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. 2016 Jan;21(1):133-48.
doi: 10.1038/mp.2014.193. Epub 2015 Feb 3.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes

H Hu  1 , S A Haas  2 , J Chelly  3   4 , H Van Esch  5 , M Raynaud  6   7   8 , A P M de Brouwer  9 , S Weinert  10   11 , G Froyen  12   13 , S G M Frints  14   15 , F Laumonnier  6   7 , T Zemojtel  2 , M I Love  2 , H Richard  2 , A-K Emde  2 , M Bienek  1 , C Jensen  1 , M Hambrock  1 , U Fischer  1 , C Langnick  10 , M Feldkamp  10 , W Wissink-Lindhout  9 , N Lebrun  3   4 , L Castelnau  3   4 , J Rucci  3   4 , R Montjean  3   4 , O Dorseuil  3   4 , P Billuart  3   4 , T Stuhlmann  10   11 , M Shaw  16   17 , M A Corbett  16   17 , A Gardner  16   17 , S Willis-Owen  16   18 , C Tan  16 , K L Friend  19 , S Belet  12   13 , K E P van Roozendaal  14   15 , M Jimenez-Pocquet  8 , M-P Moizard  6   7   8 , N Ronce  6   7   8 , R Sun  2 , S O'Keeffe  2 , R Chenna  2 , A van Bömmel  2 , J Göke  2 , A Hackett  20 , M Field  20 , L Christie  20 , J Boyle  20 , E Haan  16   19 , J Nelson  21 , G Turner  20 , G Baynam  21   22   23   24 , G Gillessen-Kaesbach  25 , U Müller  26   27 , D Steinberger  26   27 , B Budny  28 , M Badura-Stronka  29 , A Latos-Bieleńska  29 , L B Ousager  30 , P Wieacker  31 , G Rodríguez Criado  32 , M-L Bondeson  33 , G Annerén  33 , A Dufke  34 , M Cohen  35 , L Van Maldergem  36 , C Vincent-Delorme  37 , B Echenne  38 , B Simon-Bouy  39 , T Kleefstra  9 , M Willemsen  9 , J-P Fryns  5 , K Devriendt  5 , R Ullmann  1 , M Vingron  2 , K Wrogemann  1   40 , T F Wienker  1 , A Tzschach  1 , H van Bokhoven  9 , J Gecz  16   17 , T J Jentsch  10   11 , W Chen  1   10 , H-H Ropers  1 , V M Kalscheuer  1
Affiliations

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes

H Hu et al. Mol Psychiatry. 2016 Jan.

Abstract

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Apparently pathogenic CLCN4 mutations identified in the screen and functional analysis of the missense variants. (a) Pedigrees of families with CLCN4 likely pathogenic mutations. Individuals tested for co-segregation with X-linked intellectual disability (XLID) and the results are indicated, *=mutation carrier, wt=subject does not carry the mutation. (b) Current–voltage relationships of the electrogenic Cl−/H+ exchanger protein ClC-4 and its mutants expressed in Xenopus oocytes, shown as mean values of normalized steady-state currents from several oocytes (numbers indicated in figure, in parentheses: number of frogs). Compared with the strongly outwardly-rectifying currents of wild-type ClC-4,, currents were much smaller or even absent with CIC-4 mutant proteins carrying p.Gly78Ser, p.Leu221Val, p.Val536Met and p.Gly731Arg substitutions. ctr, non-injected controls; error bars, s.e.m. Two-tailed t-test was used for statistical comparisons (**P<0.01, ***P<0.001 compared with wild-type ClC-4 currents). (c) Analogous positions of amino acids mutated in ClC-4 highlighted in the crystal structure of CmClC. Amino acids are displayed as spheres in colors like in (b). The small green spheres represent Cl− ions. CLC transporters form dimers of identical subunits (shown in different shades) and include a transmembrane domain (TMD) and two cytosolic cystathionine-β-synthase (CBS) domains.
Figure 2
Figure 2
Pedigrees of families with co-segregating truncating and missense variants in novel and previously suggested candidate X-linked intellectual disability (XLID) genes validated through this study. (a) In the postsynaptic density protein CNKSR2, we observed a protein truncating variant in family P180. (b) In FRMPD4, we detected a unique protein truncating variant in family P58 with five affected males. (c) In KLHL15, we identified a protein truncating variant in family D60 with eight affected males. (d) In LAS1L, we found unique missense variants in families MRXS6 (ref. 66) and T50, both with Wilson-Turner (WTS) syndrome. (e) In RLIM, we identified missense variants in three large families D72, T11 and AU31. (f) In USPX27, we found a protein truncating variant in family D177 and a missense variant in family L75. (g) In the novel candidate XLID gene CDK16, we detected a protein truncating variant in family L56. (h) In the novel candidate XLID gene TAF1, we identified missense variants in families D185 and N67. *=mutation carrier, wt=wild type.
Figure 3
Figure 3
Effects of Clcn4 or Cnksr2 downregulation on morphology of mouse hippocampal neurons. Typical arborization of GFP-labeled neurons cultured for 18 days in vitro (DIV) after targeting by non-silencing (NS) or gene-specific shRNA (Clcn4 or Cnksr2) at 11 DIV. Quantification of transfected neurons, for total length of neuritic branches, total number of branches (a branch is considered as the segment between two branching points) and for dendritic branching complexity (levels were quantified per neuron from 1 to 6, each time a branching point is met from nucleus toward the distal part of each dendrite). Detection of co-transfections of shRNA and cDNA encoding plasmids for rescue experiments is shown as overlap of GFP (green) and Halotag (red) signals. Clcn4 experiment is shown in (a) and Cnksr2 in (c). More than 15 representative cells of each type were analyzed per experiment, with three independent experiments conducted. (b) Quantification of neuritic arborization in GFP expressing primary hippocampal neurons derived from Clcn4+/+ and Clcn4−/− mice as described above. Two independent experiments with>30 cells per genotype of five wild-type and four knock-out mice were analyzed. ClC-4-deficient neurons showed a significant reduction in the total number and total length of neuritic branches compared with wild-type cells. Average values with s.e.m. are shown (i) in histograms for neuritic length and number of branches and (ii) in curves for complexity levels of branching. Mann-Whitney and Chi2 tests were respectively used for statistical comparisons (ns: non-statistically significant, *P<0.05, **P<0.01, ***P<0.001). Scale bar represents 10 μm.
Figure 4
Figure 4
Novel X-linked intellectual disability (XLID) genes and candidates that emerged from this study encode components of key cellular protein networks. All available protein–protein interactions involving known intellectual disability (ID) proteins and the proteins likely implicated in XLID identified in this study were first extracted from the literature and then connected into a set of protein–protein interaction networks via the Ingenuity tool. Functional cellular subnetworks were extracted by using the available annotations of the interacting proteins (e.g., defined by functional category ‘translation/transcription') and by performing literature searches. (a) PSD-95 (postsynaptic density protein 95)/Ras/Rho interaction network. CNKSR2 (CNK2, MAGUIN1, validated XLID protein) that likely functions as an adapter protein or regulator of Ras signaling pathways interacts with PSD-95 in synaptosomes. FRMPD4 (Preso, validated XLID protein), which is a positive regulator of dendritic spine morphogenesis and density and is required for the maintenance of excitatory synaptic transmission, interacts with PSD-95, and together with its binding partner ARHGEF7 (βPix) localizes in dendritic growth cones. (b) Transcriptional/translational interaction network. Known protein complexes are highlighted. RNA Polymerase II (RNAPII) complex with the core component TAF1 (novel candidate XLID protein). ATN1 (known ID protein) interacts with TAF4 and negatively regulates transcription of RNAPII. Large ribosomal subunit (60S) contains RPL10 (known candidate XLID/autism protein). LAS1L (novel XLID protein) is essential for the biogenesis of the ribosomal subunit 60S. Eukaryotic translation initiation factor, EIF2S3 (novel XLID protein), is a component of the translation initiation complex and promotes binding of the initiator methionyl-tRNA to the 40S ribosomal subunit. POLDIP3 (SKAR), involved in positive regulation of translation, associates with THOC2 (novel XLID protein) as a part of the TREX complex (functioning in mRNA export), with mRNA surveillance factor UPF3B (known XLID protein), as well as with a core component of the exon junction complex, EIF4A3. CDK16 (novel candidate XLID protein) and Synapsin 1 (Syn1, known XLID protein) were shown to interact in a membrane fraction from brain. Cdk16 associates with 14-3-3 zeta in Neuro-2A cells. Mediator complex, which functions as a transcriptional coactivator, contains MED12 (known XLID protein) and MED13L (known ID protein). NIPBL (known ID protein) is involved in loading of cohesin and associates with the mediator-cohesin complex, which interfaces gene expression and chromatin structure. Histone methyltransferase MLL2 (known ID protein) associates with a core component of Pol II, POLR2B, and activates transcription. Deubiquitinating enzyme USP27X (novel XLID protein) interacts with USP22 that is required for histone deubiquitination, and which associates together with TAF10 as part of the TBP-free TAF complex (TFTC). ADRA2B, G-protein coupled receptor, by interacting with EIF2B and 14-3-3 zeta links G protein-mediated signaling network and cellular control of protein synthesis. (c) Ubiquitination interaction network. KLHL15 (validated XLID protein) with a function in protein ubiquitination interacts with a component of an ubiquitin E3 ligase, CUL3. RLIM (novel XLID protein) is an E3 ubiquitin protein ligase and associates with UBE2D1.
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