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] 3172 Human Molecular Genetics, 2015, Vol. 24, No. 11 | 3173 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×.