OMICS A Journal of Integrative Biology Volume 24, Number 3, 2020 Research Article ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2019.0192

Rett Syndrome, a Neurodevelopmental Disorder, Whole-Transcriptome, and Mitochondrial Genome Multiomics Analyses Identify Novel Variations and Disease Pathways

Mazhor Aldosary,1,* AlBandary Al-Bakheet,1,* Hesham Al-Dhalaan,2 Rawan Almass,1,{ Maysoon Alsagob,1,{ Banan Al-Younes,1,{ Laila AlQuait,1,{ Osama Mufid Mustafa,1,{ Mustafa Bulbul,1 Zuhair Rahbeeni,3 Majid Alfadhel,4 Aziza Chedrawi,2 Zuhair Al-Hassnan,3 Mohammed AlDosari,5 Hamad Al-Zaidan,3 Mohammad A. Al-Muhaizea,2 Moeenaldeen D. AlSayed,3 Mustafa A. Salih,6 Mai AlShammari,1 Muhammad Faizal-Ul-Haque,7 Mohammad Azhar Chishti,8 Olfat Al-Harazi,9 Ali Al-Odaib,1 Namik Kaya,1 and Dilek Colak9

Abstract

Rett syndrome (RTT) is a severe neurodevelopmental disorder reported worldwide in diverse populations. RTT is diagnosed primarily in females, with clinical findings manifesting early in life. Despite the variable rates across populations, RTT has an estimated prevalence of *1 in 10,000 live female births. Among 215 Saudi Arabian patients with neurodevelopmental and autism spectrum disorders, we identified 33 patients with RTT who were subsequently examined by genome-wide transcriptome and mitochondrial genome variations. To the best of our knowledge, this is the first in-depth molecular and multiomics analyses of a large cohort of Saudi RTT cases with a view to informing the underlying mechanisms of this disease that impact many patients and families world- wide. The patients were unrelated, except for 2 affected sisters, and comprised of 25 classic and eight atypical RTT cases. The cases were screened for methyl-CpG binding protein 2 (MECP2), CDKL5, FOXG1, NTNG1, and mitochondrial DNA (mtDNA) variants, as well as copy number variations (CNVs) using a genome-wide ex- perimental strategy. We found that 15 patients (13 classic and two atypical RTT) have MECP2 mutations, 2 of which were novel variants. Two patients had novel FOXG1 and CDKL5 variants (both atypical RTT). Whole mtDNA sequencing of the patients who were MECP2 negative revealed two novel mtDNA variants in two classic RTT patients. Importantly, the whole-transcriptome analysis of our RTT patients’ blood and further comparison with previous expression profiling of brain tissue from patients with RTT revealed 77 significantly Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. dysregulated genes. The gene ontology and interaction network analysis indicated potentially critical roles of MAPK9, NDUFA5, ATR, SMARCA5, RPL23, SRSF3, and mitochondrial dysfunction, oxidative stress response and MAPK signaling pathways in the pathogenesis of RTT genes. This study expands our knowledge on RTT disease networks and pathways as well as presents novel mutations and mtDNA alterations in RTT in a population sample that was not previously studied.

Keywords: Rett syndrome, neurodevelopmental and autism spectrum disorder, mitochondria, copy number variations (CNVs), MECP2, transcriptome

Departments of 1Genetics, 2Neuroscience, and 3Medical Genetics, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia. 4King Abdullah International Medical Research Centre, King Saud bin Abdulaziz University for Health Sciences, Genetics Division, Department of Pediatrics, King Abdullah Specialized Children Hospital, Riyadh, Saudi Arabia. 5Center for Pediatric Neurosciences, Cleveland Clinic, Cleveland, Ohio. 6Division of Pediatric Neurology, College of Medicine, King Saud University, Riyadh, Saudi Arabia. Departments of 7Pathology and 8Biochemistry, King Khalid Hospital, King Saud University, Riyadh, Saudi Arabia. 9Department of Biostatistics, Epidemiology, and Scientific Computing, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. *These authors contributed equally to this work. {These authors contributed equally to this work.

1 2 ALDOSARY ET AL.

Introduction The MECP2 disruption—although presenting more often with the classic RTT phenotype (Amir et al., 1999; Bienvenu irst described by an austrian pediatric neurolo- et al., 2000; Shulyakova et al., 2017)—has also been ob- Fgist >50 years ago, Rett syndrome (RTT; MIM: 312750) served in atypical RTT (Li et al., 2009; Psoni et al., 2012; is a neurodevelopmental disorder with a characteristic pro- Zappella, 1992), and MECP2 duplication syndrome (Sign- gressive course and a near-exclusive female predisposition. orini et al., 2016), and intellectual disability (Muthusamy Despite the variable rates reported, RTT has an estimated et al., 2017; Schonewolf-Greulich et al., 2016). With that, the prevalence of *1 in 10,000 live female births throughout the perplexity of the clinical genetic intersections of the disease world (Laurvick et al., 2006; Neul et al., 2010). become evident, and the compelling need for a better un- Clinically, although patients appear to have normal devel- derstanding of such interactions is necessary. opment in the first few months of life, developmental arrest Among 215 Saudi Arabian patients with neurodevelop- typically ensues at ages between 6 and 18 months, followed by mental and ASDs, we identified 33 unrelated patients, except rapid deterioration of motor and higher mental functioning in for 2 affected sisters, with RTT who were subsequently ex- the first few years of life. During this period, patients classi- amined by genome-wide transcriptome and mitochondrial cally present with acquired hand skill loss, truncal ataxia, genome variations. To the best of our knowledge, this is the and/or gait apraxia. In addition, distinctive purposeless hand- first in-depth molecular and multiomics analyses of a large wringing stereotypies become almost invariably evident dur- cohort of Saudi RTT cases with a view to informing the ing wakefulness. Besides fine and gross motor regression, underlying mechanisms of this disease that impact many RTT patients experience marked language and communica- patients and families worldwide. tion impairment manifested by the loss of verbal communi- cation, eye contact, and social interaction as well as restricted Materials and Methods interest—all of which are hallmarks of autistic disorders. Other manifestations of this syndrome include acquired Patients microcephaly, autonomic nervous system and breathing dys- All patients were examined by a geneticist, a pediatric function, sleeping disturbances, seizures, dementia and mental neurologist, and pediatric psychologist. After clinical evalu- retardation. The rapid deterioration phase tends to plateau at ation, selected patients were recruited during 2003–2019 around the fourth year of life, when improvements in behavior, under the King Faisal Specialist Hospital and Research Centre hand use, and communication are noticed. Typically, there- (KFSHRC) IRB-approved protocol (RAC Nos. 2040024, after, a period of apparent stability lasts for decades despite a 2040042, 2120022, and 2030046) and consented by their legal slow decline in motor functions being apparent. guardians for their participation in the study. This characteristic clinical picture, however, is not always The patients’ recruitment and sample collections for the present—making RTT a diagnostic challenge. To that end, study were done during 15-year period. Therefore, initial di- Hagberg (2002) reviewed and established criteria for RTT agnosis of the patients was based on Hagberg et al.’s (2002) diagnosis. According to the number of primary and supportive criteria; however after the recent adoption of Neul et al.’s criteria met, the disease is labeled as either a classic, atypical, or (2010) criteria, we re-examined all the patients’ diagnoses and mild syndrome with preserved speech variant. More recently, selected the RTT patients according to the recent criteria. In Neul J. and a large, multinational collaborative team have addition, the methods for genetic testing have evolved dra- suggested a revision of Hagberg’s criteria (Neul et al., 2010) matically during the study period. Hence, various different andreclassificationofRTTintoclassic and atypical, the latter of cytogenetic methods were used. which includes preserved speech, early seizure, and congenital The final patient cohort was comprised of 33 RTT patients variants. Due to discrepancy in the reported genetic findings so (25 classic and 8 atypical). All patients, except for two sisters far, their work highlights the notion that RTT is a clinical di- (Patients 32 and 33 in Table 1), were unrelated. Clinical

Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. agnosis independent of the identified molecular underpinnings. descriptions of the selected patients, including cases with Indeed, our understanding of this disease is evolving; al- novel MECP2, CDKL5, FOXG1 mutations and mitochon- though it was once believed to be a pervasive developmental drial DNA (mtDNA) variants, are presented in Supplemen- disorder, RTT is no longer considered a subdiagnosis of au- tary Data. All patients included in this study were females tism spectrum disorder (ASD) in the Diagnostic and Statis- except for Patient 31 (Table 1). For global expression pro- tical Manual of Mental Disorders, Fifth Edition (DSM-V)— filing, three RTT patients with MECP2 mutations (indicated emphasizing its distinct clinical course and genetic links that in Table 1 with asterisks) and age-, ethnicity-, and sex- distinguish it from ASD. It is, in fact, merely during the rapid matched healthy controls were used. The cytogenetic testing deterioration phase that patients may show autistic features was performed for 20 patients (Table 1). meeting the criteria of ASD, which then cease to exist thereafter (American Psychiatric Association, 2013; Cha- pleau et al., 2013; Percy et al., 2010). DNA isolation, polymerase chain reaction, The current understanding of the molecular genetic cor- mutation detection relations of RTT is still progressing. While RTT is classically Five milliliters of whole blood was collected into EDTA known to be caused by mutations in the X-linked methyl- tubes for genomic DNA (nuclear and mitochondrial) isola- CpG binding protein 2 (MECP2) gene, involvement of other tion. The DNA was isolated from the blood using the Pure- genes has also been reported. Gene DNA Purification Kit according to the manufacturer’s For example, CDKL5 and NTNG1 have been shown to be instructions (Gentra Systems, Inc., Minneapolis, MN, USA). culprits in some RTT cases (Archer et al., 2006a, 2006b; Polymerase chain reaction (PCR) was performed according Nawaz et al., 2016; Tao et al., 2004; Weaving et al., 2004). to standard protocols. In brief, 10 ng of genomic DNA was Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only.

Table 1. List of MECP2, CDKL5, FOXG1 Mutations and Mitochondrial DNA Variants Identified in This Study Patients RTT type MECP2 CDKL5 NTNG1 FOXG1 mtDNA References Patient 1 Classic c.763C>T, p.R255X — — — — Amir et al. (1999) Patient 2a,b Classic c.808C>T, p.R270X — — — — Cheadle et al. (2000) Patient 3b Classic c.27-1G>C — — — — This study Patient 4a,b Classic c.806delG, p.G269Afs*20 — — — — Wan et al. (1999) Patient 5b Classic c.1157_1197del, p.L386Hfs*5 — — — — Hoffbuhr et al. (2001) Patient 6b Classic — — — — — Patient 7b Classic — — — — — Patient 8b Classic — — — — MT-RNR2; m.2648T>C This study Patient 9b Classic — — — — MT-TK; m.8346delC This study Patient 10b Classic — — — — — Patient 11a,b Classic c.316C>T, p.R106W — — — — Amir et al. (1999) Patient 12b Classic — — — — — Patient 13b Classic — — — — — Patient 14b Classic — — — — — Patient 15 Classic c.808C>T, p.R270X — — — — Cheadle et al. (2000) Patient 16b Classic — — NA — NA Patient 17b Classic — — NA — NA 3 Patient 18b Classic c.502C>T, p.R168X — — — — Wan et al. (1999) Patient 19 Atypical — c.223T>C,pL75L — — — This study Patient 20b Atypical — — NA — NA Patient 21b Classic — — NA — — Patient 22b Atypical — — NA — NA Patient 23 Atypical — — NA — NA Patient 24b Atypical — — NA — — Patient 25 Atypical c.317G>A, p.R106Q — — — NA Bienvenu et al. (2000) Patient 26 Atypical c.763C>T, p.R255X — — — NA Amir et al. (1999) Patient 27 Classic — — NA — NA Patient 28 Classic c. 916C>T, p.R306C — — — NA Cheadle et al. (2000) Patient 29 Classic c.965C>T, p.P322L — — — NA Huppke et al. (2000) Patient 30 Classic c.880C>T, p.R294X — — — NA Cheadle et al. (2000) Patient 31 Atypical — — — c.578C>Ap.A193D NA This study Patient 32 Classic c.58G>C, p.G20R — — — NA This study Patient 33 Classic c.58G>C, p.G20R — — — NA This study

Numbering based on reference sequence NM_004992.3, NM_003159.2, NM_005249.3 for MECP2, CDKL5, and FOXG1, respectively. aIndicate the patients with transcriptome data. bPatients with cytogenetics test data. —, Indicates the patient is tested for the mutation but found negative; MECP2, methyl-CpG binding protein 2; mtDNA, mitochondrial DNA; MT-RNR2, mitochondrial 16srRNA; MT-TK, mt-tRNALys; NA, not available; RTT, Rett syndrome. 4 ALDOSARY ET AL.

used in the PCR (a final volume of 25 lL). The reaction was guidelines (MRC-Holland, Amsterdam, Holland). Data were run 35 cycles initiated by a 95C for 15 min denaturation step. analyzed using Coffalyser.Net software (MRC-Holland); peak The cycles included brief denaturation step at 95C for heights for each fragment were visually and quantitatively 40 sec, an annealing step at 60C for 40 sec, and an extension compared with those of control samples (four normal controls step at 72C for 40 sec, which was followed by a final ex- and one negative). Deletions were suspected when the peak tension step at 72C for 10 min. List of primer sequences used intensities differed by >30%. To ensure accurate analysis of in this study is given in Supplementary Table S1. suspected deletions of the target region, normalization of peak After that, purified PCR products were directly sequenced areas of each amplification product was performed by nor- on an ABI 3730/373XL DNA Analyzer (Applied Biosystems, malization methods described in the manufacturer’s protocol. Foster City, CA, USA). For the sequencing reaction, BigDye Terminator v3.1 kit (Applied Biosystems) was utilized ac- Array comparative genomic hybridization cording to the manufacturer’s instruction. The reactions were experiments and data analysis run for 1 min at 96C for incubation. Then, the reaction was Human Genome CGH Microarray Kit 244A (Agilent carried out 30 cycles at 95C for 10 sec, 50Cfor0.5sec,and Technologies, Santa Clara, CA, USA) was utilized for array 60C for 2 min. After that the samples were purified with Dye- comparative genomic hybridization (aCGH) experiments ac- Ex protocol before it was run on the capillary sequencer. cording to manufacturer’s instructions. Three micrograms of Collected data from the sequencer were blasted to the NCBI genomic DNA from patients and universal human genomic database and aligned with the reference sequences. Sequen- DNA (Promega, Madison, WI, USA) were used for the ex- cing analysis and contig assembly were performed using periments. Bioprime Labeling Kit (Invitrogen, Inc., Carlsbad, Lasergene-SeqMan version 6.1 (DNA Star, Inc.) software. CA, USA) was used for labeling reference and patients’ DNA separately with Cy3-dUTP and Cy5-dUTP (PerkinElmer, mtDNA mutation screening and sequence Waltham, MA, USA), respectively. Labeled DNA samples analysis of the mtDNA coding region were mixed, purified, and prepared for hybridization (38 h). The extracted genomic DNA as above was used as a tem- The slides were washed and then scanned on the DNA Mi- plate for mtDNA amplification by PCR. Samples with a DNA croarray Scanner (Agilent Technologies). The images were concentration <100 ng/lL were amplified using two rounds of extracted using the manufacturer’s Extraction software. The PCR. Amplification was carried out using 36 overlapping pairs data analyses were performed using CGH Analytics software. of primers with a similar annealing temperature (60C). In addition, they were engineered to be tagged with universal Affymetrix CytoScan 2.7M arrays M13-tails to facilitate sequencing of the PCR products (Sup- Affymetrix Cytogenetics Whole-Genome 2.7M Array plementary Table S1). The M13 forward primer sequence was (Affymetrix, Inc., Santa Clara, CA, USA) provides 2.7 million 5¢-TGTAAAACGACGGCCAGT-3¢, and the reverse primer markers, including probes capable of targeting 400,000 single M13-tag sequence was 5¢-CAGGAAACAGCTATGACC-3¢. nucleotide polymorphisms (SNPs) to enable the detection of The PCRs were performed in total volume of 25 lL containing loss of heterozygosity, uniparental disomy, and regions 1 lL DNA as template at *100 ng/lL concentration, 2.5 lLof identical by descent. The sample preparation and downstream 10 · dNTPs, 2.5 lLof10· PCR buffer, 1 lLof20lMforward workflow was performed according to the manufacturer’s primer, 1 lLof20lM reverse primer, and 0.13 lLofAmpli- guidelines and protocols. Taq Gold DNA polymerase. PCR-graded dH2O was added to make a final reaction volume of 25 lL. Affymetrix CytoScan HD arrays and data analysis PCR cycling conditions for the amplification included an initial denaturation at 95C for 10 min to activate the poly- Affymetrix’s Cytogenetics Whole-Genome CytoScan HD merase followed by 30 cycles at 94C denaturation for 45 sec, arrays (Affymetrix, Inc.) were used to check cytogenetic ab- Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. 58C primer annealing for 45 sec, and 72C primer extension normalities in one patient in the study. The HD arrays consist for 1 min. Finally, amplification was allowed for final ex- of >2 million probes, and interrogate polymorphic and non- tension at 72C for 8 min. In all cases, a positive and negative polymorphic genomic sequences in the tested DNA samples. control, which had no DNA, were included in the PCR am- From sample preparation to data analysis, all the steps in- plification setup. cluding assay preparation, scanning, image processing, geno- Sequencing was carried out as mentioned above. Results typing, as well as preliminary quality control check were done were compared with the corrected Cambridge reference se- according to the manufacturer’s protocols and guidelines. Copy quence (Brandon et al., 2005, Cheadle et al., 2000; Hoffbuhr number variation (CNV) detection was done using Affyme- et al., 2001; Huppke et al., 2000; Wan et al., 1999). All frag- trix’s ‘‘Chromosome Analysis Software’’ based on the soft- ments were sequenced in both forward and reverse directions ware’s default detection settings for high resolution. Previously at least twice for confirmation of any detected variant. Se- reported benign CNVs were excluded from the analysis. quence results were compared with the MITOMAP database (Brandon et al., 2005) and the mtDB—Human Mitochondrial Global gene expression profiling Genome Database (Ingman and Gyllensten, 2006). The PAXgene Blood RNA kit was used to extract RNA from 2.5-mL whole blood collected in PAXgene Blood RNA Multiplex ligation-dependent probe amplification assay tubes (PreAnalytiX, Hombrechtikon, Switzerland) per the SALSA multiplex ligation-dependent probe amplification manufacturer’s instructions from Saudi RTT patients with (MLPA) kit P015 MECP2 and SALSA MLPA kit P189 assays MECP2 mutation (n = 3, one sample performed in duplicate), were performed according to the manufacturer’s protocols and and age- and sex-matched normal controls (n = 8) were used RETT SYNDROME TRANSCRIPTOME AND MITOCHONDRIAL GENOME 5

for global expression profiling using Affymetrix’s Gene- in two patients: (1) m.2648T>C (in the mitochondrial Chip Human Genome U133 Plus 2.0 Arrays following the 16srRNA [MT-RNR2]) (Supplementary Fig. S2) and (2) manufacturer’s instructions and guidelines (Supplementary m.8346delC (deletion of the nucleotide C at position 8346 in Table S2). Microarray data normalization was performed the mt-tRNALys; MT-TK) gene (Supplementary Fig. S3). using GC Robust Multi-array Average (GC-RMA) algorithm The mtDNA changes identified in MECP2-patients were not (Wu and Irizarry, 2004, 2005). present in >120 ethnically matching normal controls and Significantly dysregulated genes in RTT were identified have not been previously reported in the mtDNA databases using Student’s t-test with the adjustment of probability ( p) (Ingman and Gyllensten, 2006; Ruiz-Pesini et al., 2007) values for multiple comparisons by false discovery rate ac- (Table 1). Notably, the mtDNA variations found in our pa- cording to Benjamini–Hochberg step-up procedure (Benjamini tients affect nucleotides that are highly conserved in multiple and Hochberg, 1995). Significantly modulated genes were species (Supplementary Figs. S2 and S3). defined as those with absolute fold change (FC) >2 and ad- justed p-value of <5%. For RTT brain tissue transcriptome Genome-wide gene expression profiling comparison analysis, we used publicly available dataset from We performed analyses on the whole blood genome-wide the Gene Expression Omnibus (GEO; GSE6955) for gene ex- messenger RNA (mRNA) expression profiling of RTT patients pression profile of human postmortem brains of RTT patients with MECP2 mutation and their age-, sex-, and ethnicity- (2–4, 6, and 8 years old) and age-matched normal brains using matched healthy controls using a microarray approach. Com- Affymetrix arrays. parison of transcriptomes of RTT patients with the normal The differentially expressed genes (DEGs) were identified controls revealed significant modulation of 817 probes, corre- as described previously (Ehrhart et al., 2019). The functional sponding to 637 DEGs, majority of which (72%) were down- annotation and gene ontology (GO) enrichment analyses regulated in patients compared with normal individuals with were performed using DAVID Bioinformatics Resources absolute FC >2andadjustedp-value <0.5 (Supplementary (Dennis et al., 2003) using the software’s defaults. The net- Table S3). works and functional analyses were generated through the To obtain a deeper insight into the disease pathogenesis, we use of Ingenuity Pathways Analysis (QIAGEN, Inc.). Right- performed GO enrichment and interaction network analyses by tailed Fisher’s exact test was used to calculate a p-value de- using DAVID Bioinformatics Resources (Dennis et al., 2003) termining the probability that the biological function and the Ingenuity knowledge base. Genes related to RNA post- (or pathway) assigned to that dataset is explained by chance transcriptional modification, immune and inflammatory re- alone based on the functional/pathway annotations stored in sponse (Papini et al., 2014), lipid metabolism (Kyle et al., the Ingenuity Knowledge Base. Statistical analyses were 2018; Pacheco et al., 2017), ribosomal biogenesis (Hetman and conducted with PARTEK Genomics Suite (Partek, Inc., St. Slomnicki, 2019), and protein synthesis and proteolysis (Pe- Louis, MO, USA). All statistical tests were two sided, and p- corelli et al., 2013) were significantly dysregulated in RTT value <0.05 was considered statistically significant. patients (Fig. 1A and Supplementary Tables S4 and S5). Significant canonical pathways include EIF2 signaling Results (Ehrhart et al., 2019), mitochondrial dysfunction (Colak Mutation screening and copy number changes et al., 2011; Shovlin and Tropea, 2018; Shulyakova et al., 2017; Valenti et al., 2014), oxidative phosphorylation (Shu- Of 215 Saudi patients, 33 were diagnosed as RTT lyakova et al., 2017; Valenti et al., 2014), and glucocorticoid (25 classic and 8 atypical) and were included in this study. receptor signaling (Braun et al., 2012; Nuber et al., 2005) Notably, 15 patients (13 classic and 2 atypical) were found to (Fig. 1B and Table 2). The gene interaction network analyses have MECP2 mutations, 2 of which are novel variants (c.27– indicated the alterations of mitochondrial genes and path- 1G>C and c.58G>C) (Table 1 and Supplementary Fig. S1). ways in the downstream of MECP2 (Fig. 1C). Significantly Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. The remaining 10 classic and 4 atypical RTT patients did not altered Kyoto Encyclopedia of Genes and Genomes (KEGG) have any mutation in any of the tested genes. No apparent hot pathways in RTT included those for ribosome, oxidative spots were detected in our study; however, 12 patients had phosphorylation, Alzheimer’s disease, Parkinson’s disease, previously reported mutations (Table 1). and Huntington’s disease (Table 3). No mutations in NTNG1 were detected in any of the patients. Only one atypical RTT patient had a novel FOXG1 variant Identification of significant genes in RTT patients’ (c.578C>A), and another atypical RTT case had a novel CDKL5 blood and brain tissues variant (c.223T>C). Subsequently, MLPA assays that are de- signed to identify gross changes in MECP2, NTNG1, CDKL5, We further compared the significantly expressed genes in and ARX genes and high-density genome-wide oligoarrays transcriptome of our RTT patients with the DEGs from the (aCGH arrays from Agilent Inc., GeneChip Genome-Wide previously published study on brain of RTT patients due to Cytogenetic Human 2.7M and Cyto HD Arrays) were per- MeCP2 mutations (Deng et al., 2007; Ehrhart et al., 2019). formed. These assays did not reveal any gross abnormalities or The dataset included whole-genome gene expression profile pathogenic copy variations. All patients included in this study of postmortem brains affected by RTT and that of age- were females except for one male who had the FOXG1 variant. matched normal brains in three age groups spanning the first 10 years of postnatal life (Deng et al., 2007). The comparison of our DEGs with those from the brain of RTT patients re- Sequence analysis of mtDNA vealed 77 significantly regulated genes in RTT brain and Full mtDNA sequencing of the patients (selected classic blood (adjusted p-value <0.05 and FC >2) (Supplementary RTT patients) revealed two novel mtDNA changes (Table 1) Table S6). The GO and interaction network analysis indicated Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. 6

FIG. 1. Transcriptional changes associated with RTT. (A) Over-represented GO categories and biological functions, and (B) significantly altered canonical pathways associated with DEG (up- or downregulated) in RTT patients. X-axis indicates the significance (-log p value) of the functional/pathway association that is dependent on the number of genes in a class as well as biological relevance. The threshold line represents a p value of 0.05. (C) Top scoring gene interaction network of DEGs in our RTT patients and (D) significant subnetwork that is associated with DEGs in individuals with both blood and brain tissues of RTT patients. The significant canonical pathways are indicated. Nodes represent genes, with their shape representing the functional class of the gene product, and edges indicate biological relationship between the nodes (see legend). Green (red) indicates down- (up-) regulated, in RTT compared with controls. The color intensity is correlated with fold change. CP, canonical pathway; DEGs, differentially expressed genes; GO, gene ontology; RTT, Rett syndrome. RETT SYNDROME TRANSCRIPTOME AND MITOCHONDRIAL GENOME 7

Table 2. Canonical Pathways That Are Significantly Dysregulated in Rett Syndrome Patients Canonical pathways p-value Genes EIF2 signaling 5.01E-08 PIK3C2A, RPS3A, RPL36A, RPL34, RPL22L1, RPL17, RPS21, RPL26, RPL23, EIF3E, EIF2S1, RPL7, RPL9, RPS7, RPL35, RPS27L, EIF3A, RPS17, RPL31, RPS24, ATM Mitochondrial dysfunction 1.58E-06 NDUFA4, ATP5J, COX7B, CASP3, COX6C, ATP5O, COX7C, MAPK9, NDUFB1, GPX7, UQCRB, ATP5C1, NDUFA5, SOD2, SNCA, ATP5I, UQCRQ, PSEN1 Oxidative phosphorylation 5.01E-05 ATP5J, NDUFA4, ATP5C1, NDUFA5, COX7B, COX6C, COX7C, ATP5O, NDUFB1ATP5I, UQCRQ, UQCRB Glucocorticoid receptor 7.9E-04 TAF9, TGFBR1, PIK3C2A, MAPK9, POLR2K, JAK2, IL1R2, FOS, signaling DUSP1, SMARCA2, ANXA1, CDKN1A, HSP90AA1, IL1B, STAT5B, TAF15, ATM, CREBZF Regulation of eIF4 and 7.9E-04 RPS7, RPS3A, PIK3C2A, RPS27L, EIF3A, RPS21, EIF3E, EIF2S1, RPS17, p70S6K signaling ITGA4, ATM, RPS24 Natural killer cell signaling 0.001 KIR3DL1, SH2D1A, KLRC4-LRK1/KLRK1, PIK3C2A, FCGR2A, KLRB1, NCR3, FCGR3A/FCGR3B, LCP2, ATM Systemic lupus erythematosus 0.0025 FOS, SNRPN, PIK3C2A, FCGR2A, HNRNPA2B1, IL1B, PRPF40A, IGL, signaling SNRPE, ZCRB1, SNRPD2, FCGR2B, FCGR3A/FCGR3B, ATM Role of NFAT in regulation 0.0032 RCAN1, FOS, PIK3C2A, FCGR2A, HLA-DQA1, FCER1A, IKBKAP, of the immune response HLA-DQB1, FCGR2B, FCGR3A/FCGR3B, LCP2, ATM Protein ubiquitination 0.0126 PSMA6, DNAJC10, USP1, DNAJA1, UBE2J1, UBE2B, PSMC6, USP47, pathway USP16, PSMA4, HSP90AA1, PSMA3, AMFR, USP25

potentially critical roles of MECP2, MAPK9, ATR, SMARCA5, classic and atypical RTT cases who were screened for MECP2, SRSF3, and mitochondrial dysfunction, oxidative stress re- CDKL5, FOXG1,andNTNG1 mutations, mtDNA variants, and sponse and MAPK signaling pathways in the pathogenesis of gross chromosomal abnormalities. In addition, we performed RTT (Supplementary Table S6 and Fig. 1D). genome-wide gene expression study on the selected cases. The RTT cohort included classic (n = 26) and atypical RTT (n = 7) patients who were diagnosed according to most recent diag- Discussion nostic criteria (Neul et al., 2010). RTT and its underlying mechanisms still remain poorly Recently, various genetic studies of RTT were performed understood. Clinically available new diagnostics with robust in different ethnic groups, and differing results were reported forecasting performance calls for multiomics research of the (Das et al., 2013; Hettiarachchi et al., 2019; Nasiri et al., 2019; underlying pathophysiological mechanisms. We report here Zengin-Akkus et al., 2018). For example, a recent study on original findings in this context using both whole-transcriptome Indian RTT patients evaluated 90 patients and pinpointed 19 and mitochondrial genome analyses and importantly, in a different MECP2 mutations, some of which were identified in population sample that was previously not studied. We report more than one patient (Das et al., 2013). In the same cohort, 62 here the first multiomics analysis of a large cohort of Saudi (15 classic and 47 atypical) individuals did not have any Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. Table 3. KEGG Pathway Terms That Are Enriched in Rett Syndrome Versus Control Differentially Expressed Genes

Gene KEGG pathway count % p-value Genes Ribosome 18 2.85 2.60E-09 RPL17, RPL36A, RPL35, RPL26, RPS27L, RPL22L1, RPS7, MRPL13, RPL7, RPL23, RPS3A, RPS17, RPL31, RPL34, RPL9, RSL24D1, RPS21, RPS24 Parkinson’s disease 14 2.22 3.24E-04 NDUFA4, NDUFA5, SNCA, COX7B, UBE2J1, COX7C, UQCRQ, NDUFB1, COX6C, CASP3, ATP5C1, ATP5O, UQCRB, ATP5J Alzheimer’s disease 16 2.54 3.35E-04 NDUFA4, NDUFA5, SNCA, COX7B, COX7C, MME, UQCRQ, NDUFB1, COX6C, CASP3, PSEN1, ATP5C1, IL1B, ATP5O, UQCRB, ATP5J Oxidative 13 2.06 0.001 NDUFA4, NDUFA5, COX7B, COX7C, UQCRQ, NDUFB1, PPA1, phosphorylation COX6C, ATP5C1, ATP5O, ATP5I, UQCRB, ATP5J Huntington’s 15 2.38 0.003 NDUFA4, NDUFA5, POLR2K, COX7B, COX7C, CREB5, UQCRQ, disease NDUFB1, SOD2, COX6C, CASP3, ATP5C1, ATP5O, UQCRB, ATP5J Spliceosome 12 1.90 0.003 SFRS7, SF3B1, TCERG1, SR140, PQBP1, SLU7, SNRPD2, SNRPE, HNRNPU, RBM25, SFRS3, PRPF40A 8 ALDOSARY ET AL.

MECP2 mutation (Das et al., 2013). Interestingly, more hits in phrenia (Takahashi et al., 2010), autism and intellectual MECP2 were found in a study focused on Sri Lankan classic disability (Al-Owain et al., 2013; Diaz-Beltran et al., 2016; RTT patients (Hettiarachchi et al., 2019). Of 16 cases studied, Hu et al., 2009; Kong et al., 2012, 2013; Muthusamy et al., 8 patients had two common mutations (p. R168X and 2017), and RTT (Colak et al., 2011; Papini et al., 2014; p.T158M) in addition to another 4 patients carrying p.R255X Sanfeliu et al., 2019) in otherwise difficult-to-obtain target and p.R133C mutations. tissue (i.e., brain), especially with the ethical issues sur- Rest of the cohort had single hit in MECP2, and interestingly rounding sampling of such tissues. Hence, we performed all the patients carried previously reported MECP2 mutations gene expression profiling experiments on the blood samples (Hettiarachchi et al., 2019). A recent study focusing on Iranian of selected classic RTT patients versus those of age–sex classic and atypical patients (n = 24) identified an MECP2 matching controls. mutation in 54% of the patients (9 classic and 4 atypical) with Furthermore, we identified 77 overlapping genes between 10 different MECP2 mutations (Nasiri et al., 2019). Similarly, brain and blood of RTT patients due to MeCP2 mutations MECP2 mutation was detected in 28 Greek classic RTT pa- using previously published study on gene expression profiling tients (n = 40). Among them three common RTT mutations of postmortem brain of RTT patients. Significantly dysregu- (p.R106W, p.R133C, and p.T158M) were found in 12 pa- lated genes as well as their networks of interaction may harbor tients. Another common mutation (p.R306C) was also identi- valuable information regarding the disease pathogenesis (Al- fied in three patients. In the same cohort, an MECP2 mutation Harazi et al., 2016, 2019; Kori et al., 2019; Podder and Latha, was detected in seven atypical RTT patients (n = 34). 2014). The network analysis of RTT-associated genes indi- Low and high frequencies of MECP2 mutations in classic cated potentially important roles of genes involved in mito- and atypical RTT patients in various ethnic groups have been chondrial dysfunction and oxidative stress response, including reported to date. On the one hand, only 7 of 18 (39%) RTT NDUFA5, MAPK9, ATP5PO, MAF, FOS, and SAPK in the patients in New Zealand were found to have MECP2 muta- pathogenesis of the RTT. Indeed, recent studies revealed mi- tions (Raizis et al., 2009); on the other hand, MECP2 muta- tochondrial dysfunction on the examined RTT transcriptomes tions were identified in *88% of Chinese RTT cases (Colak et al., 2011; Shovlin and Tropea, 2018; Shulyakova (n = 121) (Li et al., 2007b). Even higher frequency rates were et al., 2017; Valenti et al., 2014). also reported due to exclusion of MECP2-negative cases Functional and pathway analyses also revealed that the (Zengin-Akkus et al., 2018). One of the main reasons con- DEGs were involved in mitochondrial dysfunction pathways tributing to such variation is perhaps due to the patient se- (Shovlin and Tropea, 2018; Shulyakova et al., 2017), oxi- lection and application of differing diagnostic criteria. In our dative phosphorylation (Shulyakova et al., 2017; Valenti cohort of 33 Saudi RTT patients, we detected MECP2 mu- et al., 2014), metabolic processes (Hayek et al., 2017; Kyle tations in 15 patients (13 classic and 2 atypical), and 2 of the et al., 2018; Sanfeliu et al., 2019), and immune and inflam- variants are novel (c.27–1G>C and c.58G>C). matory response (De Felice et al., 2016; Papini et al., 2014; Interestingly, despite its documented role in RTT patho- Sanfeliu et al., 2019), EIF2 signaling (Ehrhart et al., 2019), genesis particularly in atypical form (Ariani et al., 2008; Mari ribosome and mTOR signaling pathways (Olson et al., 2018). et al., 2005), CDKL5 and FOXG1 mutations remain relatively Several recent studies highlighted that RTT brain and blood rare. For example, in the same Chinese cohort, only a sin- display deregulation of protein synthesis, ribosomal protein, gleton was found to be positive out of 121 RTT patients and mTOR signaling pathways (Olson et al., 2018; Pecorelli (Li et al., 2007b). Similarly, Evans et al. (2005) screened 94 et al., 2013; Ricciardi et al., 2011). patients with RTT or RTT-like syndrome and found only 3 To further investigate the role of other genes/pathways in females with mutations in CDKL5. Similar to our study, a the pathogenesis of RTT, particularly among the MECP2- large Hungarian cohort was screened for MECP2, CDKL5, negative cases, we raised the possibility of the involvement of and FOXG1 mutations (Hadzsiev et al., 2011). the mtDNA, as the results from previous studies on fibroblast

Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. While only 42 patients (27.6%) were identified carrying an and muscle samples from patients with RTT indicated mito- MECP2 mutation, only 2 CDKL5 and no FOXG1 mutations chondrial abnormalities in number and size (Cardaioli et al., were detected in the remaining individuals (Hadzsiev et al., 1999). Indeed, our full mitochondrial genome sequence of the 2011). Similarly, screening of Japanese atypical cases for DNA samples from MECP2-negative patients revealed two FOXG1 revealed only two positive hits (Takahashi et al., novel mitochondrial changes in the MT-RNR2 and the MT-TK 2012). Indeed, our mutation screening revealed only a novel genes (m.2648T>C and m.8346delC). The changes affect the FOXG1 and another unreported CDKL5 variant in our RTT highly conserved nucleotides based on the genomic sequences patient cohort. Role of these variants in RTT pathogenesis of different species (Supplementary Figs. S2 and S3). needs further investigations. Previous reports identified mtDNA changes in several There is accumulating evidence indicating similarities and diseases of central nervous system and in RTT (Lewis et al., shared mechanisms between nerve and blood vessel wiring 1994; Pinto and Moraes, 2014; Shulyakova et al., 2017). and function (Carmeliet and Tessier-Lavigne, 2005; Diaz- Several mtDNA mutations have been identified in the (MT- Beltran et al., 2016; Eichmann et al., 2005; Sanfeliu et al., TK) gene causing myoclonus epilepsy with ragged-red fibers 2019). Such parallelism and resemblance were recently em- syndrome (MERRF) (Arenas et al., 1999), diabetes (Ka- ployed for genome-wide gene expression studies of neuro- meoka et al., 1998), mitochondrial myopathy, encephalopa- logical and psychiatric diseases (Coimbra et al., 2006; Kurian thy, lactic acidosis, and stroke-like episodes (MELAS) et al., 2009; Sanfeliu et al., 2019; Saris et al., 2009; Sullivan (Sakuta et al., 2002), epilepsy (Ahadi et al., 2008), and car- et al., 2006; Tang et al., 2005). diomyopathy (Akita et al., 2000). Similar approaches were also strategically utilized to in- In addition, other mtDNA changes have been described in vestigate complex neuropsychiatric diseases such as schizo- the MT-RNR2 causing migraine, cyclic vomiting syndrome RETT SYNDROME TRANSCRIPTOME AND MITOCHONDRIAL GENOME 9

(Boles et al., 2009), MELAS (Hsieh et al., 2001; Li et al., cases recruited over a decade from a population who was not 2007a), myopathy (Coulbault et al., 2007), Alzheimer’s and studied before and reports novel molecular findings. Indeed, Parkinson’s diseases (Li et al., 2019; Shoffner et al., 1993). the novel mutations, mtDNA variants, and mitochondrial This confirms the involvement of these genes in the pathoge- pathways in RTT patients that we have provided in this study nicity of different clinical phenotypes, which may strengthen have potential to be important for the disease pathogenesis that their potential involvement in RTT as well. may impact many patients and families worldwide. Indeed, MeCP2-308 mouse model of RTT was used to investigate the pathogenic role of mitochondria in RTT Conclusions (De Filippis et al., 2012). Results revealed a defect in respi- The study adds to a growing body of knowledge on RTT, and ratory chain complex II activity, which subsequently caused might inform and stimulate research and discoveries on neu- an overproduction of hydrogen peroxides, decreased mem- rodevelopmental and ASDs broadly. We report novel MECP2, brane potential, and decreased ATP production in the brains FOXG1, and CDKL5 variants, and novel mtDNA variants in a of RTT mice (De Filippis et al., 2015), hence providing clear large cohort of Saudi patients with RTT as well as gene net- evidence for mitochondrial involvement in RTT mice. works and pathways associated with RTT. Taken together, this Our findings revealed novel mtDNA variants in RTT pa- work offers new insights on genotype–phenotype associations tients. This raises the possibility of speculating that MT- and calls for further studies of RTT in the light of mitochondrial RNR2 and MT-TK may be hot spots for mitochondrial involvement, particularly in MECP2-negative patients. changes in RTT patients. The mitochondrial mutations de- scribed here may play a potential role in the pathogenesis of Web Sources RTT, as other mutations caused morphological impairment of mitochondria in RTT patients (Cardaioli et al., 1999). www.mitomap.org/MITOMAP Moreover, our transcriptome analysis of RTT revealed www.mtdb.igp.uu.se/ involvement of mitochondrial dysfunction and oxidative https://blast.ncbi.nlm.nih.gov/Blast.cgi phosphorylation. Several other studies also linked oxidative https://david.ncifcrf.gov stress and mitochondrial dysfunction to RTT (Colak et al., www.ncbi.nlm.nih.gov/search 2011; Filosa et al., 2015; Shovlin and Tropea, 2018; Shu- http://dgv.tcag.ca/dgv/app/home lyakova et al., 2017; Valenti et al., 2014). However, in- www.genecards.org volvement of the novel mtDNA variants in RTT is still http://asia.ensembl.org/index.html questionable since it lacks functional evidence; hence, such https://genome-euro.ucsc.edu/index.html involvement requires further investigation and confirmation. The variants identified in the investigated MECP2-patients Acknowledgments may play a role in the RTT clinical phenotypes by affecting the We thank the patients and their family members for their mitochondrial proteins’ translation, therefore impacting pro- participation in this study. We express our gratitude to Prof. tein synthesis. Given that parts of the ribosome bind to transfer Dr. Pinar T. Ozand (deceased) and Dr. Michael Nester (re- RNAs (tRNAs), mRNAs, in addition to initiation, elongation, tired) for their generous contributions to this study. We also and termination sites (Dahlberg, 1989; Noller, 1993; Wool thank Ibrahim Hamza Kaya, College of Medicine, Alfaisal et al., 1990), one would expect that mutations in any of the University for carefully editing the article. RNA genes subsequently would affect the pathway of protein synthesis. Moreover, we cannot exclude the role of mito- Author Disclosure Statement chondria in the RTT pathogenesis through defective respira- The authors declare they have no conflicting financial tory chain complex activity, free radical production, or interests. deficiency in the mitochondrial ATP production, which was

Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. indeed observed in an RTT animal model (De Filippis et al., Funding Information 2015), or mainly implicated in the pathogenicity of other neurological diseases (Breuer et al., 2013; Sai et al., 2012). This research was conducted through funds provided by King The limitation of the study is that since it has been carried out Faisal Specialist Hospital and Research Centre (KFSHRC Re- for a long period of time (since early 2000), we have used search Advisory Council Projects No. 2040024; No. 2110006), various different methods for cytogenetic testing. For example, King Salman Centre for Disability Research (Grant No. 02-R- the first recruited cases were collected during the early 2000s 0029-NE-02-AU-1 to N.K.; Grant No. 2180 004 to N.K.), and and high-density array CGH, SNP arrays, and related micro- National Plan for Science, Technology and Innovation program array testing were not available at that time as diagnostic tools. (NSTIP/KACST) (Grants 2110006 to D.C. and 14-MED2007- Therefore, some of these cases were mostly screened for 20 to N.K.). M.A.S. was supported by the Researchers Sup- MECP2 mutations using Sanger Sequencing and screened either porting Project number (RSP-2019/38), King Saud University, using MLPA or different types of low-density arrays. However, Riyadh, Saudi Arabia. The funders had no role in study design, the cases recruited later on were analyzed with HD arrays. data collection and analysis, decision to publish, or preparation Moreover, we could not have samples from some of the of the article. earlier cases to perform further experiments particularly for Supplementary Material certain genetic testing, such as gene expression studies and microarray studies for chromosomal abnormalities. Therefore, Supplementary Data we were able to perform cytogenetic testing for 20 patients Supplementary Figure S1 (of 33). However, that being said, this study is the first in-depth Supplementary Figure S2 molecular and multiomics analyses of a large cohort of RTT Supplementary Figure S3 10 ALDOSARY ET AL.

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Downloaded by 37.224.16.109 from www.liebertpub.com at 03/01/20. For personal use only. blood-based gene expression signatures. Schizophr Res 119, CT ¼ computed tomography 210–218. DEGs ¼ differentially expressed genes Takahashi S, Matsumoto N, Okayama A, et al. (2012). FOXG1 EEG ¼ electroencephalogram mutations in Japanese patients with the congenital variant of FC ¼ fold change Rett syndrome. Clin Genet 82, 569–573. GO ¼ gene ontology Tang Y, Gilbert DL, Glauser TA, Hershey AD, and Sharp FR. KEGG ¼ Kyoto Encyclopedia of Genes and Genomes (2005). Blood gene expression profiling of neurologic dis- MECP2 ¼ methyl-CpG binding protein 2 eases: A pilot microarray study. Arch Neurol 62, 210–215. MELAS ¼ mitochondrial myopathy, encephalopathy, Tao J, Van Esch H, Hagedorn-Greiwe M, et al. (2004). Muta- lactic acidosis, and stroke-like episodes tions in the X-linked cyclin-dependent kinase-like 5 (CDKL5/ MLPA ¼ multiplex ligation-dependent probe STK9) gene are associated with severe neurodevelopmental amplification retardation. Am J Hum Genet 75, 1149–1154. MRI ¼ magnetic resonance imaging Valenti D, De Bari L, De Filippis B, Henrion-Caude A, and mRNA ¼ messenger RNA Vacca RA. (2014). Mitochondrial dysfunction as a central mtDNA ¼ mitochondrial DNA actor in intellectual disability-related diseases: An overview MT-RNR2 ¼ mitochondrial 16srRNA of Down syndrome, autism, Fragile X and Rett syndrome. MT-TK ¼ mt-tRNALys Neurosci Biobehav Rev 46(Pt 2), 202–217. PCR ¼ polymerase chain reaction Wan M, Lee SS, Zhang X, et al. (1999). Rett syndrome and RTT ¼ Rett syndrome beyond: Recurrent spontaneous and familial MECP2 muta- SNP ¼ single nucleotide polymorphism tions at CpG hotspots. Am J Hum Genet 65, 1520–1529.