Mutations in DCPS and EDC3 in Autosomal Recessive Intellectual

Human Molecular Genetics, 2015, Vol. 24, No. 11 3172–3180

doi: 10.1093/hmg/ddv069 Advance Access Publication Date: 20 February 2015 Original Article Downloaded from ORIGINAL ARTICLE Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA http://hmg.oxfordjournals.org/ decapping in neurodevelopment Iltaf Ahmed1,2,†, Rebecca Buchert3,†, Mi Zhou5,†, Xinfu Jiao5,†, Kirti Mittal1, Taimoor I. Sheikh1, Ute Scheller3, Nasim Vasli1, Muhammad Arshad Raﬁq1, 6 1 7 2 M. Qasim Brohi , Anna Mikhailov , Muhammad Ayaz , Attya Bhatti , at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 Heinrich Sticht4, Tanveer Nasr8,9, Melissa T. Carter10, Steffen Uebe3, André Reis3, Muhammad Ayub7,11, Peter John2, Megerditch Kiledjian5,*, John B. Vincent1,12,13,* and Rami Abou Jamra3,*

1Molecular Neuropsychiatry and Development Lab, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8, 2Atta-ur-Rehman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan, 3Institute of Human Genetics and 4Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen 91054, Germany, 5Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA, 6Sir Cowasji Jehangir Institute of Psychiatry, Hyderabad, Sindh 71000, Pakistan, 7Lahore Institute of Research and Development, Lahore 51000, Pakistan, 8Department of Psychiatry, Mayo Hospital, Lahore 54000, Pakistan, 9Department of Psychiatry, Chaudhary Hospital, Gujranwala 52250, Pakistan, 10Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada M5G1X8, 11Division of Developmental Disabilities, Department of Psychiatry, Queen’s University, Kingston, Ontario, Canada K7L 3N6, 12Department of Psychiatry and 13Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 2J7

*To whom correspondence should be addressed. Tel: +1 8484453306; Fax: +1 7324451794; Email: [email protected] (M.K.); Tel: +1 4165358501 x6487; Fax: +1 4169794666; Email: [email protected]; [email protected] (J.B.V.); Humangenetisches Institut, Schwabachanlage 10, Erlangen 91054, Germany. Tel: +49 91318526477; Fax +49 91318523232; Email: [email protected] (R.A.J.)

Abstract There are two known mRNA degradation pathways, 3′ to 5′ and 5′ to 3′. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3′ to 5′ exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3

† These authors contributed equally to this study. Received: December 15, 2014. Revised and Accepted: February 16, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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stimulates DCP2, which decaps mRNAs at the beginning of the 5′ to 3′ degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further afﬁrm an emerging theme linking aberrant mRNA metabolism to neurological defects.

Introduction to light and eye movement. No history of other brain issue or epi- Downloaded from lepsy was reported by their parents. Individual IV-7 also has mus- Gene expression levels in biological systems are highly regulated cle hypotonia, motor functions are severely delayed and she is to ensure optimal cell function. Hence, RNA levels are speciﬁcally unable to walk. The affected individuals recognize their parents regulated by either transcription rates or mRNA stability and deg- and homes and have a rather shy demeanor but are generally radation. In particular, mRNA degradation provides an important cooperative. III-2 and IV-3 sometimes show aggressive behavior. – mechanism for rapid response to cellular events (1 3). In mam- http://hmg.oxfordjournals.org/ III-2 can speak in sentences, albeit slurred, and he shows some malian cells, two main exonucleolytic mRNA decay pathways autistic features. The other two show little or no speech develop- have been described. In the 3′ to 5′ decay pathway, the poly (A) ment. Magnetic resonance imaging of the brain of individual IV-3 tail is initially removed by deadenylation followed by degradation at the age of 17 was unremarkable (Supplementary Material, of the mRNA body from the 3′ end by the exosome exonuclease Fig. S1). complex. The remaining monomethylguanosine mRNA cap Family B, from Syria, includes three healthy and two affected (m7G cap) structure is subsequently hydrolyzed by the scavenger children, born to healthy ﬁrst-degree cousins (Fig. 2). We exam- decapping enzyme [DCPS (MIM610534)] (4,5). The 5′ to 3′ decay ined the affected siblings at ages of 12 (IV-2) and 7 years (IV-4). pathway also starts with the deadenylation of the poly (A) tail, Pregnancy, delivery and birth parameters of both children were then the m7G cap is decapped by the decapping enzyme 2

reported to be normal by the parents and the midwife. Both chil- at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 [DCP2 (MIM 609844)] or the NUDT16 decapping enzyme (6), fol- dren developed unremarkably in the first year of life. Then, a lowed by 5′ to 3′ exoribonucleolytic decay of the remaining slower cognitive development in comparison to their healthy sib- mRNA by XRN1 (MIM 607994) (7,8). Several proteins are required lings was observed. Nevertheless, motor development was unre- for efficient DCP2 activity including DCP1A [decapping enzyme 1 markable. At the time of examination, both children present with A (MIM 607010)], EDC4 [enhancer of mRNA decapping 4 (MIM mild intellectual disability with an estimated IQ of 50–70. Sleep- 606030)], DDX6 [DEAD/H Box 6 (MIM 600326)] and EDC3 [enhancer ing pattern is unremarkable, and neither child has epilepsy. of mRNA decapping 3 (MIM 609842)]. This decapping complex is Further, IV-4 has sensory hearing loss (right ear: 35–40 dB; left conserved in all eukaryotes (9,10). Thus, a change in mRNA deg- ear: not measurable). Wearing a hearing aid has not improved radation rates or specificity is expected to have numerous effects his language and pronunciation. He also has sectoral hetero- on the expression levels of various pathways and disturb the cel- chromia iridum. Other than this, the affected children have no lular balance and function. dysmorphological facial features (Fig. 2). At the time of examin- Here, we report on mutations in DCPS and EDC3 in two fam- ation, IV-2 had normal height (145 cm at age of 12 years, −0.7 ilies (Family A and Family B, respectively), with autosomal reces- SD) and a reduced head circumference (50.5 cm, −3 SD), and IV- sive intellectual disability. In vitro biochemical assays support the 4 had normal growth parameters and a reduced head circum- reduced activities for both DCPS variants and for the EDC3 ference (48 cm, −3.6 SD). The healthy parents were in the lower variant. normal spectrum regarding height and head circumferences (father 54 cm, mother 52 cm). For Family A, 500K SNP microarray genotyping (Affymetrix) Results was performed for the affected individuals III-2 and IV-3 and un- In Family A, from the Azad Kashmir region of Pakistan, there are affected individual III-3. Pathogenic copy number variants were three affected individuals in three different branches (Fig. 1), all excluded using dChip (13). Positional mapping was conducted born to consanguineous parents. We examined the affected using dChip (http://www.hsph.harvard.edu/cli/complab/dchip/) individuals at ages of 21 years (III-2), 17 years (IV-3) and 10 and HomozygosityMapper (http://www.homozygositymapper. years (IV-7). The pregnancy and delivery were reported to be nor- org) software (14,15) under the hypothesis of a founder mutation. mal for III-2 and IV-3; however, IV-7 was born premature, in the Microsatellite markers were used to corroborate autozygosity, 28th week of pregnancy. For all three affected persons, motor de- including in the right-hand branch of the pedigree (i.e. in IV-7). velopment milestones were achieved late (III-2 and IV-3) or not No common region of homozygosity could be identified for all af- achieved at all (IV-7), also all have intellectual disability, ranging fected individuals, but we identified a single candidate region of from mild to severe [III-2: mild to moderate (IQ: 45–55); IV-3: mod- 11.3 MB on chromosome 11 that is shared by individuals III-2 and erate (IQ: 35–45); IV-7: severe (IQ: <35)]. No clear facial dysmorphic IV-3. Exome sequencing was performed using DNA from individ- features or abnormalities of skin, hair, joints, vision or hearing ual III-2. Details on sequencing procedure and bioinformatics were found. A prominent upper jaw is noted in III-2 and IV-3; processing are described elsewhere (16). Whole-exome sequen- however, photographs were not available to determine whether cing (WES) resulted in an average of 91-fold read coverage. this is a family trait. Head circumference is in the normal range 93.75% of the target sequences were covered with a depth of at for III-2 and IV-3 (56 and 55 cm, respectively) but below the least 20×, and 97.97% were covered with a depth of at least 10×. third centile for IV-7 (48 cm) (11) and heights for III-2, IV-3 and Filtering revealed one homozygous candidate mutation in the IV-7 were 157.5, 147, and 119.5 cm, respectively. For IV-3 and IV- decapping scavenger gene, DCPS (NM_014026.3): chr11:126 208 7, this is below the second centile for Pakistani children (12) (no 295G>A;c.636+1G>A. This variant is highly conserved, predicted data is available for the Pakistani adult population). Neurological to prevent correct splicing (17), and is not reported in the investigation showed normal cranial nerves, pupillary response Exome Aggregation Consortium browser (ExAC; Cambridge, MA, 3174 | Human Molecular Genetics, 2015, Vol. 24, No. 11 Downloaded from http://hmg.oxfordjournals.org/ at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016

Figure 1. Pedigree of Pakistani Family A. Individuals indicated with black shaded symbols are affected with apparent NS-ARID and photographs of affected individuals III-2, IV-3 and IV-7.

USA; http://exac.broadinstitute.org; Dec 2014). A G to T at this pos- Exome sequencing was performed using DNA from individual ition, however, is reported in 1 out of 126 460 alleles. Reverse tran- IV-2, as described elsewhere (21). WES resulted in an average scription (RT)–PCR followed by Sanger sequencing revealed a of 43.2-fold read coverage. 57.1% of the target sequences were shift to a cryptic splice site 45 bp downstream from the canonical covered with a depth of at least 20× and 67.4% were covered splice donor with correct splicing to exon 5. This maintains the with a depth of at least 10×. The candidate mutation in EDC3 reading frame and results in the addition of 15 amino acids (5′ (NM_025083.3): chr15:74 967 305A>G;c.161T>C; p.Phe54Ser on Val-Lys-Val-Ser-Gly-Trp-Asn-Val-Leu-Ile-Ser-Gly-His-Pro-Ala 3′) chromosome 15q24.1 was the only variant located in a candidate to the DCPS protein (Supplementary Material, Fig. S2). Affected region, is highly conserved is and predicted in silico to be dam- individuals III-2 and IV-3 are homozygous for this variant, aging (17–19). It is not reported in the ExAC browser or in >700 while individual IV-7 is heterozygous. We thus sequenced in-house exomes. As the coverage was low, we repeated sequen- all coding exons of DCPS using Sanger sequencing and identified cing of the coding elements (CDS) in the candidate regions (496 in individual IV-7 a second heterozygous candidate muta- genes) by a custom targeted sequencing using DNA from individ- tion in DCPS (NM_014026.3): chr11:126 215 441C>T; c.947C>T; ual IV-4. We achieved a mean coverage of 299 reads, at least 20× p.Thr316Met. This variant was genotyped across the family, coverage for 80.8% of the target sequence and at least 10× for and only the mother (III-11) was identified with this allele (het- 84.6%. Combining reads from exome and CDS custom targeted erozygous). This variant (rs137941190) is conserved, is predicted sequencing, 85.4% of the CDS target region was covered at least to be deleterious (17–19) and is present in the ExAC browser at a 10× in at least one of the siblings. No additional candidate var- low frequency of 0.001263. iants were identified using this approach. For Family B, SNP microarrays (Affymetrix SNP Chip 6.0) were To gain further information on potential pathogenic effect of run for both affected individuals and their healthy siblings, and the identified variants, we performed molecular modeling using pathogenic copy number variants were excluded using Affyme- Modeler 9.9 (22). In the altered DCPS protein carrying a 15-residue trix Genotyping Console Software (Version 3.0.2). Positional map- (DCPSIns15) insertion, the loop connecting the amino acids lysine ping was performed using HomozygosityMapper (15)underthe (p.Lys207) and tyrosine (p.Tyr217) that build the active decapping hypothesis of a founder mutation as described in detail in a pre- site (23) is extended (Supplementary Material, Fig. S3A). As a con- vious study (20). Mapping for Family B revealed two candidate re- sequence, the active site becomes distorted and the long loop gions of a total length of 33 Mb on chromosomes 15 and 19. also interferes with the DCPS homodimer interface. Therefore, Human Molecular Genetics, 2015, Vol. 24, No. 11 | 3175 Downloaded from http://hmg.oxfordjournals.org/ at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016

Figure 2. Pedigree of Syrian family B. Individuals indicated with black shaded symbols are affected with apparent NS-ARID and pictures of the affected persons: There are no significant facial characteristics of the affected individuals, the affected boy has heterochromia iridum and wears a hearing aid. Both show mild ID. we expect the dimer to have reduced stability and enzymatic and DCPSWT and DCPSIns15 alleles in the III-3 heterozygous activity. Molecular modeling of the homodimeric DCPS also cells (Fig. 3C). Western blot analysis of lymphoblastoid cell predicted that p.Thr316 forms stabilizing water-mediated inter- lysates confirmed the generation of DCPSIns15 proteininthe actions with the glutamine at position p.314 and with the gluta- homozygous individuals with the complete absence of DCPSWT mate at position p.303 (Supplementary Material, Fig. S3C). These protein and, most importantly, also revealed greatly reduced polar interactions can no longer be formed in altered DCPS with a steady-state levels of DCPSIns15 in both homozygous and hetero- methionine at position p.316; thus, DCPS homodimer stability zygous cells (Fig. 3D). may be decreased. Molecular modeling of EDC3 with the amino We also assayed the propensity of EDC3 wild-type (EDC3WT) acid serine at position p.54 instead of phenylalanine predicted and EDC3 with the p.Phe54Ser variant (EDC3F54S) to stimulate that the substitution at p.54, which is located within the LSm do- DCP2 decapping in vitro. As expected, EDC3WTenhanced DCP2 main, leads to a disruption of the domain structure due to a decapping activity by 2-fold while the same was not observed poorly packed hydrophobic core. Energetic calculations indicate with EDC3F54S.EDC3F54S even demonstrated inhibitory activity a loss of 4–5 kcal/mol of stabilization energy for p.Ser54, suggest- at higher concentrations (Fig. 4). This suggests that DCP2 decap- ing that the domain might entirely unfold (Supplementary ping is impaired in affected individuals. Material, Fig. S4). Since the LSm domain is crucial for the interaction between EDC3 and DCP2, we anticipate that this variant Discussion will significantly impair protein function. Functional decapping assays were carried out to test whether Taken together, in two families with a non-specific form of intel- the respective mutations adversely affect decapping. The scaven- lectual disability, we identified biallelic variants in DCPS or EDC3. ger decapping activity of recombinant proteins corresponding In silico parameters and molecular modeling suggested deleteri- to DCPS wild-type (DCPSWT), DCPS with the 15 amino acid inser- ous effects of all variants. Functional analyses validated this by tion (DCPSIns15) and DCPS with the p.Thr316Met substitution revealing an impairment of protein function. (DCPST316M) were tested. Decapping activity from the DCPSIns15 In cells, a disruption of DCPS decapping activity is expected and DCPST316M variants was almost completely ablated relative to lead to the accumulation of m7GpppN cap structures, which to DCPSWT (Fig. 3A). Similarly, proteins extracted from lympho- can sequester the nuclear cap binding protein and decrease the blastoid cell lines from affected individuals homozygous for the efficiency of first intron splicing (25) and potentially alter cyto- splice site variant (III-2 and IV-3) were assayed and shown to plasmic mRNA translation by sequestering the cytoplasmic have significantly reduced or no detectable mRNA decapping ac- cap-binding protein (26). In addition, at least in Saccharomyces cer- tivity in comparison to their unaffected heterozygous siblings evisiae and Caenorhabditis elegans, DCPS orthologs contribute to (III-3 and IV-2) (Fig. 3B). RT–PCR of total cellular RNA derived Xrn1-mediated exonucleolytic decay (27–29). Whether DCPS ful- from the lymphoblastoid cells revealed the presence of mRNA fills a similar role in mammalian cells remains to be determined. corresponding to DCPSIns15 allele in the two homozygous cells Genomic mRNA and proteomic analysis of the individuals 3176 | Human Molecular Genetics, 2015, Vol. 24, No. 11 Downloaded from http://hmg.oxfordjournals.org/ at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016

Figure 3. Functional analysis of DCPS variants. (A) DCPS in vitro decapping assay. mRNA decapping was assayed using recombinant full-length DCPS wild-type (DCPSWT)or altered [splice mutation/45 bp insertion (DCPSIns15) and p.Thr316Met(DCPST316M)] DCPS protein in pET28a (Novagen, EMD Biosciences) vector expressed and purified from Escherichia coli BL21(DE3) cells. Migration of the input cap structure (m7GpppG) or m7GMP product (m7Gp) is designated, with the red ‘p’ denoting the location of the 32P. The percent decapping is indicated at the bottom of the panel. (B) DCPS activity in lymphoblastoid cell lines. Lymphoblastoid cell lines from homozygous affected (III-2 and IV- 3) and heterozygous unaffected (III-3 and IV-2) family members were used for decapping assays. Decapping products and labeling are as in (A). (C)RT–PCR analysis for DCPS. RT–PCR across the DCPS splice mutation (from exons 3 to 6) was performed using mRNA from patients (III-2 and IV-3) and unaffected family member (III-3), plus cartoon indicating exon usage. (D) Western Blot of DCPS. Wild-type and variant DCPS protein from the indicated lymphoblast cell lines were detected by western blotting to demonstrate migration of the variant DCPS protein in homozygous and heterozygous individuals. A wild-type lymphoblastoid cell line is shown to indicate the position of wild-type DCPS. expressing homozygous DCPSIns15 will enable us to uncover yet to be determined. An alternative explanation is that, while the underlying molecular defect attributed to expression of this low levels of mRNA decapping are important in all tissues, neural variant protein. development and/or function may be more sensitive to disrup- In an effort to generate a Dcps null mouse, we found that Dcps tion of decapping activity than other tissues. Further, we con- is an essential gene and its deletion is embryonic lethal (M. Kiled- sider it unlikely that fully null homozygous mutations in DCPS jian, unpublished observations), demonstrating a role for Dcps will be identified in a clinical context and that bi-allelic muta- protein in embryogenesis. CRISPR screens of human genes also tions would always be hypomorphic or with restricted effect on indicate the requirement of DCPS for cellular viability (30), under- specific isoforms or functions of the protein. lining the importance of this protein in fundamental cellular EDC3 interacts with and stimulates DCP2 (31). The identified functions. The viability of individuals with a decapping-compro- alteration in EDC3 is situated in the interacting LSm domain mised DCPS suggests that the decapping activity of DCPS is only and, thus, possibly disturbs this interaction. Our functional ana- important for normal cognition, while an additional decapping- lyses showed that the altered EDC3 does not enhance DCP2 activ- independent function of DCPS may also be required for normal ity and at high concentration even inhibits DCP2 activity. development and survival. However, this additional function is Therefore, EDC3F54S is compromised in its ability to stimulate Human Molecular Genetics, 2015, Vol. 24, No. 11 | 3177

A. Nevertheless, the phenotypes of the patients with variants in EDC3 and in DCPS are rather similar, although DCPS and EDC3 are involved in different pathways of RNA decapping and at different stages of RNA degradation. The role of both genes in mRNA degradation and thus regulation of gene expression suggests DCPS and EDC3 as excellent candidate genes for a neurodevelopmental phenotype. Regulation of gene expression is crucial for neuronal development and function.

As an example, this has been shown in mutations in the transcrip- Downloaded from tion factor TAF2 (32). Further regulators of mRNA stability have been implicated in cognitive function. Mutations in VCX3A(MIM 300533), a gene associated with the inhibition of mRNA decapping (33), were reported to segregate with X-linked non-syndromic intellectual disability (34). Although a more recent study of X-linked

ichthyosis [XLI (MIM 308100)] and intellectual disability suggests http://hmg.oxfordjournals.org/ that deletion of VCX3A alone is insufﬁcient to result in intellectual disability and may involve a dosage compensation of VCX paralogs (35), VCX3A is involved in neuritogenesis and is an inhibitor of DCP2 decapping (33). Consistent with a role for decapping regulation in neuronal function, enhanced mRNA decapping induced by silencing of VCX3A results in decreased primary and secondary neurite projections, while exogenous expression of VCX3A in rat primary hippocampal neurons promotes neurite

arborization (33). A role for proper mRNA turnover in cognitive at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 function is further evident in individuals with mutations in the non-sense-mediated mRNA decay pathway gene, UPF3B (MIM 300298), a protein involved in mRNA quality control (36). Although at present we only have identified disruptions in either DCPS or EDC3 in two families, the presence of compound heterozygous variants in DCPS in an affected person in Family A supports a role for DCPS as the causative agent for intellectual disability in these individuals. Moreover, the identification of a second family, of Jordanian origin, with intellectual disability caused by a homozygous mutation in DCPS, leading to truncation of a major isoform and missense change in a minor isoform, re- inforces the significance of DCPS for neuronal function (C. Ng Figure 4. Functional analysis of EDC3 variant. EDC3-DCP2 decapping assay. Wild- et al., this issue). The presence of obvious physical anomalies type EDC3 enhances DCP2-mediated decapping in vitro, while a similar and neuromuscular phenotype in the Jordanian family suggests enhancement was not observed with the Phe54Ser variant (EDC3F54S), and that a relatively wide clinical spectrum can be anticipated for dif- inhibition of decapping was detected at the highest concentration tested. fi Decapping assays were carried out with 32P-cap-labeled RNA with 10 n DCP2 ferent types and locations of mutations in DCPS. Future identi - and the indicated amounts of EDC3 proteins as previously described (24). (A)A cation of additional individuals with intellectual disability and representative decapping assay is shown and labeled as in the legend to disruptions in DCPS, EDC3 or other components of the decapping Figure 3A. A schematic of the substrate with the m7Gppp denoting the cap and machinery would further substantiate a role for these genes in the line representing the RNA is shown at the bottom. (B) The average of three cognitive ability. independent decapping assays in the presence of the different EDC3 proteins is In conclusion, we report the investigation of two families with shown with error bars representing ±standard deviation. apparent non-specific autosomal recessive intellectual disability, leading to the identification of mutations in DCPS and EDC3. DCP2 decapping in the affected individuals, leading to aberrant Both genes are involved in RNA metabolism and function in either accumulation of a subset of mRNAs. This may have adverse mRNA scavenger decapping or mRNA decapping. At present, the consequences on normal neural function, and this dysregulation molecular consequence of defects in these genes is not known, of mRNA is a possible cause of intellectual disability in Family B. yet they share a physiological consequence of adverse overall cog- The individuals examined in this report have intellectual dis- nition in individuals harboring the mutant alleles. Future identifi- ability as a common phenotype. Nevertheless, there are some cation of the downstream target mRNAs or proteins altered in the significant differences in further details. Individual IV-7 of Family cells of affected individuals will provide insights into the contribu- A with the compound heterozygote DCPS mutation also presents tion of DCPS and EDC3 in gene expression and cognitive function. with a neuromuscular phenotype with extremely delayed motor development. Although this may be a result of the different genotype, it is also possible that prematurity (birth in the 28th Materials and Methods pregnancy week) is a contributory factor for this phenotypic Patient ascertainment and clinical information variability. It cannot be excluded that a variant in an intronic or regulatory region at DCPS, or an independent mutation in This study was approved by the Ethics Committees of the Univer- another gene may also play a role in this individual. sities of Bonn and Erlangen-Nürnberg in Germany, the Centre for The individuals with EDC3 mutations in Family B present with Addiction and Mental Health in Toronto, Canada, and the a milder form of intellectual disability than individuals in Family National University of Science and Technology, Islamabad, 3178 | Human Molecular Genetics, 2015, Vol. 24, No. 11

Pakistan. Informed written consent was obtained from all exam- sequencing was used to conﬁrm a change in splice donor usage ined persons before obtaining blood samples. Blood was drawn at exon 4/intron 4 for the splice variant allele. for genetic studies, and genomic DNA was extracted by standard methods. Neurological and medical assessment, as well as as- Molecular modeling for the mutations in DCPS and EDC3 sessment of the level of ID, was performed on affected members by a team of experienced clinicians. For Family A, this involved Modeling of the mutations was based on the Dcps crystal struc- assessment by a clinical psychologist experienced in assessment ture (38). The p.T316M mutation was generated with SwissModel of intellectual disability in this culture (T.N.) using the Vineland (39), and the p.Q212_L213Ins15 mutation was modeled with Mod-

Adaptive Behaviour Scale (second edition) (37). Photographs of Loop (40). RasMol (41) was used for structure analysis and visual- Downloaded from family members were assessed by an experienced clinical geneti- ization. ClustalW2 comparison of DCPS Thr316 across species cist for dysmorphic features. shows conservation through most vertebrates, also to archaeo- chordate Branchiostoma ﬂoridae (lancelet), also fruit ﬂy and nema- Homozygosity-by-descent mapping tode (see Supplementary Material). Molecular modeling using GeneSilico fold recognition meta-server (42) and Modeler9.9 (22)

For Family A, SNP microarrays (Affymetrix 500K NspI, Santa http://hmg.oxfordjournals.org/ using the closest related hydrolase (PDB code: 3LP5) as template Clara, CA, USA) were run for affected individuals III-2 and IV-3, highlighted the detrimental effect of the exchange at position and an unaffected individual III-3. Microarray genotyping was 54 on the structure of EDC3 (Fig. 3). Phenylalanine 54 is located performed as a service by the London Regional Genome Centre within the LSm domain and is highly conserved across evolution. (www.lrgc.ca). Genotypes were analyzed for HBD regions using ClustalW2 comparison of EDC3 54Phe across species shows it to dCHIP software (14) and HomozygosityMapper (15). Microsatellite be present across vertebrates all the way to fish, but not including markers were used to corroborate autozygosity, and the 11.3 Mb lancelet (see Supplementary Material). The amino acid substitu- region on 11q24.1-q25 was the sole locus not excluded using this tion at position p.54 leads to a disruption of the domain structure approach. due to a poorly packed hydrophobic core. Energetic calculations For Family B, SNP microarrays (Affymetrix SNP Chip 6.0, Santa indicate a loss of 4–5 kcal/mol of stabilization energy, suggesting at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 15, 2016 Clara, CA, USA) were run for all affected and their healthy sib- that the domain might entirely unfold upon Phe54Ser mutation. lings. HomozygosityMapper (15) was used for homozygosity Since the LSm domain is crucial for the interaction between EDC3 mapping. and DCP2, we anticipate that this variant will significantly impair protein function. WES and targeted sequencing For Family A, WES was performed using the SOLiD 4 (Life Tech- DCPS mRNA decapping assay nologies, Carlsbad, CA, USA) platform and SureSelect Human All Exon 50 Mb (Agilent technologies, Santa Clara, CA, USA) The DCPS gene was amplified from affected and unaffected DNA capture array, according to the manufacturers’ recom- individuals from Family A by RT–PCR. PCR products were purified mended protocols. Details on the sequencing procedures and after gel electrophoresis followed by elution from the gel using bioinformatics processing are described elsewhere (16). Sanger the QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands). WT sequencing was used to confirm a homozygous variant identified The full-length wild-type (DCPS ), and mutant (splice mutation/ Ins15 T316M as a potential splice site mutation in DCPS and its segregation in 45 bp insertion: DCPS ) and Thr316Met (DCPS ) DCPS the family. Both III-2 and IV-3 were homozygous for the variant. cDNAs were initially cloned into the pDRIVE vector (Qiagen) No other homozygotes were identified in the family. Individual using primers 5′-ACCACAACGGGGCCAAAGGCAGTAAC-3′ (for- IV-7 was compound heterozygous for this variant and the ward) and 5′-ACTCCTCCCCCAATCCCACACATCTG-3′ (reverse). Thr316Met variant. PCR amplification of the full-length inserts with primers with For Family B, exome sequencing was performed using DNA BamHI and XhoI restriction sites was performed prior to subclon- from individual IV-2. DNA was enriched using the SureSelect ing into the corresponding restriction enzyme sites within vector Human All Exon 50Mb Kit (Agilent technologies) and paired- pET28a (Novagen, EMD Biosciences, Danvers MA, USA) and con- end sequenced on a SOLiD 4 instrument (Life Technologies). Cus- firmed by sequencing. Recombinant proteins expressed from the tom targeted enrichment was performed using a custom targeted pET28a constructs were generated in Escherichia coli BL21(DE3) capture kit (Agilent technologies) for all coding elements (CDS) cells induced with 0.5 m IPTG and purified according to the in the candidate regions (496 genes) using DNA from individual manufacturer (Novagen, EMD Biosciences), except that 300 m IV-4. The library was then sequenced on a SOLiD 4 platform. urea and 0.5% TritonX-100 was included in the binding buffer. Details on sequencing procedure and bioinformatics processing The nickel column was then washed twice using wash buffer are described elsewhere (21). PCR and Sanger sequencing were with 800 m urea and 0.5% TritonX-100 and once using wash done according to standard protocols to exclude technical buffer without urea and TritonX-100. Protein eluted from the artifacts and to test for segregation. nickel column was dialyzed against PBS and concentrated by Centricon centrifugal filter columns (Amicon, EMD Biosciences, Danvers MA) and stored at −80°C in 10% glycerol. Protein concen- mRNA analysis for DCPS trations were determined by Bradford assay. Generation of la- Whole blood mRNA from members of Family A was extracted beled RNA and cap structures: unlabeled, uncapped RNA from blood collected in Tempus™ RNA tubes (Life Technologies) corresponding to the pcDNA3 polylinker spanning the SP6 and using TRIzol. After first-strand cDNA synthesis using SuperScript T7 promoters (pcP) was generated from SP6 RNA polymerase III Reverse Transcriptase (RT) (Invitrogen, Carlsbad, CA, USA) and transcribed RNA, 32P-cap labeled and cap structure isolated as random hexamers (Fermentas, Waltham, MA, USA), the DCPS previously described (24). DCPS wild-type and mutant proteins gene was amplified by RT–PCR. Primers from exon 2 to 5 were at the indicated concentrations were incubated with 10 fmol of used: 5′-CACTTGTTCCCTCCAAGACAA-3′ (2F), 5′-TAAGGTCGCG- 32P-labeled methylated-capped RNA (32P m7Gp*ppG) substrate TAGGGATCTG-3′ (5R), to generate a 352 bp amplicon. Sanger and decapping products resolved by polyethyleneimine (PEI) Human Molecular Genetics, 2015, Vol. 24, No. 11 | 3179

cellulose thin-layer chromatography (TLC) (Sigma-Aldrich, St Erlanger Leistungsbezogene Anschubsﬁnanzierung und Nach- Louis, MO, USA), as described previously (24). This buffer condi- wuchsförderung (ELAN; 13-05-10-1-Abou Jamra) to R.A.J., and tion separated the input cap structure from the resulting NIH grant GM067005 to M.K. m7GMP generated by the DCPS decapping activity. References DCPS in vitro decapping assays 1. Adjibade, P. and Mazroui, R. (2014) Control of mRNA turnover: Lymphoblast cell lines were established from whole blood from implication of cytoplasmic RNA granules. Semin. Cell Dev. Biol., homozygous affected (III-2 and IV-3) and heterozygous unaffect- 34,15–23. Downloaded from ed (III-3 and IV-2) family members by Epstein-Barr virus trans- 2. Garneau, N.L., Wilusz, J. and Wilusz, C.J. (2007) The highways formation. Decapping assays were carried out with 5 µg total and byways of mRNA decay. Nat. Rev. Mol. Cell Biol., 8, 113–126. cell extract derived from family member lymphoblasts. Proteins 3. Wu, X. and Brewer, G. (2012) The regulation of mRNA stability or extract were incubated with the labeled cap structures in dec- in mammalian cells: 2.0. Gene, 500,10–21.  –   apping buffer (10 m Tris HCl at pH 7.5, 100 m KCl, 2 m MgCl2, 4. Liu, H. and Kiledjian, M. (2006) Decapping the message: a be-

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