 mutations [START_REF] Stamberger | STXBP1 encephalopathy: A neurodevelopmental disorder including epilepsy[END_REF][START_REF] Mignot | STXBP1-related encephalopathy presenting as infantile spasms and generelized tremor in three patients[END_REF] . We finally classified this variant as likely pathogenic because i) it met biological criteria for class 4 variants, ii) an increasing number of STXBP1 variants are identified in patients without seizures (7% in the largest series published so far [START_REF] Stamberger | STXBP1 encephalopathy: A neurodevelopmental disorder including epilepsy[END_REF] ), iii) one reported patient with epileptic encephalopathy had a variant similarly affecting one isoform only [START_REF] Milh | Epileptic and non-epileptic features in patients with early onset epileptic encephalopathy and STXBP1 mutations[END_REF] .

Unexpected variants found in genes not associated with human diseases

The TruSight One gene panel contains genes known for human diseases but also some genes considered as good candidates for human diseases (at the time of its design), including UNC80, PHIP and RORA.

We identified biallelic truncating mutations in UNC80 in patient #36 with global developmental delay, microcephaly, marked hypotonia and chorea/dystonia.

The clinical significance of this result remained briefly uncertain until the publication of an article reporting biallelic variants in UNC80 in patients with a Accepted Article similar phenotype [START_REF] Shamseldin | Mutations in UNC80, Encoding Part of the UNC79-UNC80-NALCN Channel Complex, Cause Autosomal-Recessive Severe Infantile Encephalopathy[END_REF] . Likewise, the de novo heterozygous truncating variant identified in PHIP in patient #49 with syndromic ID became the likely cause of his NDD after the publication of two other patients 28 .

We found a de novo heterozygous truncating variant in RORA in patient #50 with ataxia, epilepsy and severe ID. RORA encodes the retinoic-acid orphan receptor alpha expressed in the brain [START_REF] Cook | Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism[END_REF] and is intolerant to loss-of-function variants (pLi 0.95 in ExAC http://exac.broadinstitute.org). Most patients with heterozygous deletions of this gene have ID and epilepsy [START_REF] Yamamoto | Overlapping microdeletions involving 15q22.2 narrow the critical region for intellectual disability to NARG2 and RORA[END_REF] . However, no deleterious point variant in RORA has been reported to date. Available data suggest that the heterozygous truncating variant found in patient #50 may be the cause of her NDD. Thus, the TruSight One panel may provide excellent candidate variants even for a few "candidate" genes.

Diagnosis rate

We obtained a global diagnostic yield of 25.9%. The diagnostic yield of WES for all types of genetic diseases, most of which are developmental disorders, is 25-32% when index cases only are studied [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][START_REF] Yang | Molecular findings among patients referred for clinical whole-exome sequencing[END_REF] and raises to 30-38.5% with trios 7,19 , mainly because variants are discovered in "new" genes. Thus, our results are lower but close to those obtained with WES performed with index cases only.

However, the diagnosis rate is obviously limited with ME because new genes involved in NDD are regularly identified.

We identified a pathogenic mutation in 4/7 patients with NDEG but this number is too small to discuss the efficiency of ME in this clinical context. The 32% of diagnoses in patients with ID versus 3.7% in those with ASD is related to our Accepted Article classification of NDD. Some patients meeting the ADI criteria for ASD were classified in the SID and NSID groups because of early developmental delay suggesting that ASD was a manifestation of their NDD rather than the NDD itself. As examples, this was the case for patient #2 with a variant in ADNP and for patient #32 with a variant in SYNGAP1. Patients with ASD in our study roughly corresponds to the "essential" ASD group defined in a previous article [START_REF] Tammimies | Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder[END_REF] in which trio-based WES revealed a pathogenic variant in 2/64 patients (3.1%).

This result is close to ours and suggests that careful selection of patients with NDD may help defining subgroups of patients with a higher probability of achieving a molecular diagnosis.

Proposition of a rational use of ME in a context of economic constraints

Despite the diagnostic efficiency of WES for patients with NDD, some genetic centers cannot use it in their daily practice because of its high cost. The TruSight One gene panel covers 12 Mb of the genome while WES kits cover about 60 Mb. When studying trios with the ME, 36 Mb are sequenced, which is 60% of the coverage for one WES. Given that the cost per base is set with a given sequencing kit and that the cost of library preparations is comparable between kits, ME in trios (for one patient) represents a 40% saving on sequencing reagents compared to WES in index cases at constant depth.

The diagnostic yield is markedly increased by the sequencing strategy of trios versus index cases with WES [START_REF] Farwell | Enhanced utility of family-centered diagnostic exome sequencing with inheritance model-based analysis: results from 500 unselected families with undiagnosed genetic conditions[END_REF] . The aim of our study was not to compare trios versus index cases (or duos) with ME. As demonstrated by the previous series of ME [START_REF] Pajusalu | Large Gene Panel Sequencing in Clinical Diagnostics -Results from 501 Consecutive Cases[END_REF] , the "index case" strategy may reveal pathogenic variants when specific

Accepted Article

genes are suspected. Because of the low rate of diagnoses obtained with index cases (or duos) in our first sequencing series of patients without clinical diagnosis, we decided to use trio-based sequencing when possible. Though the cost of sequencing is three times higher than with index cases, this strategy greatly facilitates the downstream analyses and variant interpretation by allowing to detect de novo and compound heterozygous variants and reduces the cost of Sanger cosegregation analyzes.

We conclude that for centers that do not use WES for routine diagnoses, trio- F : female, M : male, ht : heterozygous, hi : hemizygous, hm : homozygous, cht : compound heterozygous, AD : autosomal dominant, AR : autosomal recessive, XL : X-linked

  sequencing

(

  ARID1B, STXBP1, SCN2A, SYNGAP1, TCF4, ANKRD11, ADNP and ATP1A3). As expected, six of these genes are among the most frequently mutated in the DDD study reporting pathogenic variants found by WES in individuals with developmental disorders 7 .Most gene panels are targeted to the molecular investigation of patients with particular phenotypes. Because of the large clinical and molecular Accepted Article heterogeneity of NDD, the search for its etiology requires testing with panels comprising hundreds of genes or an informative clinical examination allowing the targeting of specific panels. Using ME sequencing, we identified the etiology of NDD in 53/56 patients without definite clinical diagnoses. The corresponding disorders had not been suggested by physicians because characteristic features of the disease were absent or too mild to be noted, or because the syndrome was not recognized. As examples, patient #3 with ANKRD11 variant had a mild KBG phenotype, patient #7 had a variant in ATRX but a nonsyndromic phenotype, even after a reverse phenotyping, patient #10 had a variant in FOXP1 but the corresponding syndrome was poorly known, patient #15 had a phenotype suggestive of Lesch-Nyhan disease but with a mildly (and overlooked) elevated uricemia, patient #25 had PANK2 variants but a brain MRI that did not show the characteristic "eye-of-the-tiger" sign before the analysis. These clinical pitfalls were overcome using ME. The >30% of diagnostic yield in patients with SID and in those with NSID suggests that ME could be used as a second-line genetic test in patients without clinical diagnoses instead of sequential studies on smaller panels chosen on the basis of clinical signs. It is of note that the secondary evaluation of many patients of our series allowed a retrospective clinical (or radiological) validation of the suspected variant based on subtle, most of the time overlooked, clinical signs. This pleads for a close dialogue between molecular and clinical geneticists, as anticipated by Hennekam and Biesecker[START_REF] Hennekam | Next-generation sequencing demands next-generation phenotyping[END_REF] .We identified variants in genes associated with phenotypes different from those of our patients. Variants in SCN2A (MIM 182390) and STXBP1 (MIM 602926)

  based ME may be considered as a useful alternative strategy to investigate NDD. The rate of diagnoses can be further improved by selecting patients with the highest likelihood of achieving a molecular diagnosis, i.e. those with ID or

	Accepted Article Accepted Article	13 22 34 35 14 15 16 17 18 19 20 21 33 23 24 25 26 27 28 29 30 31 32 36 37 38 39	NDEG. M GRIA3 F GRIN2B M MICU1 M TCF4 M TCF4 F HNRNPU M HPRT M KDM5C F KDM6A F KIF1A M LARS2 F MED13L M MFSD8 M SYNGAP1 F NAA10 M NFIX M PANK2 M POGZ M RAI1 M SATB2 M SCN1A F SCN2A M SCN8A M SYNGAP1 M UNC80 M UPF3B M ZEB2 M ZMYND11	hi ht cht ht ht ht hi hi ht ht cht ht cht ht ht ht cht ht ht ht ht ht ht ht cht hi ht ht	NM_007325.4:c.1964T>C,ChrX(GRCh3 7):g.122561878T>C, p.Phe655Ser NM_006077.3:c.1048C>T,Chr10(GRCh 37):g.74183021G>A, p.Gln350*, NM_001243226.2:c.2039G>A,Chr18(G RCh37):g.52896224C>T, p.Arg680His maternal inherited NM_006077.3:c.40del,Chr10(GRCh37): g.74326512del, p.Ala14Leufs*20 , paternal inherited NM_001243226.2:c.2263_2264del,Chr 18(GRCh37):g.52895514_52895515del , p.Ser755Leufs*57 NM_000834.3:c.1966C>T,Chr12(GRCh 37):g.13761581G>A, p.Gln656* NM_031844.2(HNRNPU): c.16delinsATT,Chr1(GRCh37):g.245027 594delinsAAT, p.Val6Ilefs*4 NM_000194.2:c.47G>T,ChrX(GRCh37): g.133607408G>T, p.Gly16Val NM_004187.3:c.2482C>T,ChrX(GRCh3 7):g.53227706G>A, p.Arg828* NM_001291415.1:c.2988+1G>C,ChrX( GRCh37):g.44936072G>C, p.? NM_001244008.1:c.920G>A,Chr2(GRC h37):g.241715306C>T, p.Arg307Gln NM_015340.3:c.1987C>T,Chr3(GRCh3 7):g.45557711C>T, p.Arg663Trp and NM_015340.3:c.371A>T,Chr3(GRCh37) :g.45458981A>T, p.Asn124Ile NM_015335.4:c.5588+1G>A,Chr12(GR Ch37):g.116413319C>T, p.? NM_152778.2:c.1444C>T,Chr4(GRCh3 7):g.128841898G>A, p.Arg482*, maternally inherited and NM_152778.2:c.416G>A,Chr4(GRCh37 NM_006772.2:c.3190C>T,Chr6(GRCh3 7):g.33411519C>T, p.Gln1064* paternally inherited ):g.128864930C>T, p.Arg139His, NM_003491.3:c.384T>G,ChrX(GRCh37) :g.153197526A>C, p.Phe128Leu NM_001271043.2:c.97del,Chr19(GRCh 37):g.13135880del, p.Ala33Leufs*32. NM_153638.2:c.1235+1G>T,Chr20(GR Ch37):g.3891478G>T, p.?, paternally inherited and NM_153638.2:c.1561G>A,Chr20(GRCh 37):g.3899342G>A, p.Gly521Arg NM_015100.3:c.1810G>T,Chr1(GRCh3 7):g.151384217C>A,p.Glu604* NM_030665.3:c.2966_2969del,Chr17( GRCh37):g.17699228_17699231del, p.Lys989Serfs*74 NM_001172509.1:c.1627del,Chr2(GRC h37):g.200173596del, p.Arg543Alafs*3 NM_001165963.1:c.5726C>T,Chr2(GR Ch37):g.166848059G>A, p.Thr1909Ile NM_021007.2:c.2558G>A,Chr2(GRCh3 7):g.166198975G>A, p.Arg853Gln NM_014191.3:c.4394A>T,Chr12(GRCh 37):g.52183177A>T, p.Asp1465Val NM_006772.2:c.490C>T,Chr6(GRCh37) :g.33400564C>T, p.Arg164* NM_032504.1:c.2399del,Chr2(GRCh37 ):g.210690698del, p.Leu800Trpfs*19, paternal inherited and NM_032504.1:c.4150G>T,Chr2(GRCh3 7):g.210752852G>T, p.Glu1384*, maternal inherited NM_080632.2:c.846+1G>A,ChrX(GRCh 37):g.118974608C>T, p.? NM_014795.3:c.3170G>A,Chr2(GRCh3 7):g.145147493C>T, p.Cys1057Tyr NM_006624.5:c.76C>T,Chr10(GRCh37) :g.226028C>T, p.Arg26Trp	maternally inherited (mosaicism 10% reads in blood) de novo biparental de novo de novo de novo maternally inherited maternally inherited de novo de novo biparental transmission apparently de novo germline mosaicism (sister and brother affected) de novo biparental transmission de novo de novo biparental transmission de novo de novo de novo de novo de novo de novo, possible paternal mosaicism (3/251 reads in blood) de novo biparental maternally inherited de novo de novo	XL AD AR AD AD AD XL XL XL AD AR AD AD AR XL AD AR AD AD AD AD AD AD AD AR XL AD AD	Mental retardation, X-linked, syndromic, wu type Epileptic Myopathy with Pitt-Hopkins syndrome extrapyramidal signs Pitt-Hopkins syndrome encephalopathy, early infantile, 27 registered as Phenotype in ClinVar Lesch-Nyhan syndrome Mental retardation, X-linked, syndromic, Claes-Jensen type Kabuki syndrome 2 Mental retardation, autosomal dominant 9 Perrault syndrome Mental retardation and distinctive facial features with or without cardiac defects autosomal retardation, Mental Ceroid lipofuscinosis, dominant 5 neuronal, 7 Ogden syndrome Sotos syndrome 2 Neurodegenerati on with brain iron accumulation 1 White Sutton syndrome Smith-Magenis syndrome Glass syndrome Dravet syndrome Epileptic encephalopathy, early infantile, 11 Epileptic encephalopathy, early infantile, 13 Mental retardation, autosomal Hypotonia, infantile, with psychomotor retardation and characteristic facies 2 Mental retardation, X-linked, syndromic 14 Mowat-Wilson syndrome Mental retardation, autosomal dominant 30
								dominant 5

Table 2 .

 2 Likely pathogenic variants identified with ME in 17 patients with NDD and partial diagnoses made in two.

	Patient #	Sex	Mutated gene	Status	Variants	Inheritance	Variant type	OMIM/ClinVar phenotype
	40	F	ARID1B	ht	NM_020732.3:c.5025+1G>A,Chr6(GRC h37):g.157525131G>A, p.?	de novo	AD	Coffin Siris syndrome 1
								Cerebellar
								ataxia, areflexia,
	41	F	ATP1A3	ht	NM_152296.4:c.2224G>T,Chr19(GRCh 37):g.42479820C>A, p.Asp742Tyr	de novo	AD	pes cavus, optic atrophy and
								sensoryneural
								hearing loss
	42	M ATP1A3	ht	NM_001256214.1:c.499A>G,Chr19(GR Ch37):g.42490279T>C, p.Met167Val	de novo	AD	CAPOS syndrome
								Spastic
	43	F	AP4S1	hm	NM_007077.4:c.289C>T,Chr14(GRCh3 7):g.31542174C>T, p.Arg97*	biparental transmission	AR	paraplegia 52, autosomal
								recessive
								Cerebellar
	44	M CAMTA1	ht	NM_015215.3:c.2863C>T,Chr1(GRCh3 7):g.7737742C>T, p.Arg955Trp	de novo	AD	ataxia, nonprogressive, with mental
								retardation
					NM_006348.3:c.2324C>T,Chr7(GRCh3
	45	F	COG5	cht	7):g.106851608G>A, p.Pro775Leu, paternally inherited (class 4) and NM_006348.3:c.1508dup,Chr7(GRCh3 7):g.106924076dup, p.Gly505Trpfs*3	biparental transmission	AR	Congenital disorder of glycosylation, type IIi
					maternally inherited (class 5)
	46	F	FOXG1	ht	NM_005249.4:c.545C>T,Chr14(GRCh3 7):g.29237030C>T, p.Pro182Leu	de novo	AD	Rett syndrome congenital variant)
	47	M	PEX16	hm	NM_057174.2:c.104T>G,Chr11(GRCh3 7):g.45939259A>C, p.Leu35Arg	biparental inheritance	AR	Peroxysome biogenesis disorder 8B
					NM_004523.3:c.862_871del,Chr10(GR			Microcephaly,
	48	F	KIF11	ht	Ch37):g.94373206_94373215del,	de novo	AD	lymphoedema,
					p.Ile288Profs*3			retinal dysplasia
								Registered as a
					NM_017934.5:c.3892C>T,			phenotype in
	49	M	PHIP	ht	Chr6(GRCh37):g.79664960G>A,	de novo	AD	ClinVar
					p.Arg1298*			(syndromic mild
								ID)
	50	F	RORA	ht	NM_134260.2:c.1118del,Chr15(GRCh3 7):g.60795790del, p.Arg373Profs*17	de novo	AD	none

Webster E, Cho MT, Alexander N et al. De novo PHIP-predicted deleterious variants are associated with developmental delay, intellectual disability, obesity, and dysmorphic features. Cold Spring Harb Mol Case Stud 2016; 2: a001172.

ACKNOWLEDGMENTS

This work was funded by Assistance Publique des Hôpitaux de Paris (APHP), by a grant from the Rennes University Hospital (AAP innovations médicales, 2014) and la Fondation Groupama; Christel Depienne and Caroline Nava are supported as members of the Bio-Psy Labex.

We would like to thank the families for their participation in the study. We are grateful to Artem Kim for carefully reading this manuscript. We also thank our technicians in La Pitié-Salpêtrière (Corinne Mach, Valérie Olin, Claude Estrade, Aurélie Lafitte, Sabina Karagic, Elodie Lejeune) and in Rennes (Sophie Marzelière, Valérie Perrault) for their contribution to this work.

The authors declare they have no competing financial interests in relation to the work described.

ETHICAL STATEMENT

The study was carried out in accordance with the policies of La Pitié-Salpêtrière and Rennes University Hospital.

Accepted Article

F : female, M : male, ht : heterozygous, hi : hemizygous, hm : homozygous, cht : compound heterozygous, AD : autosomal dominant, AR : autosomal recessive, XL : X-linked