Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

Targeted genes and capture design

The 217 selected genes include 99 genes associated to XLID and 118 genes located on autosomes, implicated in dominant (45), recessive (66) or complex (7) forms of ID (see online supplementary table S8, further justification on gene selection is given in online supplementary methods). We targeted all protein-coding exons of these genes, including 20 bp of intronic flanking sequences. The overall size of targeted regions is 1.034 Mbp. Corresponding 120 bp RNA baits were designed using SureDesign (https://earray.chem.agilent.com/suredesign/).

Library preparation and sequencing

DNA samples were extracted from peripheral blood or saliva. Sequencing libraries were prepared as described previously,¹³ performing individual in-solution SureSelect capture reaction for each DNA sample (Agilent, Santa Clara, California, USA). Paired-end sequencing (2×101-bp) was performed on an Illumina HiSeq 2000/2500, multiplexing up to 16 samples per sequencing lane.

Bioinformatic pipeline and variant ranking

Read mapping, variant calling and annotation was performed as described previously.¹³ Detected variants (short indels and single nucleotide variants (SNVs)) were ranked by VaRank (an in-house developed script), which incorporates the annotations retrieved by alamut-HT (putative effect on the protein, conservation scores, splice site predictions, allelic frequency in the 106 patients and in control cohorts such as Exome Variant Server (EVS) or 1000 genomes). Candidate variants were selected when harbouring a frequency compatible with the incidence of the disease: expectedly accounting for less than 0.1% of all ID cases, aka resulting in a disease frequency <0.002%.¹⁴ Using the EVS population as a subset of the general population, candidate variants were thus retrieved when reported in EVS: with a minor allele frequency lower than 0.45% for variants in autosomal-recessive genes (ie, with a frequency of homozygotes <0.002%), in no more than one carrier for variants in autosomal-dominant genes or in no more than one male for variants in X-linked genes (as we cannot exclude, although it is unlikely, that a particular carrier from the general population might have mild ID). We concomitantly excluded variants present more than twice in the cohort of 106 patients. Remaining variants predicted as potentially pathogenic and fitting with the mode of inheritance associated to the affected gene were further tested for validation (see online supplementary table S9).

CNVs detection pipeline

Putative heterozygous/homozygous/hemizygous structural variants or CNVs were highlighted using the previously described method based on a depth-of-coverage comparison between the index sample and eight other random samples from the same sequencing lane.¹³ For the X-chromosome, coverage was normalised according to the number of X-chromosomes of the patient.

Cohort description and diagnostic yield

Patients harboured various degrees of cognitive impairment, although with a higher proportion of moderate or severe forms (46% and 42% respectively; table 1). The cohort was highly enriched in males. Among male probands, 68% were sporadic cases, the remaining had familial history of cognitive impairment mainly evocative of an X-linked mode of transmission (table 1). We detected certainly causative mutations in 26/106 patients (table 2; see online supplementary figures S2–S19), leading to an overall diagnostic yield of 25% for the entire cohort (from 23% for sporadic cases to 31% for familial cases). Unexpectedly, the diagnostic yield appears unrelated to the severity of ID in patients (table 1).

Sixteen mutations are located in genes of the X-chromosome: 14 point mutations or small indels (in ATRX, CUL4B, DMD, HCFC1, IL1RAPL1, IQSEC2, KDM5Cx2, MECP2x2, MAOA, PHF8, SLC9A6, SLC16A2), as well as two larger pathogenic events (one hemizygous complex rearrangement in MECP2, one hemizygous exon deletion in FMR1; see online supplementary figures S17 and S13). We identified 10 de novo point mutations or small indels in genes involved in autosomal-dominant/haploinsufficient forms of ID (in DYRK1Ax2, GRIN1, MED13L, RAI1, SHANK3, SYNGAP1, SLC2A1, TCF4x2). In four other patients, we identified potentially causative mutations (in NLGN3, PQBP1, SLC2A1 and TCF4; see online supplementary figures S20, S21, S7 and S9, respectively), whose implication in cognitive impairment has to be further confirmed. Finally, missense variants that appeared at first likely to be pathogenic, notably based on the very high evolutionary conservation of the affected residues or on previous publications, appeared excluded as causal after further segregation analysis (in FLNA, FMR1, HUWE1 or MECP2, see online supplementary figure S22).

Atypical type of mutations

Among the 26 certainly causative mutations identified, some were surprising by the nature of the mutation itself. In a boy with severe encephalopathy, epilepsy, hypotonia and microcephaly, we detected a highly complex rearrangement in exon #4 of MECP2, involving a 139 bp deletion flanked by the insertion of two sequences in inverted orientation derived from intron #2 still keeping the reading frame downstream of the rearrangement. Such event is inherited from the proband's mother who presents with speech delay and dyslexia (see online supplementary figure S17). This exon is known to be the one accumulating most mutations in patients, especially the 3′ half, which is a recombination hotspot and the target of several deletions/duplications and less frequently inversions.^16–18

We report here for the first time an intragenic deletion affecting FMR1 outside of the promoter/exon #1 region that is the target of the fragile-X syndrome CGG-expansion. Very few point mutations have been reported in coding regions. We identified a complete deletion of the last exon of FMR1 in one patient and his two affected brothers unevenly presenting with clinical features of fragile-X syndrome (see online supplementary figure S13).

We also identified a patient carrying a maternally inherited 10 bp deletion causing a frameshift in exon #7 of IL1RAPL1, while unexpectedly his affected brother bears a de novo deletion of the full exon highlighting that affected relatives may carry distinct mutations. We propose that small 10 bp deletion may have created a sequence conformation favouring further instability and leading to the larger deletion observed in the second brother (see online supplementary figure S14). Indeed, a large proportion of IL1RAPL1 causative mutations are intragenic exon deletions or pericentric inversions,¹⁹ supporting that IL1RAPL1 region is highly susceptible to recombination events.

Lastly, we identified a de novo Pro578Arg missense mutation in GRIN1 in a male proband with severe ID, hypotonia, feeding disorders and very poor speech but no epilepsy, a phenotype similar to that associated to GRIN1 missense mutations in some patients.^{20 21} A maternal uncle presented with similar features except for the poor speech and hypotonia (see online supplementary figure S3), initially suggesting an associated X-linked mode of inheritance. The finding of a de novo deleterious missense mutation excludes this latter hypothesis, further highlighting the prevalence of phenocopies in cognitive disorders.

Genotype–phenotype correlations: from expected to unexpected

The majority of certainly causative mutations were identified in patients whose clinical phenotype was retrospectively consistent with previous reports (table 2). For instance, the proband carrying a truncating mutation in IQSEC2 presents with severe ID, no speech, motor developmental delay, severe epilepsy, strabismus and autistic features (see online supplementary figure S15), which matches the recently proposed clinical spectrum associated to mutations in this gene.^{22 23} Likewise, DYRK1A was originally found disrupted by translocations or deleted in several patients with ID and microcephaly.^24–26 More recently, truncating mutations in this gene were shown to cause ID associated with primary microcephaly (sometimes borderline at −2 SD), growth retardation, developmental delay, facial dysmorphic traits, seizures and major feeding difficulties, with or without associated autism.^27–31 We report here two novel de novo truncating mutations in patients with similar clinical features but no epilepsy (see online supplementary figure S2). Nonetheless, in a few other cases (eg, mutations in RAI1 or MECP2), the probands lacked some clinical features, thus the corresponding diagnosis of Smith-Magenis (MIM 182290) or Rett (MIM 312750) syndrome was not evoked by experienced clinical geneticists (see online supplementary figures S5 and S17). For instance, among the three patients with MECP2 mutations, one female had the classic Rett phenotype, the other female proband presented with non-classic Rett phenotype (no regression episode, no hand-flapping), while the rarity of reports of MECP2 encephalopathies in adolescent males precluded suspicion of the involvement of that gene in a patient (APN-3) who had undergone rather extensive prior genetic testing

A few detected mutations were unexpected as they were detected in patients whose phenotype did not match previous descriptions. For instance, mutations in TCF4 mainly cause Pitt–Hopkins syndrome (PHS, MIM 610954) characterised by severe motor retardation, absence of speech, characteristic dysmorphic traits, autistic features, intestinal problems and hyperventilation.³² We here describe truncating TCF4 mutations in two patients, one with clinical manifestations highly suggestive of PHS, the other with less syndromic manifestations and no dysmorphic traits (see online supplementary figure S9). TCF4 mutations were already reported in patients with non-syndromic ID, suggesting that such mutations were likely to be underdiagnosed.³³

Another patient and his affected brother both carry a distal frameshift mutation in DMD affecting the major muscle transcript encoding the dystrophin protein associated to Duchenne or Becker muscular dystrophy (DMD, MIM 310200; BMD, MIM 300376), and the brain-specific isoform Dp71. The index case presents with moderate ID, psychomotor retardation, no speech, behavioural disorders, dysmorphic traits but strikingly no muscular phenotype (see online supplementary figure S12). His brother harbours a milder phenotype with additional hypotonia and cerebellar dysplasia. Both harbour borderline-high CPK levels. The association of cognitive impairment with DMD/BMD has been extensively reported and correlated to truncating mutations affecting Dp71, yet never in the absence of a muscular phenotype.^34–39 Our findings extend the recent report of a large family with affected males carrying an in-frame single amino acid deletion associated to mild ID and no muscular phenotype.⁴⁰

Confirmation of candidate genes for cognitive disorders

Some selected genes were only candidate ID or ASD genes at the time of the design, with single pieces of evidence in the literature. The identification of additional mutations in patients with similar phenotype definitively confirms their implication in cognitive disorders.

We reported a damaging missense affecting the function of the Monoamine Oxidase A enzyme (MAOA),^{41 42} which replicated for the first time in 20 years the implication of MAOA in autism/ID associated to significant behavioural disorders.^{41 42} We also identified a probably pathogenic missense variant in NLGN3 in a male and his cousin, both presenting with ID and autism (see online supplementary figure S20). In silico predictions, high conservation of the mutated residue across all neuroligin paralogs, and familial analysis are altogether in favour of a pathogenicity of this missense change. A definitive functional effect of this missense still needs to be demonstrated to clearly establish the diagnosis. The implication of this gene was never replicated since the initial publication,⁴³ although screened in several cohorts with comparable phenotypes.^44–50

Similarly, we identified a novel truncating point mutation in MED13L confirming the implication of this gene in ID (see online supplementary figure S4). Disruption of MED13 L was initially associated with transposition of the great arteries (TGA), associated to ID in a single case with a chromosomal translocation.⁵¹ A homozygous missense mutation was then identified in two siblings from a consanguineous family presenting with non-syndromic ID, suggesting an implication of the gene in autosomal-recessive forms of ID justifying its selection in our panel.⁵ A total of five patients were more recently described with de novo intragenic CNVs or point mutations affecting MED13L, delineating a recognisable MED13L-haploinsufficiency syndrome characterised by hypotonia, moderate ID, variable cardiac defects, facial hypotonia and dysmorphic traits.^{52 53} Our patient with the MED13L mutation presents with concordant phenotype such as an open mouth appearance and muscular hypotonia but no cardiac defects. Due to the initially proposed autosomal-recessive mode of inheritance associated to ID, at first we did not consider this heterozygous truncating mutation as causative, as it may also have been the case for a heterozygous splicing mutation identified earlier in a large-scale exome sequencing study in one male with ASD.²⁸ Altogether, these findings suggest that ID associated to MED13L-haploinsufficiency syndrome is a relatively frequent condition.^{5 53}

Ambiguous mode of inheritance in ID-associated genes: the example of DEAF1

As for MED13L, the mode of inheritance associated to some genes is ambiguously described in literature. We identified an heterozygous variant affecting splicing in DEAF1 inherited from the asymptomatic mother in a patient presenting with severe ID, developmental delay, poor speech, pain resistance, dysmorphic features and aggressive behaviour (figure 1), while the gene had been proposed as associated to autosomal-dominant forms of ID.^{7 8} The recent report of two additional individuals carrying de novo missense mutations narrowed the associated phenotype to moderate/severe ID, speech impairment, behavioural problems, high pain threshold, dysmorphic features and abnormal walking pattern, hence highly similar to the one of our proband.⁵⁴ Although in vitro validation studies suggest that the reported missense variants lead to an impaired function of DEAF1, the authors concluded that they presumably act as dominant-negatives incapacitating both normal and mutant proteins since truncating variants had been observed in asymptomatic individuals.⁵⁴ In parallel, a homozygous missense mutation clustering in the same SAND-domain with all three de novo missense mutations was reported in members of a consanguineous family presenting with ID, microcephaly and white matter abnormalities, therefore suggesting a possible autosomal-recessive mode of inheritance.⁵⁵ The pathogenic mechanism associated to DEAF1 mutations is therefore unclear. Due to the highly similar clinical features of the herein reported proband and of probands carrying de novo missense mutations, the splice variant detected here may contribute to the phenotype of our patient, possibly through a recessive mode of inheritance (ie, acting in trans with another heterozygous variant) since haploinsufficiency appears tolerated in healthy individuals. Altogether those findings either suppose a similar phenotype for autosomal-dominant and autosomal-recessive mode of inheritance associated to DEAF1 mutations, or a universal autosomal-recessive mode of inheritance with a second variant that has not been yet identified, alike what was finally proven for Thrombocytopenia-absent radius (TAR) syndrome for instance.⁵⁶

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Figure 1

Inherited disrupting splice variant in DEAF1: what contribution to the phenotype? (A) Pedigree showing the maternally inherited splice variant in DEAF1 (c.290-3C>G); (B) prediction scores for the effect of the herein described variant on splicing (prediction scores for acceptor splice sites (ASS) as computed by SpliceSite Finder, MaxEntScan, NNsplice, GeneSplicer and Human Splicing Finder for the consensus ASS with either the wild-type or the mutated allele); (C) localisation of the variant, and its resulting effect on splicing in vitro (minigene construct): 90% of abnormal transcripts: 80% with entire exon #2 skipped, and 10% using the alternative ASS c.290-16 both leading to a frameshift and a premature stop codon.

Unsupportive evidences for proposed ID-associated mutations and genes

The identification in patients of a previously reported ID mutation should be considered with caution: indeed, as already discussed in previous publications, some ‘false-positive’ mutations are present among variants annotated as ‘pathogenic’ in dbSNP, OMIM or in published reports.^{13 14} We here question the causative effect of a few previously proposed mutations (see online supplementary table S4). For instance, one missense variant identified in FLNA (c.3872C>T , p.Pro1291Leu) was previously reported in a patient with FG syndrome (MIM 300321).⁵⁷ This variant was identified here in a patient with a different phenotype (see online supplementary figure S22) and is also reported in one male in EVS (presumably not presenting with cognitive disorders), raising doubt about its pathogenicity.

The identification of non-segregating truncating variants (ie, detected both in patients and healthy relatives) can also challenge the implication of genes in X-linked and autosomal-dominant forms of ID. In one family, we identified a frameshift variant in SRPX2 in a male proband. It is most likely inherited from the deceased asymptomatic maternal grandfather since it was also detected in the mother and in three maternal aunts yet absent from the maternal grandmother. Despite recent functional evidences regarding the role of SRPX2 in brain development,^{58 59} its definitive implication in cognitive disorders has already been questioned following the presence of the initially proposed mutations in EVS¹⁴ and in control individuals,⁶⁰ and subsequently to the identification of a missense mutation in GRIN2A co-segregating with the epileptic status in the initial SRPX2 family.⁶¹ In another family, we identified a nonsense variant in SHROOM4 in a male proband yet also in his unaffected brothers, a finding that further challenges the implication of SHROOM4 in X-linked cognitive disorders (see online supplementary table S5, figure 2).¹⁴

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Figure 2

Truncating variants not or ambiguously co-segregating with ID. (A) Pedigree of patient APN-13 carrying a frameshift variant in SRPX2 (c.602del, p.Ala201Valfs*10) demonstrating the likely inheritance from the asymptomatic (deceased) maternal grandfather, yet a putative germinal mosaicism of a de novo mutation cannot be excluded.; (B) predicted functional domains of SRXP2 from Pfam indicating locations of the herein identified mutation (in red) and those previously described. DUF4174: domain of unknown function; (C) pedigree showing the non-segregating nonsense variant in SHROOM4 (c.3772C>T; p.Gln1258*) in the family of patient APN-86; (D) location of the premature stop codon, which would disrupt the ASD2 domain of the protein.

This study was supported by grants and fellowships from Fondation pour la Recherche Médicale, Agence de Biomedecine and Fondation Jerome Lejeune, APLM and CREGEMES. We thank the students who were involved in this project: Sébastien Kirsch, Audrey Creppy, Inès Bekkour and Grace Gan. We thank Nadège Calmels, Valérie Biancalana, Elsa Nourisson and all other members of the Genetic Diagnostic Laboratory of the Nouvel Hopital Civil (Strasbourg) for their help with patients’ DNA sample selection and preparation. We thank Cecile Pizot for the development of VaRank. We thank Damien Sanlaville, Christine Coubes, Delphine Héron, Sophie Naudion, James Lespinasse and Marie-Line Bichon for their contribution to the recruitment of patients, and all medical interns or genetic counsellors who participated in this project. Finally, we warmly thank all patients and their families for their implication in this study.