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NUDT21-spanning CNVs lead to neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation

  1. Vincenzo A Gennarino
  2. Callison E Alcott
  3. Chun-An Chen
  4. Arindam Chaudhury
  5. Madelyn A Gillentine
  6. Jill A Rosenfeld
  7. Sumit Parikh
  8. James W Wheless
  9. Elizabeth R Roeder
  10. Dafne DG Horovitz
  11. Erin K Roney
  12. Janice L Smith
  13. Sau W Cheung
  14. Wei Li
  15. Joel R Neilson
  16. Christian P Schaaf  Is a corresponding author
  17. Huda Y Zoghbi  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Texas Children's Hospital, United States
  3. Cleveland Clinic Children's Hospital, United States
  4. University of Tennessee Health Science Center, United States
  5. Instituto Nacional de Saude da Mulher, da Criança e do Adolescente Fernandes Figueira, Brazil
  6. Howard Hughes Medical Institute, Baylor College of Medicine, United States
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Cite this article as: eLife 2015;4:e10782 doi: 10.7554/eLife.10782

Abstract

The brain is sensitive to the dose of MeCP2 such that small fluctuations in protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2 levels to brain function, little is known about its regulation. In this study, we report eleven individuals with neuropsychiatric disease and copy-number variations spanning NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2 protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in the MECP2 3′ UTR, resulting in an enrichment of inefficiently translated long mRNA isoforms. Furthermore, normalization of NUDT21 via siRNA-mediated knockdown in duplication patient lymphoblasts restores MeCP2 to normal levels. Ultimately, we identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via altered alternative polyadenylation.

https://doi.org/10.7554/eLife.10782.001

eLife digest

The X-chromosome carries a number of genes that are involved in a child's intellectual development. One of these genes encodes a protein called MeCP2, which is important for brain function after birth. Mutations in the MECP2 gene cause a disorder known as Rett syndrome. At around 18 months of age, affected children begin to lose the cognitive and motor skills that they had previously acquired. Individuals with extra copies of this gene also show cognitive impairments. For both diseases, individuals with levels of the MeCP2 protein that are the most different from those found in healthy individuals also show the most severe symptoms.

To produce the protein that is encoded by a particular gene, enzymes inside the cell must first make a copy of that gene using a molecule called messenger ribonucleic acid (or mRNA). This mRNA is then used as a template to assemble the protein itself. In the case of MECP2, two different mRNA templates are produced: a long version and a short version. A gene called NUDT21 makes a protein that regulates whether the long or short version of MECP2 mRNA is made.

Gennarino, Alcott et al. have now discovered that people with too many, or too few, copies of the NUDT21 gene have intellectual disabilities and altered levels of MeCP2 protein. Specifically, individuals with extra copies of NUDT21—and thus higher levels of the corresponding protein—produce more of the long MECP2 mRNA. The production of proteins from this long mRNA is less efficient than from the short mRNA; therefore, these individuals have lower levels of MeCP2 protein. The opposite is true for individuals who lack a copy of the NUDT21 gene.

To confirm these data, Gennarino, Alcott et al. grew cells in the laboratory from patients with extra copies of the NUDT21 gene and found that reducing the production of its protein returned the levels of the MeCP2 protein back to normal. These findings show that alterations in the NUDT21 gene cause changes in the level of MeCP2 protein in cells and leads to neuropsychiatric diseases.

https://doi.org/10.7554/eLife.10782.002

Main text

Methyl CpG-binding protein 2 (MeCP2) binds methylated cytosines (Lewis et al., 1992, Guo et al., 2014) and affects the expression of thousands of genes (Chahrour et al., 2008). MECP2 loss-of-function is the predominant cause of Rett syndrome, a postnatal neurological disorder that typically manifests around 18 months of age with developmental regression (Amir et al., 1999; Ravn et al., 2005; Pan et al., 2006). MECP2 mutations additionally cause other neurological disorders, such as non-specific autism (Carney et al., 2003), Angelman-like syndrome (Imessaoudene et al., 2001), and intellectual disability (Orrico et al., 2000). And in the absence of MECP2 mutations, decreased MeCP2 expression has been detected in the brains of patients with autism, Angelman syndrome, and Prader–Willi syndrome (Samaco et al., 2004; Nagarajan et al., 2006).

Duplications within Xq28 involving MECP2 account for 1% of X-linked intellectual disability (del Gaudio et al., 2006; Friez et al., 2006; Lugtenberg et al., 2006; Meins et al., 2005; Van Esch et al., 2005; Cox et al., 2003) and triplications encompassing MECP2 lead to a more severe phenotype (del Gaudio et al., 2006). Mouse studies have established that MECP2 alone within the duplicated and triplicated region is sufficient to cause all the neurological phenotypes of the duplication and triplication syndromes (Collins et al., 2004; Samaco et al., 2012). Notably, even small changes in MeCP2 protein level lead to neurocognitive deficits and behavioral abnormalities, and the severity of the phenotype correlates with the level of MeCP2 (Chao and Zoghbi, 2012).

MECP2 is distinctive for its long 3′ UTR of about 8.5 kb that contains two predominant poly-adenylation (p(A)) sites: a proximal p(A) site located just after the last exon, and a distal p(A) site approximately 8 kb from the final exon, which result in short and long messenger ribonucleic acid (mRNA) isoforms, respectively (Coy et al., 1999; Shahbazian et al., 2002). 3′ UTRs are important for fine-tuning transcript and protein levels because they contain binding sites for regulatory molecules such as RNA-binding proteins and microRNAs (miRNAs), (Bartel, 2009, Gennarino et al., 2012; Gennarino et al., 2015) which induce degradation of the mRNA or inhibit its translation. Thus, long isoforms allow for greater regulation due to an increased number of regulatory elements. The long 3′ UTR of MECP2 harbors more than 50 putative miRNA-binding sites, including the miRNAs known to bind MECP2—miR-483-5p, miR-132 and miR-155—and reduce MeCP2 protein abundance (Klein et al., 2007; Kuhn et al., 2010; Han et al., 2013). The proximal p(A) site of MECP2 is increasingly used throughout postnatal development, resulting in more short mRNA isoforms and a concomitant increase in protein (Balmer et al., 2003), which may be driven by the loss of regulatory binding sites in its 3′ UTR. These observations highlight the complex and precise regulation of MeCP2 in the human brain.

Pre-messenger RNA cleavage factor Im (CFIm) regulates 3′ UTR length by mediating alternative poly-adenylation (APA) (Millevoi and Vagner, 2010). It binds pre-messenger mRNA as a heterotetramer that consists of a NUDT21-encoded CFIm25 dimer and any two of the paralogs CFIm59 or CFIm68 (or its splice variant, CFIm72) (Kim et al., 2010; Yang et al., 2011). In vitro, CFIm25 or CFIm68 knockdown leads to a transcriptome-wide increase in proximal APA site usage, whereas knockdown of other p(A) proteins, such as CFIm59, CPSF, CSTF, or CFIIm, has minimal effects on APA (Gruber et al., 2012; Masamha et al., 2014). Several models have been proposed to explain the role of CFIm25 in APA, but the exact mechanism remains unknown. A total of 59 genes have increased proximal p(A) site usage when comparing low- to high-NUDT21 expressing glioblastoma tumors, and of these, 24 show the same effect as that following CFIm25 knockdown in HeLa cells. MECP2 is the most affected, and its protein levels increase accordingly (Masamha et al., 2014). Moreover, Clip-seq data show enriched CFIm25 binding of the MECP2 3′ UTR near its p(A) sites (Gruber et al., 2012). Based on this, we hypothesized that CFIm25 regulates MeCP2 in humans by changing the ratio of short to long mRNA isoforms and that NUDT21 gain- or loss-of-function will be correlated with neuropsychiatric disease.

To support our hypothesis, we sought patients with copy-number variation (CNV) of NUDT21. We identified eight individuals with duplications and three with deletions spanning NUDT21, all of whom had undergone clinical chromosome microarray testing. Of these, five consented to provide a detailed medical history (Figure 1 and Table 1), and four provided blood samples for molecular studies (three with NUDT21-spanning duplications and one with a deletion).

Subjects with NUDT21-spanning copy-number variations (CNVs).

(A) Five intrachromosomal rearrangements of chromosome 16q including the NUDT21 gene, identified by clinical array comparative genomic hybridization. Duplications shown in blue, deletion in red. Del, deletion; Dup, duplication; Mb, megabases. The striped bars indicate that the copy variant is not drawn to scale. (B) Array plots of oligonucleotide arrays on subjects 1, 2, and 4. The array plots of subject 3 and 5 were unavailable due to the closure of the Signature Genomics microarray laboratory in June 2014. Black dots indicate probes with normal copy-number, green dots indicate copy-number gain, and red dots indicate copy-number loss. Solid and dotted lines respectively define the minimum and maximum expected boundaries of the CNVs.

https://doi.org/10.7554/eLife.10782.003
Table 1

Molecular and clinical characteristics of four individuals with NUDT21 duplication and one individual with NUDT21 deletion

https://doi.org/10.7554/eLife.10782.004
Subject 1Subject 2Subject 3Subject 4Subject 5
SexFMMFF
Age10 years5 years15 years13 years8 years
Deletion/duplicationDuplicationDuplicationDuplicationDeletionDuplication
Coordinateschr16: 46,500,741-64,653,093chr16: 46,500,741-58,539,422chr16: 55,725,264-57,533,101chr16: 56,344,856-63,521,523chr16: 55,919,145-56,619,283
Size18.15 Mb12.04 Mb1.81 Mb7.18 Mb0.70 Mb
ZygosityHeterozygousHeterozygousMosaic marker chromosome (present in 45% of cells)HeterozygousHeterozygous
InheritanceDe novoDe novoDe novoUnknownNot maternal*
Additional CNVs52 kb duplication of chr6: 152, 716, 341-152, 768, 421 (maternal)NoneNone144 kb duplication of chr17: 427, 284-571, 275 (inheritance unknown)None
Developmental delay/intellectual disabilityIntellectual disability (clinical impression: moderate)Significant developmental delayIntellectual disabilityIntellectual disability (full scale IQ 53)Intellectual disability
Developmental regressionYes, at 2 years of ageNoYesNoNo
Autism spectrum disorderYesNoYesNoYes
EpilepsySingle, isolated seizure at 5 years of ageNoNoIntractable, symptomatic partial onset seizuresNo
ADHDYesYesUnknownYesNo
  1. F, female; M, male; chr, chromosome; MB, megabases; CNVs, copy number variants; kb, kilobases; ADHD, attention deficit hyperactivity disorder.

  2. *

    Father not available.

Lymphoblastoid cell lines are commonly used in patient-specific molecular and functional studies of neurological disease (Sie et al., 2009). We, therefore, established immortalized lymphoblastoid cell lines of the four individuals and tested them for relative changes in MeCP2 protein and mRNA levels, and APA site usage. As hypothesized, in the duplication patients, we found ∼60% more CFIm25 and ∼50% less MeCP2 protein (Figure 2A), while in the deletion patient, we found ∼50% less CFIm25 and ∼50% more MeCP2 protein (Figure 2B), when compared to 13 age-matched control subjects. In order to prove that NUDT21 alone can regulate MeCP2 levels, we performed an siRNA-mediated knockdown of NUDT21 and assessed for changes in MeCP2 protein abundance. We found that NUDT21 knockdown increases MeCP2 in patient-derived lymphoblastoid cells, and that reducing it to wild-type levels in the NUDT21-duplication patients rescues MeCP2 protein levels to that of the healthy controls (Figure 2C). To our knowledge, this is the first time that patient-derived lymphoblastoid cells have been transfected or transduced with siRNAs and that the functional consequence of a candidate gene within a larger CNV has been validated in vitro.

Figure 2 with 1 supplement see all
CFIm25 regulates MeCP2 protein levels in patient-derived lymphoblastoid cells with NUDT21 CNVs.

(A) Representative western blot picture for three duplication patients compared to four age-matched controls showing the increase of CFIm25 and decrease of MeCP2 protein levels. (B) Representative western blot picture for one deletion patient compared to four age-matched controls showing the decrease of CFIm25 and increase of MeCP2 protein levels. Quantification of protein levels for both CFIm25 and MeCP2 from three duplication patients and one deletion patient compared to a total of 13 age-matched controls are shown below the corresponding western blot. Data represent mean ± SEM from a total of six technical replicates. Data were normalized to GAPDH protein levels. (C) Western blot and its relative quantification showing that knockdown of NUDT21 by siRNA-NUDT21 nucleofection increases MeCP2 in control and duplication subjects, and normalizing CFIm25 in duplication patients rescue MeCP2 to control levels. Data represent mean ± SEM from four age-matched control and three duplication cases. Data were normalized to GAPDH protein levels. **p < 0.01, ***p < 0.001. M, mosaic patient.

https://doi.org/10.7554/eLife.10782.005

We then set out to understand how NUDT21 CNVs lead to altered MeCP2 protein levels by investigating their effects on MECP2 mRNA. Quantitative RT-PCR showed that total MECP2 mRNA was elevated in both the duplication cases and the deletion patient (Figure 3A). However, using primer pairs that detected only the long 3′ UTR isoform of MECP2, we found that duplication patients have increased long MECP2 (Figure 3A), indicating that elevated CFIm25 increases distal APA site usage. Conversely, the deletion patient showed a decrease in the long MECP2 isoform, further indicating that NUDT21 levels correlate with MECP2 3′ UTR length (Figure 3A). Northern blot analysis of RNA from the lymphoblastoid cells confirmed the switching between long and short MECP2 isoforms for both duplication and deletion patients (Figure 3—figure supplement 1). Our analysis of the MECP2 mRNA levels in NUDT21-spanning duplication and deletion subjects shows that CFIm25 regulates the ratio of short to long mRNA isoforms (Figure 3A).

Figure 3 with 2 supplements see all
NUDT21 mRNA levels correlate with inefficiently translated long MECP2 mRNA.

(A) RNA quantification by quantitative RT-polymerase chain reaction (qRT-PCR) from lymphoblastoid cells of NUDT21 duplication and deletion patients. The bar graph shows the total mRNA fold change of NUDT21, total MECP2, and long MECP2 for the three duplication patients and one deletion patient compared to 13 age-matched controls. Data represent mean ± SEM from five independent experiments. Data were normalized to GAPDH mRNA levels. (B) Relative polyribosomal and non-poyribosomal enrichment of total and long MECP2 mRNA isoforms of NUDT21 duplication patients compared to age-matched controls. Data represent mean ± SEM from a total of three control and duplication cases. Data were normalized to ACTB mRNA levels. (C) Proposed model showing that duplication and deletion patients have more or less CFIm25, respectively leading to a relative increase in long and short MECP2 3′ UTR isoforms. In both cases, there is an accumulation of mRNA: in the deletion patient, this leads to more MeCP2 protein, but in the duplication patients, it results in less MeCP2 protein due to a translational block from the CFIm25-mediated increase in long MECP2 isoforms and putative binding of miRNAs or RNA-binding proteins to the 3′ UTR. *p < 0.05, **p < 0.01, ***p < 0.001.

https://doi.org/10.7554/eLife.10782.007

To clarify how an increase in MECP2 mRNA may still lead to decreased MeCP2 protein in the NUDT21-duplication patients, we performed a polyribosome fractionation assay to assess translation efficiency of total and long MECP2 mRNA (Figure 3—figure supplement 2). We found that the NUDT21-duplication patients translate MECP2 less efficiently than healthy controls, with a ∼50% reduction in total MECP2 mRNA in the polyribosome fraction and a dramatic enrichment of the long isoform in the non-polyribosome fraction (Figure 3B). These data support the hypothesis that NUDT21-duplication patients have decreased MeCP2 protein despite increased mRNA because NUDT21 promotes distal APA site usage and the elevated RNA pool consists largely of the inefficiently translated long MECP2 isoform (Figure 3C).

The CFIm25 subunit of pre-messenger RNA CFIm represents the first identified post-transcriptional protein regulator of MeCP2 and provides important insights into MeCP2 regulation during normal development and disease.

The presented cases of individuals with NUDT21 CNVs and subsequent MeCP2 level changes suggest NUDT21 as a novel candidate gene for intellectual disability and neuropsychiatric disease. Their CNVs, however, are non-recurrent, and all affect several genes in addition to NUDT21, so that the actual effect of the NUDT21 dosage change alone on phenotype cannot be determined at this point. CNVs affecting only NUDT21 or point mutation cases of NUDT21 would provide important insight. However, we found no NUDT21-spanning CNVs in 30,466 healthy controls (Pinto et al., 2007; Simon-Sanchez et al., 2007; Zogopoulos et al., 2007; International Schizophrenia C, 2008; Jakobsson et al., 2008; Itsara et al., 2009; Kirov et al., 2009; Shaikh et al., 2009; Conrad et al., 2010; International HapMap et al., 2010; Vogler et al., 2010; Banerjee et al., 2011; Campbell et al., 2011; Cooper et al., 2011; Genomes Project et al., 2012), while among our group of 87,200 patients who underwent clinical chromosome microarray analysis, mostly for neurodevelopmental disorders, we identified 11 NUDT21-spanning CNVs (p = 0.025, one-tailed Chi square test without Yates correction), suggesting that CNVs involving NUDT21 are indeed pathogenic. Moreover, of the four other genes common to all the patients' CNVs, only one is associated with neurological disease, BBS2, but it is autosomal recessive and is characterized by distinctive features not seen in the patients we studied.

The clinical presentation of the three individuals with increased NUDT21 copy number and decreased MeCP2 protein levels is not that of classic Rett syndrome, which is not surprising since individuals with Rett lack the protein in 50% of their cells, whereas the duplication of NUDT21 causes a partial decrease in all cells. However, these individuals suffer from intellectual disability and autism spectrum disorder, and two of the three individuals manifested significant developmental regression, which is a rare clinical phenomenon and considered a hallmark of Rett syndrome. The identification of additional patients will help us better define the phenotypic overlap and differences between NUDT21-duplication and Rett syndrome, and for NUDT21 deletion or loss-of-function and MECP2 duplication syndrome.

The clinical and molecular data presented provide insight into the post-transcriptional regulation of MECP2, and identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric disease. Further, we developed a new way of validating the role of individual genes within pathogenic CNVs by effectively transfecting patient-derived lymphoblastoid cells. Ultimately, this study provides an example of how the complexity of a CNV goes well beyond the affected chromosomal domain and the genes affected within. The better we understand gene networks, gene-protein, and protein–protein interactions, the better we will be positioned to identify the molecular bases of various neuropsychiatric disorders and design treatment strategies for the affected individuals.

Materials and methods

Human subjects

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Of approximately 52,000 patients referred to the Baylor College of Medicine (BCM) Medical Genetics Laboratory (MGL) for clinical array comparative genomic hybridization (aCGH) analysis between April 2007 and February 2013, six probands with copy number variants affecting NUDT21 were identified: five duplication cases and one deletion. We also screened the database of Signature Genomics Laboratories, based on approximately 35,200 samples submitted for clinical aCGH between November 2007 and February 2013, and identified three duplication cases and two deletions. The search was limited to copy-number variants <20 megabases. The clinical labs shared our contact information with the referring providers of all eleven cases, and we were subsequently contacted by five families who expressed interest in participating in this research study. Their copy-number variants had been detected by clinical aCGH on the following platforms: CMA-HR + SNP V9.1.1, Baylor College of Medicine (subject 1); Oligo V8.1.1, Baylor College of Medicine (subject 2); SignatureChipOS v2.0, Signature Genomics (subject 3); and Oligo V6.5, Baylor College of Medicine (subject 4). Following informed consent, approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine, we performed a comprehensive chart review of medical records and neuropsychological testing. A venous blood sample was provided by the probands in order to establish immortalized lymphoblastoid cell lines. All individuals with NUDT21 copy-number variants were enrolled in a research study approved by the Institutional Review Board of Baylor College of Medicine (H-25466). The consent form specifically allows for sharing of medical information and physical exam findings. All individuals whose lymphoblastoid cell lines were enrolled as controls had been enrolled in a research study approved by the Institutional Review Board of Baylor College of Medicine (H-25531) as unaffected sibling controls.

Lymphoblastoid cell culture

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Venous blood of the recruited probands was drawn into ACD solution A tubes. Buffy coat was prepared, lymphocytes were pelleted, and transformed with Epstein–Barr virus and cyclosporin A following standard procedures. Cell lines were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (Atlanta Biological, Flowery Branch, GA, United States) and 1% penicillin/streptomycin. Cell cultures were maintained at 37°C in a humidified incubator supplemented with 5% CO2. Medium was renewed every 2 to 3 days to maintain the cell density between 1 × 105 and 2 × 106 cells/ml.

Lymphoblastoid nucleofection

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Lymphoblastoid cells were nucleotransfected with 300 nM of either Ambion small interfering RNA siNUDT21 (s21771) or siScramble (4390843) using Amaxa 4D-Nucleofector and P3 Primary Cell 4D-Nucleofector X Kit (Lonza, Cologne, Germany, cat# V4XP-3024). Cells (2 × 107) were centrifuged at 200×g for 10 min at room temperature, then resuspended in 100 μl P3 Primary Cell 4D-Nucleofector Solution with the added supplement. In microfuge tubes, 300 nM siRNA was mixed with the suspended cells and transferred to Nucleocuvette Vessels prior to nucleofection. Cells were nucleofected using the program FI-115 (efficiency). Post-nucleofection, cells were resuspended with 0.5-ml pre-warmed media (RPMI, 10% FBS, and 1% antibiotic–antimycotic) and transferred to T25 flasks. After 6 hr, medium was removed and replaced with fresh media. Cells were incubated at 37°C with 5% CO2 for 48 hr prior to protein extraction and western blot analysis. The nucleofection protocol was optimized by transfecting 2 × 107 lymphoblastoid cells with 2 μM of siGLO Green transfection indicator (Dharmacon, Lafayette, CO, United States) or Control (empty) either using the program FI-115 (efficiency) or EO-115 (functionality). Cells were later incubated at 37°C with 5% CO2 and collected at different time points from 12 to 72 hr for cytometric analysis using a BD LSR FORTESSA (BD Biosciences, San Jose, CA, United States) according to manufacture's instructions. We determined that the efficiency protocol was preferable because the nucleofection rate was higher, and the survival rate was acceptable. We extracted protein at 48 hr post-nucleofection because the survival was satisfactory, and it was the latest time point at which nucleofected small RNA was nearly 100%, which would allow the siRNA the most time to act (Figure 2—figure supplement 1).

Western blot

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Lymphoblastoid cell suspension cell cultures were collected at 6 × 106 confluence and processed for protein extraction. Cell pellets were lysed with modified RIPA buffer (180 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 2% SDS, and complete protease inhibitor cocktail (Roche, France)) by pipetting them up and down with a p1000 tip and then placed at room temperature rotisserie shaker for 10 min followed by boiling for 5 min. Then the lysates were repeatedly passed through a 27-G needle with a syringe to reduce viscosity due to DNA breakage, followed by centrifugation at 13,000 rpm at room temperature for 15 min. Proteins were quantified by Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States) and resolved by high resolution NuPAGE 4–12% Bis-Tris Gel (Life Technologies, Waltham, MA, United States) according to the manufacturer's instruction. Antibodies: Mouse α-NUDT21 (1:500, Santa Cruz Biotechnology, Dallas, TX, United States, sc-81109); rabbit α-serum MeCP2 N-terminus (1:5000, Zoghbi Lab, #0535); mouse α-GAPDH (1:10,000, Millipore, Billerica, MA, United States, CB1001-500UG).

RNA extraction and quantitative real-time PCR

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Lymphoblastoid cell suspension cell cultures were collected at 6 × 106 confluence and processed for RNA extraction. Total RNA was obtained using the miRNeasy kit (Qiagen, Netherlands) according to the manufacturer's instructions. RNA was quantified using the NanoDrop 1000 (Thermo Fisher, Waltham, MA, United States). Quality of RNA was assessed by gel electrophoresis. cDNA was synthesized using Quantitect Reverse Transcription kit (Qiagen, Netherlands) starting from 1 μg of DNase-treated RNA. Quantitative RT-polymerase chain reaction (qRT-PCR) experiments were performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States) with PerfeCta SYBR Green FastMix, ROX (Quanta Biosciences, Gaithersburg, MD, United States). Real-time PCR results were analyzed using the comparative Ct method normalized against the housekeeping gene GAPDH (Vandesompele et al., 2002). The range of expression levels was determined by calculating the standard deviation of the ΔCt (Pfaffl, 2001). To ensure efficacy of the genomic DNA elimination, we ran negative control samples in the qRT-PCR that did not have reverse transcriptase (-RT) in the cDNA synthesis reaction.

Northern blot assay

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Total RNA was obtained from lymphoblastoid cell suspension cell cultures as mentioned above. Total RNA (15 μg) was separated on 1.2% formaldehyde agarose gel and transferred to Hybond-N+ nylon membrane (Amersham, United Kingdom), followed by UV crosslinking with Stratalinker 2400 (Agilent Technologies, Santa Clara, CA, United States). MECP2 cDNA probe (1431 bp), which contains a majority of exon1 and exon2, was synthesized by random primed DNA labeling kit (Roche, France) according to the manufacturer's instructions. The blots were hybridized using ULTRAhyb hybridization buffer (Applied Biosystems, Waltham, MA, United States) according to the manufacturer's instructions. In brief, the blots were hybridized with radiolabeled probes (106 cpm/ml) in ULTRAhyb hybridization buffer for 2 hr at 68°C. The blots were then washed with 2× SSC, 0.05% SDS for 20 min at room temperature, followed by washing with 0.1× SSC, 0.1% SDS at 50°C for 10 min. The blots were exposed for 72 hr for analysis. For loading control, the blots were then stripped in boiling 0.2% SDS and hybridized with GAPDH probe.

Polysomal profiling

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1 × 106 lymphoblastoid cells were treated with either 100 µg/ml cycloheximide (Sigma–Aldrich, St. Louis, MO, United States) for 60 min at 37°C before being washed with cold PBS containing 100 µg/ml Cycloheximide or Puromycin. The cells were then lysed in polysome lysis buffer (10 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% (vol/vol) Triton X-100, 0.5% (wt/vol) deoxycholate, 1000 U/ml RNasin, 2 mM DTT and 100 µg/ml Cycloheximide in DEPC-treated water), incubated on ice for 10 min and centrifuged at 16,000×g at 4°C to obtain the clarified post-nuclear lysate. Post-nuclear clarified lysates (50 OD units) were layered on top of 10–50% sucrose gradients and centrifuged at 28,000 rpm (100,000×g) for 4 hr at 4°C using an SW41 rotor (Beckman, Fullerton, CA, United States). Sucrose gradients were fractionated using a BR-184 tube piercer and a syringe pump (Brandel, Gaithersburg, MD, United States) fitted with a UA-6 UV detector (Teledyne ISCO, Lincoln, NE, United States). Digital data were collected during fractionation using the DI-158U USB data acquisition device (DATAQ Instruments, Akron, OH, United States). The digital data were processed using the Peak Chart Data Acquisition Software and UV absorption at 254 nm was plotted vs time (from top to bottom or fraction number of sucrose gradients) (Figure 3—figure supplement 2). TRIzol LS reagent (Life Technologies, Carlsbad, CA, United States) was used to extract RNA from the various polysome fraction and total lysate aliquots as per the manufacturer's recommendations.

Primers

In order to unambiguously distinguish spliced cDNA from genomic DNA contamination, specific exon primers were designed to amplify across introns of the genes tested. The primers for all target genes tested were designed with Primer3 v.0.4.0 (Koressaar and Remm, 2007). Primer sequences:

qRT-PCR

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Human GAPDH: Forward, 5′-CGACCACTTTGTCAAGCTCA-3′; Reverse, 5′-TTACTCCTTGGAGGCCATGT-3′

Human MECP2: Forward, 5′-GATCAATCCCCAGGGAAAAGC-3′; Reverse, 5′-CCTCTCCCAGTTACCGTGAAG-3′

Human long 3′ UTR: Forward, 5′-CATCCCGTGCTTTTAAGGAA-3′; Reverse, 5′-CATTCTCCCCAGCTCTTCAG-3′

Human short 3′ UTR: Forward, 5′-TCTGACAAAGCTTCCCGATT-3′; Reverse, 5′-CCAACTACTCCCACCCTGAA-3′

Northern blot

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Human MECP2: Forward, 5′-GAAGAAAAGTCAGAAGACCAGGACCT-3′; Reverse, 5′-GCTAACTCTCTCGGTCACGGG-3′

Human GAPDH: Forward, 5′-ACCACAGTCCATGCCATCAC-3′; Reverse, 5′-CACCACCCTGTTGCTGTAGCC-3′

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Decision letter

  1. Harry C Dietz
    Reviewing Editor; Johns Hopkins University School of Medicine, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled “Individuals with NUDT21 CNVs have neuropsychiatric disease and altered MeCP2 abundance via alternative polyadenylation” for consideration at eLife. Your full submission has been evaluated by Stylianos Antonarakis (Senior Editor) and three peer reviewers, one of whom is a member of our Board of Reviewing Editors. One of the three reviewers, Stephen Warren, has agreed to share his identity.

Based on our discussions, the individual reviews below and policy regarding submissions to eLife where additional significant experimentation is required, we regret to inform you that this submission of your work will not be considered further for publication. However, we would be willing to consider a substantially revised manuscript that includes additional experimentation to address all major concerns detailed below.

While all reviewers found the association between NUDT21 CNVs and neuropsychiatric illness interesting and potentially important, enthusiasm was reduced based upon the small number of cases that were considered in detail, prior knowledge regarding the relationship between NUDT21 dosage and expression of long and short forms of MECP2 mRNA with specific implication of alternative polyA site utilization (Masamha et al., 2014). There were also specific experimental concerns about certain aspects of the functional studies in your manuscript. Foremost among these were the discordant performance of controls in the mRNA studies shown in Figure 2 which substantially influences data interpretation, and the lack of studies to specifically assess for a contribution of altered mRNA stability. Given that the CNVs under consideration span many genes, both Reviewers 1 and 2 suggested targeted complementation experiments in the deletion and/or duplication cells via recombinant expression and/or siRNA-based methods, respectively. Also both reviewers asked for more clinical information about the additional CNV patients where cell lines were not available to try to increase knowledge and confidence regarding the phenotypic spectrum associated with NUDT21 CNVs.

Reviewer #1:

This interesting manuscript describes and characterizes the functional consequence of CNVs spanning NUDT21 in patients with forms of intellectual disability (ID; 3 duplications and 1 deletion). NUDT21 encodes CFIm25, a component of the heterotetrameric pre-messenger RNA cleavage factor I (CFIm) that is known to influence alternative polyadenylation (APA) site usage. Prior work suggests that increased CFIm25 dosage favors distal APA site usage for many transcripts, prominently including those for MECP2. This often (generally ?) associates with decreased protein expression, presumably due to the inclusion of additional negative regulatory 3'UTR elements such as those that encode binding sites for miRNAs that can function to decrease mRNA stability or (more commonly) translational efficiency. The reverse is true for reduced CFIm25 dosage. Importantly, either decreased or increased MeCP2 expression is known to cause ID with or without autistic features of variable severity.

Using patient lymphoblast lines, the authors show that NUDT21 duplication or deletion CNVs associate with decreased or increased expression of MeCP2, respectively. Curiously, both types of CNVs correlate with increased MECP2 mRNA expression. The authors describe apparent differences in APA site bias that might reconcile these findings and support their hypothesis that NUDT21 CNVs represent a previously unrecognized etiology of ID.

In general, this is a quality study with very interesting and provocative findings. I do, however, have a number of questions and concerns that limit my enthusiasm for this manuscript in its present form. These are detailed below.

1) My major concern with this manuscript relates to the mRNA data presented in Figure 2. While the protein data nicely document increased CFIm25 and decreased MeCP2 protein expression in duplication patients, with the reverse in the deleted patient (n=1 concerns noted), the mRNA data are more ambiguous. Specifically, the northern blot data shown in 2D appear to show an increase in the short (proximal APA site usage) isoform in both the duplicated and deletion patient(s), although this is not specifically quantified. The duplication and deletion patient(s) appear to show increased or decreased distal APA site usage respectively, in keeping with hypothesis, but this is only true when each CNV type is correlated with their respective controls. The difference in the performance of the controls in each arm of this experiment is striking and without apparent basis, with essentially no usage of the distal APA site in the duplication blot and substantial (essentially that of the duplication patients) in the deletion blot. Perusal of the Methods suggests that this does not relate to exposure time or any other definable experimental parameter. If the deletion lane were compared to the controls for the duplication blot, then no apparent difference between deletion and duplication patients would be observed, leaving in question the molecular basis for the striking difference in protein expression between the groups. Even at face value, the (somewhat) higher total mRNA expression and the equivalent long/short isoform ratio shown for the deletion patient (when compared to controls) fails to fully explain the striking increase in MeCP2 protein expression that is evident from the blot in Figure 2B (and inadequately captured in the quantitative data shown below). It would be valuable to show a single northern blot with controls, the duplication patients and the deletion patient, with independent quantification of each parameter (long, short, total, ratio) to facilitate direct comparisons.

2) Given the authors’ original hypothesis that the long isoform would show lower stability, I am not sure that a simple measurement of steady state mRNA abundance adequately captures the potential complexity of the situation. It seems possible, even likely, that an attempt at transcriptional compensation is confounding the situation. In this light, it would be useful to measure mRNA half-life, given the authors’ stated goal to understand the mechanism of altered regulation of gene expression. This could be accomplished by measuring steady-state abundance of the various mRNA isoforms in close temporal sequence with administration of a transcriptional inhibitor such as actinomycin D or DRB. It seems possible that a higher than normal level of transcription might contribute to altered APA site usage in a manner at least partially independent from the absolute level of CFIm25, due to saturation of other trans factors.

3) The authors assume that observations made using lymphoblasts are representative of those in “all cells”. Is this necessarily the case? Could additional evidence be provided?

4) It is quite obvious that there are many genes in the described CNVs that might, in theory, contribute to MeCP2 and/or phenotypic expression. This might be addressed by attempts at compensatory overexpression of CFIm25 in cells from the deleted patient and/or siRNA-mediated knockdown in cells from the duplicated patients.

5) While it is stated that there is redundancy between CFIm59, CFIm68 and CFIm72, it is also stated that isolated deficiency of CFIm68 increases proximal APA site usage. In this light, it seems possible that CNVs that alter CFIm68 expression would also lead to relevant phenotypes. Has this been assessed?

6) The authors vaguely describe additional patients with CNVs encompassing NUDT21, but provide no additional details regarding phenotype, the nature of the CNVs (deletions or duplications), or their range. Despite the fact that cell lines were only available for the 4 described patients, this additional information could prove very relevant and informative. Also, have other CNVs in this region that do not involve NUDT21 been associated with ID? Among patients with NUDT21-spanning CNVs and ID, were other obvious causes of phenotype identified?

Reviewer #2:

Gennarino et al. identify patients with large CNVs including NUDT21 and show that MECP2 protein quantity correlates inversely with NUDT21, strongly linking dosage shifts to usage of alternate polyadenylation site in MeCP2 (i.e. NUDT21 binds in complex to proximal site, leading to a longer 8.5 kbp 3'UTR, preferential degradation and reduced protein abundance). To test the relationship, the authors established lymphoblastoid lines from 3 duplication and 1 deletion patients and then assess MECP2 protein and RNA abundance site confirming the expectation.

The paper is clearly and concisely written. It is technically sound and the discovery of NUDT21 is important in providing insight into the post-transcriptional regulation of MECP2, and of potential future therapeutic value with respect to Rett patients. It is a nice twist and use of CNVs to test specific hypotheses about gene networks.

The number of ID and DD patients is rather limited (3 duplication and 1 deletion); one of the subjects is mosaic (45%) drawing correlations is limited with such a low sample size. Because of its mosaic nature, duplication subject 3 should be indicated on Figure 2.

The relationship of high and low NUDT21 expression in GBM tumors has already been shown to result in shifts in abundance of long and short MECP2 transcripts presumably as a result of preferential PA site usage (see Figure 4, Masamha et al., 2014). The results presented here are not that novel or unexpected from a molecular perspective.

The CNVs are massive ranging in size from 1.8 Mbp to 18.1 Mbp affecting the dosage in many genes. Even the smallest region of overlap appears to involve more than a dozen genes (see Figure 1). How can the authors be so sure that other genes involved in polyadenylation site usage are not affected? Wouldn't a rescue experiment be more informative and specific (i.e. targeted knockdown of the NUDT21 in duplication patients or increase expression of NUDT21 in deletion patients).

The large size of the CNV limits clinical relevance at least with respect to the Rett phenotype. Why not report the phenotype of all 11 patients at least in general terms? I understand that only 4 patients consented for additional follow-up but isn't there even general properties of the clinical presentation that might provide additional insight?

The CNV burden is borderline significance (p=0.035). It appears that both deletion cases and duplication cases were lumped together in the burden test. It seems more appropriate to consider them separately.

Reviewer #3:

This manuscript from the Zoghbi laboratory presents a story that begins with published observation that pre-messenger RNA cleavage factor I subunit CFIm25 knockdown results in marked changes in MECP2 protein levels and p(A) site usage. The authors cleverly propose that human mutations modulating the abundance of CFIm25 could alter MECP2 levels leading to an abnormal phenotype as it is well established that MECP2 levels are tightly controlled in humans. Accordingly, the authors searched clinical aCGH databases of ∼94,000 samples and identified 11 CNVs involving NUDT21 which codes for CFIm25. Of the 11, 4 families were ascertained for follow-up studies. Using lymphoblastoid cells of three duplications and one deletion, the authors convincingly show an inverse correlation between NUDT21 copy number and MECP2 levels by altering p(A) usage sites, with the long 3'UTR showing low MECP2 levels compared to the short 3-UTR. The authors make a reasonable hypothesis that the long 3'UTR has many more regulatory sites influencing translation than the short isoform.

This is a very well written paper with convincing data that represent one of the first post-transcriptional regulators of MECP2, which is notable for its need to be rigorously controlled. Moreover, it points to NUDT21 mutations as potentially important causes of neuropsychiatric disease. It is therefore an important contribution.

https://doi.org/10.7554/eLife.10782.010

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

We are happy to report that we have been able to address the reviewers’ concerns and suggestions, which were quite useful in helping substantiate our hypothesis and strengthen the narrative. Briefly, we report more cases and provide a more detailed description of the phenotypic spectrum of patients with NUDT21 CNVs. Notably, we now demonstrate that normalizing NUDT21 in lymphoblastoid cells from NUDT21 duplication patients restores MeCP2 levels to normal range. We also present a clearer molecular mechanism for how NUDT21 regulates MeCP2 in patients with neuropsychiatric disorders. Performing a polyribosome fractionation assay on lymphoblastoid cells derived from NUDT21 duplication patients, we discovered that the increased long isoform of MECP2 is highly enriched in the untranslated fraction of lymphoblastoid cells.

Reviewer #1:

1) My major concern with this manuscript relates to the mRNA data presented in Figure 2. While the protein data nicely document increased CFIm25 and decreased MeCP2 protein expression in duplication patients, with the reverse in the deleted patient (n=1 concerns noted), the mRNA data are more ambiguous. Specifically, the northern blot data shown in 2D appear to show an increase in the short (proximal APA site usage) isoform in both the duplicated and deletion patient(s), although this is not specifically quantified. The duplication and deletion patient(s) appear to show increased or decreased distal APA site usage respectively, in keeping with hypothesis, but this is only true when each CNV type is correlated with their respective controls. The difference in the performance of the controls in each arm of this experiment is striking and without apparent basis, with essentially no usage of the distal APA site in the duplication blot and substantial (essentially that of the duplication patients) in the deletion blot. Perusal of the Methods suggests that this does not relate to exposure time or any other definable experimental parameter. If the deletion lane were compared to the controls for the duplication blot, then no apparent difference between deletion and duplication patients would be observed, leaving in question the molecular basis for the striking difference in protein expression between the groups. Even at face value, the (somewhat) higher total mRNA expression and the equivalent long/short isoform ratio shown for the deletion patient (when compared to controls) fails to fully explain the striking increase in MeCP2 protein expression that is evident from the blot in Figure 2B (and inadequately captured in the quantitative data shown below). It would be valuable to show a single northern blot with controls, the duplication patients and the deletion patient, with independent quantification of each parameter (long, short, total, ratio) to facilitate direct comparisons.

This point is well taken. We agree with the reviewer that it would be better to show a single northern blot image with controls, duplications, and deletions all together. We did the experiment and have included that image (Author response image 1), which replicates our previous findings. However, we want to emphasize that the quantification presented in the first manuscript was not exclusively of the image shown, but from all three experiments performed. Importantly, we did not compare the CNV data with the controls on the blot alone, but with all the controls in every experiment, as indicated in the figure legend. We presented the northern blot quantification as a ratio of the long and short MECP2 isoforms in the original text because the isoform switching was most pertinent to our narrative. However, we have included the independent quantification of each isoform in Author response image 1. It has proven challenging to capture both the short and long isoforms on the same blot due to differing transfer efficiencies and required exposure times. We therefore decided to move the northern blot to the supplemental materials. We believe that the qPCR data are more compelling, and the new insight gained from the polyribosome assay nicely explains the fate of the RNA

Author response image 1
Northern blot assay from patient-derived lymphoblast cells.

Northern blot assay showing that duplication and deletion patients respectively have more long or short MECP2 isoforms (left panel). Quantification of three duplication patients and one deletion compared to 7 age-matched controls (right panel). Data represent mean ± SEM. The p values were calculated by Student's t-test comparing controls with duplication patients. *p<0.05. M=mosaic patient.

https://doi.org/10.7554/eLife.10782.012

2) Given the authors’ original hypothesis that the long isoform would show lower stability, I am not sure that a simple measurement of steady state mRNA abundance adequately captures the potential complexity of the situation. It seems possible, even likely, that an attempt at transcriptional compensation is confounding the situation. In this light, it would be useful to measure mRNA half-life, given the authors’ stated goal to understand the mechanism of altered regulation of gene expression. This could be accomplished by measuring steady-state abundance of the various mRNA isoforms in close temporal sequence with administration of a transcriptional inhibitor such as actinomycin D or DRB. It seems possible that a higher than normal level of transcription might contribute to altered APA site usage in a manner at least partially independent from the absolute level of CFIm25, due to saturation of other trans factors.

As recommended, we performed an RNA stability assay and found that the long MECP2 isoform has a slightly longer half-life (5.8 hours) compared to total MECP2 (5.1 hours) in patient-derived lymphoblastoid cells. This could partially account for the elevated MECP2 in the duplication patients due to their elevated levels of long MECP2, but we cannot exclude other possibilities such as a feedback mechanism. Given the small difference in the half-life, and that it does not change any of the paper’s conclusions, we have decided not to include it in the revised text. As mentioned above, we believe the ribosomal data are more critical to the conclusions of the paper.

3) The authors assume that observations made using lymphoblasts are representative of those in “all cells”. Is this necessarily the case? Could additional evidence be provided?

Patient-derived lymphoblastoid cells are often used for patient-specific molecular and functional studies. Gene expression in lymphoblastoid cells covers a wide range of metabolic pathways in common with neurons. While they may not be representative of “all cells”, they represent an important model system, due to the minimal invasiveness of obtaining them, and the ease of cell maintenance. See Sie L. et al., J Neurosci Res., 2009 (PMID:19224581) for a review.

4) It is quite obvious that there are many genes in the described CNVs that might, in theory, contribute to MeCP2 and/or phenotypic expression. This might be addressed by attempts at compensatory overexpression of CFIm25 in cells from the deleted patient and/or siRNA-mediated knockdown in cells from the duplicated patients.

We thank the reviewer for this suggestion. We first looked for patients with point mutations in NUDT21 or smaller CNVs and were able to identify one with a duplication of only 700 kb, reducing our minimal region to just five genes. Additionally, while it is not standard to transfect patient-derived lymphoblastoid due to low efficiency and cell survival following transfection, we tried a novel approach using nucleofection, and were successful. We adapted a T-cell protocol from Lonza, and as can be seen in Figure 2C and Figure 2–figure supplement 1, nucleofection of anti-NUDT21 siRNA in control lymphoblastoid cells decreases NUDT21 while increasing MeCP2 as in the deletion patient. More importantly, normalizing NUDT21 levels in the NUDT21 duplication patient lymphoblastoid cells restores MeCP2 levels to the normal range. These compelling data prove that NUDT21 regulates MeCP2 in our patients.

5) While it is stated that there is redundancy between CFIm59, CFIm68 and CFIm72, it is also stated that isolated deficiency of CFIm68 increases proximal APA site usage. In this light, it seems possible that CNVs that alter CFIm68 expression would also lead to relevant phenotypes. Has this been assessed?

We have found patients with CNVs spanning CPSF7 (which encodes CFIm59) and CPSF6 (which encodes CFIm68) with phenotypes similar to those of the NUDT21 CNV patients, and we have contacted their physicians for clinical data and blood samples. However, it took 15-18 months to receive the samples for the NUDT21 CNV patients, and will take a comparable amount of time to receive those. Thus, we will happily follow up with a Research Advance when that study is complete.

6) The authors vaguely describe additional patients with CNVs encompassing NUDT21, but provide no additional details regarding phenotype, the nature of the CNVs (deletions or duplications), or their range. Despite the fact that cell lines were only available for the 4 described patients, this additional information could prove very relevant and informative. Also, have other CNVs in this region that do not involve NUDT21 been associated with ID? Among patients with NUDT21-spanning CNVs and ID, were other obvious causes of phenotype identified?

We were able to obtain a detailed clinical history for one additional duplication case, which we now include in the manuscript. We also described the remaining CNVs by type, as the reviewer suggested, in the revised manuscript. However, we were unable to obtain clinical information beyond the indication for aCGH testing available in the databases because the primary physician did not respond to our queries. As we now mention in the revised text, none of the other genes in the minimal region of overlap are associated with neurological disease when heterozygous. Additionally, there are no individuals in the patient databases with CNVs of adjacent genes that do not also span NUDT21. All this together, along with the rescue experiment in the duplication lines, supports our conclusion that NUDT21 alters MeCP2 levels, and, hence, is the driver of pathogenicity.

Reviewer #2:

[…] The number of ID and DD patients is rather limited (3 duplication and 1 deletion); one of the subjects is mosaic (45%) drawing correlations is limited with such a low sample size. Because of its mosaic nature, duplication subject 3 should be indicated on Figure 2.

We have labeled the mosaic patient on the figures. We thank the reviewer for the helpful suggestion.

The relationship of high and low NUDT21 expression in GBM tumors has already been shown to result in shifts in abundance of long and short MECP2 transcripts presumably as a result of preferential PA site usage (see Figure 4, Masamha et al., 2014). The results presented here are not that novel or unexpected from a molecular perspective.

It is true that the inverse relationship between NUDT21 and MeCP2 via polyadenylation has been established in tumor cells; however, we make several novel contributions. We identify NUDT21 as a candidate intellectual disability and autism gene, and show that patients with NUDT21 CNVs have altered MeCP2. Importantly, in this manuscript we elucidate the mechanisms by which NUDT21 regulates MeCP2, i.e. by increasing the long mRNA isoform, which we then show is inefficiently translated.

The CNVs are massive ranging in size from 1.8 Mbp to 18.1 Mbp affecting the dosage in many genes. Even the smallest region of overlap appears to involve more than a dozen genes (see Figure 1). How can the authors be so sure that other genes involved in polyadenylation site usage are not affected? Wouldn't a rescue experiment be more informative and specific (i.e. targeted knockdown of the NUDT21 in duplication patients or increase expression of NUDT21 in deletion patients).

We thank the reviewer for suggesting this experiment. As mentioned above in response to Reviewer 1, it is not common to transfect lymphoblastoid cells to target a putative causal gene in CNV patients due to low transfection efficiency and survival of lymphoblastoid cells upon transfection. However, we attempted the experiment by optimizing a Lonza nucleofection protocol originally designed for T-cells. We are happy to report, as shown in Figure 2C and Figure 2–figure supplement 1, that we succeeded in this experiment. We nucleofected lymphoblastoid cells from control and NUDT21 duplication patients with small interfering RNA against NUDT21 and found that decreasing the NUDT21 levels in control patients increases MeCP2 like in the deletion patient, while normalizing its level in duplication patients completely restores MeCP2 protein levels. Because we could not find examples of successful transfections of patient-derived lymphoblastoid cells in the literature, we have provided a detailed protocol.

The large size of the CNV limits clinical relevance at least with respect to the Rett phenotype. Why not report the phenotype of all 11 patients at least in general terms? I understand that only 4 patients consented for additional follow-up but isn't there even general properties of the clinical presentation that might provide additional insight?

We were able to obtain a detailed clinical history from one additional patient with a duplication of only 700 kb. However, all we know about the remaining patients is their indication for aCGH testing, which is incomplete. We have put as much information in the manuscript as we could successfully obtain from referring physicians.

The CNV burden is borderline significance (p=0.035). It appears that both deletion cases and duplication cases were lumped together in the burden test. It seems more appropriate to consider them separately.

To support our hypothesis that NUDT21 dosage change leads to neuropsychiatric disease, we believe it is unnecessary to separate the duplications and deletions in the burden test, because we are showing that the brain is sensitive to the level of NUDT21 such that both increases and decreases lead to neuropsychiatric disease. Additionally, since Signature Genomics is no longer in business, we cannot sort their database to get precise numbers for their total duplication and deletion cases. We also reduced the number of patients in our denominator by 6,300 by excluding prenatal cases for whom we could not know if they would have had neuropsychiatric disease.

https://doi.org/10.7554/eLife.10782.011

Article and author information

Author details

  1. Vincenzo A Gennarino

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    VAG, Design the experiments and Performed the experiments, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Callison E Alcott
    Competing interests
    No competing interests declared.
  2. Callison E Alcott

    1. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    2. Program in Developmental Biology, Baylor College of Medicine, Houston, United States
    3. Medical Scientist Training Program, Baylor College of Medicine, Houston, United States
    Contribution
    CEA, Design the experiments and Performed the experiments, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Vincenzo A Gennarino
    Competing interests
    No competing interests declared.
  3. Chun-An Chen

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    C-AC, Contributed to molecular work and lymphoblast cultures, Acquisition of data
    Competing interests
    No competing interests declared.
  4. Arindam Chaudhury

    1. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, United States
    2. Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, United States
    Contribution
    AC, Performed the polyribosome experiments, Acquisition of data
    Competing interests
    No competing interests declared.
  5. Madelyn A Gillentine

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    MAG, Contributed to molecular work and lymphoblast cultures, Acquisition of data
    Competing interests
    No competing interests declared.
  6. Jill A Rosenfeld

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Contribution
    JAR, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5664-7987
  7. Sumit Parikh

    Center for Child Neurology, Cleveland Clinic Children's Hospital, Cleveland, United States
    Contribution
    SP, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  8. James W Wheless

    Department of Pediatric Neurology, Neuroscience Institute and Tuberous Sclerosis Clinic, Le Bonheur Children's Hospital, University of Tennessee Health Science Center, Memphis, United States
    Contribution
    JWW, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  9. Elizabeth R Roeder

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Department of Pediatrics, Baylor College of Medicine, Houston, United States
    Contribution
    ERR, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  10. Dafne DG Horovitz

    Depto de Genetica Medica, Instituto Nacional de Saude da Mulher, da Criança e do Adolescente Fernandes Figueira, Rio de Janeiro, Brazil
    Contribution
    DDGH, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  11. Erin K Roney

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Contribution
    EKR, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  12. Janice L Smith

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Contribution
    JLS, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  13. Sau W Cheung

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Contribution
    SWC, Examined the patients and provided clinical data
    Competing interests
    No competing interests declared.
  14. Wei Li

    Division of Biostatistics, Dan L Duncan Cancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States
    Contribution
    WL, Shared CFIm25 data prior to publication, Contributed unpublished essential data
    Competing interests
    No competing interests declared.
  15. Joel R Neilson

    1. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, United States
    2. Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, United States
    Contribution
    JRN, Analyzed the polyribosome experiments
    Competing interests
    No competing interests declared.
  16. Christian P Schaaf

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    Contribution
    CPS, Serves as communicating author for all clinical data, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    schaaf@bcm.edu
    Competing interests
    No competing interests declared.
  17. Huda Y Zoghbi

    1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    2. Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States
    3. Program in Developmental Biology, Baylor College of Medicine, Houston, United States
    4. Department of Pediatrics, Baylor College of Medicine, Houston, United States
    5. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States
    Contribution
    HYZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    hzoghbi@bcm.edu
    Competing interests
    HYZ: Senior editor, eLife.

Funding

Howard Hughes Medical Institute (HHMI)

  • Huda Y Zoghbi

National Institutes of Health (NIH) (5R01NS057819)

  • Huda Y Zoghbi

Baylor College of Medicine IDDRC (1 U54 HD083092)

  • Huda Y Zoghbi

Rett Syndrome Research Trust

  • Huda Y Zoghbi
  • Callison E Alcott

Joan and Stanford Alexander Family

  • Christian P Schaaf

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are deeply indebted to the patients and their families for their willingness to participate in our research study. We would like to thank Pawel Stankiewicz for helpful contributions on patient data, Zhandong Liu from the Neurological Research Institute (NRI) Bioinformatics Core for his insight and expertise in interpreting the CLIP-seq data set, and Genevara Allen of the NRI Biostatistics Core for consultation and discussion. We thank Barbara G Cochran for critical reading of the manuscript. We would like to thank the Howard Hughes Medical Institute (HHMI–HYZ), the National Institutes of Health (NIH) (5R01NS057819) (HYZ), and the Rett Syndrome research trust (HYZ) for funding this research. The project was also supported by Baylor College of Medicine IDDRC Grant Number 1 U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health. CPS is generously supported by the Joan and Stanford Alexander Family.

Ethics

Human subjects: All individuals with NUDT21 copy number variants were enrolled in a research study approved by the Institutional Review Board of Baylor College of Medicine (H-25466). The consent form specifically allows for sharing of medical information and physical exam findings. All individuals whose lymphoblastoid cell lines were enrolled as controls had been enrolled in a research study approved by the Institutional Review Board of Baylor College of Medicine (H-25531) as unaffected sibling controls.

Reviewing Editor

  1. Harry C Dietz, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: August 11, 2015
  2. Accepted: August 26, 2015
  3. Accepted Manuscript published: August 27, 2015 (version 1)
  4. Accepted Manuscript updated: August 28, 2015 (version 2)
  5. Version of Record published: September 29, 2015 (version 3)

Copyright

© 2015, Gennarino et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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