A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI‐NET sequencing

Article18 January 2023Open Access Source DataTransparent process A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing Nasrinsadat Nabavizadeh Nasrinsadat Nabavizadeh orcid.org/0000-0002-1629-6956 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Conceptualization, Data curation, Software, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Annkatrin Bressin Annkatrin Bressin orcid.org/0000-0002-3170-4823 Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Data curation, Software, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Mohammad Shboul Mohammad Shboul orcid.org/0000-0003-3286-1422 Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan Contribution: Methodology, Writing - review & editing Search for more papers by this author Ricardo Moreno Traspas Ricardo Moreno Traspas orcid.org/0000-0003-2607-107X Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Poh Hui Chia Poh Hui Chia Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Supervision, Methodology, Writing - original draft Search for more papers by this author Carine Bonnard Carine Bonnard Model Development, A*STAR Skin Research Labs (A*SRL), Singapore City, Singapore Contribution: Formal analysis, Methodology, Project administration, Writing - review & editing Search for more papers by this author Emmanuelle Szenker-Ravi Emmanuelle Szenker-Ravi Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Burak Sarıbaş Burak Sarıbaş orcid.org/0000-0003-4329-0418 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Validation, Methodology Search for more papers by this author Emmanuel Beillard Emmanuel Beillard orcid.org/0000-0002-2546-7614 Department of Biopathology, Centre Léon Bérard, Lyon, France Contribution: Methodology, Writing - review & editing Search for more papers by this author Umut Altunoglu Umut Altunoglu Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Methodology Search for more papers by this author Zohreh Hojati Zohreh Hojati orcid.org/0000-0003-4831-0123 Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran Contribution: Methodology Search for more papers by this author Scott Drutman Scott Drutman St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA Contribution: Validation, Methodology Search for more papers by this author Susanne Freier Susanne Freier Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Methodology Search for more papers by this author Mohammad El-Khateeb Mohammad El-Khateeb National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Rajaa Fathallah Rajaa Fathallah National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Jean-Laurent Casanova Jean-Laurent Casanova St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France Imagine Institute, University of Paris, Paris, France Howard Hughes Medical Institute, New York, NY, USA Pediatric Hematology and Immunology Unit, Necker Hospital for Sick Children, Paris, France Contribution: Methodology Search for more papers by this author Wesam Soror Wesam Soror National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Alaa Arafat Alaa Arafat National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Nathalie Escande-Beillard Nathalie Escande-Beillard orcid.org/0000-0002-7706-1608 Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Institute of Molecular and Cell Biology, A*STAR, Singapore City, Singapore Contribution: Resources, Validation, Methodology, Writing - review & editing Search for more papers by this author Andreas Mayer Corresponding Author Andreas Mayer [email protected] orcid.org/0000-0002-4532-9382 Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Bruno Reversade Corresponding Author Bruno Reversade [email protected] orcid.org/0000-0002-4070-7997 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Institute of Molecular and Cell Biology, A*STAR, Singapore City, Singapore Department of Paediatrics, National University of Singapore, Singapore City, Singapore Smart-Health Initiative, BESE, KAUST, Thuwal, Kingdom of Saudi Arabia Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Nasrinsadat Nabavizadeh Nasrinsadat Nabavizadeh orcid.org/0000-0002-1629-6956 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Conceptualization, Data curation, Software, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Annkatrin Bressin Annkatrin Bressin orcid.org/0000-0002-3170-4823 Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Data curation, Software, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Mohammad Shboul Mohammad Shboul orcid.org/0000-0003-3286-1422 Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan Contribution: Methodology, Writing - review & editing Search for more papers by this author Ricardo Moreno Traspas Ricardo Moreno Traspas orcid.org/0000-0003-2607-107X Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Poh Hui Chia Poh Hui Chia Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Supervision, Methodology, Writing - original draft Search for more papers by this author Carine Bonnard Carine Bonnard Model Development, A*STAR Skin Research Labs (A*SRL), Singapore City, Singapore Contribution: Formal analysis, Methodology, Project administration, Writing - review & editing Search for more papers by this author Emmanuelle Szenker-Ravi Emmanuelle Szenker-Ravi Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Contribution: Validation, Methodology, Writing - review & editing Search for more papers by this author Burak Sarıbaş Burak Sarıbaş orcid.org/0000-0003-4329-0418 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Validation, Methodology Search for more papers by this author Emmanuel Beillard Emmanuel Beillard orcid.org/0000-0002-2546-7614 Department of Biopathology, Centre Léon Bérard, Lyon, France Contribution: Methodology, Writing - review & editing Search for more papers by this author Umut Altunoglu Umut Altunoglu Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Contribution: Methodology Search for more papers by this author Zohreh Hojati Zohreh Hojati orcid.org/0000-0003-4831-0123 Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran Contribution: Methodology Search for more papers by this author Scott Drutman Scott Drutman St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA Contribution: Validation, Methodology Search for more papers by this author Susanne Freier Susanne Freier Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Methodology Search for more papers by this author Mohammad El-Khateeb Mohammad El-Khateeb National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Rajaa Fathallah Rajaa Fathallah National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Jean-Laurent Casanova Jean-Laurent Casanova St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France Imagine Institute, University of Paris, Paris, France Howard Hughes Medical Institute, New York, NY, USA Pediatric Hematology and Immunology Unit, Necker Hospital for Sick Children, Paris, France Contribution: Methodology Search for more papers by this author Wesam Soror Wesam Soror National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Alaa Arafat Alaa Arafat National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan Contribution: Methodology Search for more papers by this author Nathalie Escande-Beillard Nathalie Escande-Beillard orcid.org/0000-0002-7706-1608 Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Institute of Molecular and Cell Biology, A*STAR, Singapore City, Singapore Contribution: Resources, Validation, Methodology, Writing - review & editing Search for more papers by this author Andreas Mayer Corresponding Author Andreas Mayer [email protected] orcid.org/0000-0002-4532-9382 Max Planck Institute for Molecular Genetics, Berlin, Germany Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Bruno Reversade Corresponding Author Bruno Reversade [email protected] orcid.org/0000-0002-4070-7997 Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey Institute of Molecular and Cell Biology, A*STAR, Singapore City, Singapore Department of Paediatrics, National University of Singapore, Singapore City, Singapore Smart-Health Initiative, BESE, KAUST, Thuwal, Kingdom of Saudi Arabia Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Nasrinsadat Nabavizadeh1,2,3,†, Annkatrin Bressin4,†, Mohammad Shboul5, Ricardo Moreno Traspas1, Poh Hui Chia1, Carine Bonnard6, Emmanuelle Szenker-Ravi1, Burak Sarıbaş1,3, Emmanuel Beillard7, Umut Altunoglu3, Zohreh Hojati2, Scott Drutman8, Susanne Freier4, Mohammad El-Khateeb9, Rajaa Fathallah9, Jean-Laurent Casanova8,10,11,12,13, Wesam Soror9, Alaa Arafat9, Nathalie Escande-Beillard3,14, Andreas Mayer *,4 and Bruno Reversade *,1,3,14,15,16 1Laboratory of Human Genetics & Therapeutics, Genome Institute of Singapore, A*STAR, Singapore City, Singapore 2Division of Genetics, Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran 3Medical Genetics Department, Koç University School of Medicine, Istanbul, Turkey 4Max Planck Institute for Molecular Genetics, Berlin, Germany 5Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan 6Model Development, A*STAR Skin Research Labs (A*SRL), Singapore City, Singapore 7Department of Biopathology, Centre Léon Bérard, Lyon, France 8St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, Rockefeller University, New York, NY, USA 9National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan 10Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM U1163, Necker Hospital for Sick Children, Paris, France 11Imagine Institute, University of Paris, Paris, France 12Howard Hughes Medical Institute, New York, NY, USA 13Pediatric Hematology and Immunology Unit, Necker Hospital for Sick Children, Paris, France 14Institute of Molecular and Cell Biology, A*STAR, Singapore City, Singapore 15Department of Paediatrics, National University of Singapore, Singapore City, Singapore 16Smart-Health Initiative, BESE, KAUST, Thuwal, Kingdom of Saudi Arabia † These authors contributed equally to this work as first authors *Corresponding author. Tel: +49 30 8413 1264; E-mail: [email protected] *Corresponding author. Tel: +966 545352602; E-mail: [email protected] EMBO Mol Med (2023)15:e16478https://doi.org/10.15252/emmm.202216478 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA-seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA-seq, we delineated a deep intronic TAPT1 mutation (c.1237-52 G>A) that segregated with the disease. Using SI-NET-seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre-mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein-null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease-relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non-coding variants, as well as in illuminating the molecular mechanisms of human diseases. Synopsis This study highlights the significance of non-coding variants to uncover rare Mendelian human disorders and the power of combined transcriptomic analyses in identifying molecular pathological pathways. Patients presented with congenital, severe osteogenesis imperfecta and progeroid appearance. RNA-seq and direct Sanger sequencing in patients' fibroblasts revealed a private TAPT1 deep intronic mutation leading to a protein-null allele. TAPT1 deep intronic mutation triggers out of frame exon 12 skipped transcripts that are subject to nonsense-mediated decay (NMD). Integrative RNA-seq and SI-NET-seq analyses uncovered a dysregulation in collagen and extracellular matrix (ECM) pathways consistent with the patients' phenotypes. Introduction Whole exome sequencing (WES) targets less than 2% of our genome, whereas the majority of non-coding sequences are still understudied. These crucial sequences for gene regulation are to a large extent transcribed and form a significant portion of our genome which are also susceptible to harbor variants responsible for human diseases (Djebali et al, 2012; Khan et al, 2017; Chen et al, 2019; Jamshidi et al, 2019). Indeed, from the more than 4,000 Mendelian phenotypes reported to date, of which approximately 50% still lack the identification of the underlying genetic cause (Chong et al, 2015). This speaks to the necessity to further explore non-coding sequences. Whole-genome sequencing (WGS) provides a more comprehensive method to cover the full genome, however, a key challenge to its implementation is the prioritization of the vast amount of non-coding variants identified. This barrier to interpretation is in part driven by the lack of annotated information in intronic and intergenic regions which together comprise up to 98% of our genome. RNA-sequencing (RNA-seq) has proven to be a powerful complementary approach to overcome these hurdles by revealing the functional impact of the genetic variants at the transcriptome level. The use of RNA-seq in conjunction with WGS permits cross-referencing of endogenous RNA levels and splicing events to help prioritize disease-causing mutations at the DNA level (Cummings et al, 2017; Evrony et al, 2017; Kremer et al, 2017). Here we report the study of six affected children from two consanguineous Jordanian families that presented with a congenital syndrome consisting of osteogenesis imperfecta (OI), severe developmental delay and neonatal progeria. By combining homozygosity mapping, RNA-seq and targeted Sanger sequencing, we identified an intronic homozygous variant (c.1237-52 G>A) in TAPT1 (MIM612758) which entirely segregated with the disease. Using patient-derived fibroblasts, our downstream characterization methods including an in vitro splicing assay showed how this private non-coding mutation aggravates skipping of exon 12 leading to a TAPT1 protein-null allele. TAPT1 which codes for a predicted transmembrane protein is involved in ER/Golgi pathways, human Cytomegalovirus (HCMV) infection and primary ciliogenesis (Baldwin et al, 1996, 2000; Jonikas et al, 2009; Symoens et al, 2015; LaMonte et al, 2016, 2020; Zhang et al, 2017a, 2017b). Our functional studies using patient-derived TAPT1-knockout cells could not detect patent anomalies in the pathways previously linked to TAPT1, indicating that its precise molecular function has yet to be ascertained. Notwithstanding, our RNA-seq and SI-NET-seq analyses revealed a role for TAPT1 in collagen and ECM biology, which is consistent with the clinical presentation of our patients. Overall, our study highlights the capacity of applying robust transcriptomic approaches to prioritize disease-causing genes and understand the underlying pathogenesis of Mendelian disease. Results A severe recessive progeroid syndrome with osteogenesis imperfecta We investigated six severely-affected children from two consanguineous Jordanian families (Fig 1A and B) manifesting growth retardation, short stature, multiple bone deformities, lipodystrophy and neonatal progeria. The patients from both families had various craniofacial abnormalities including prominent forehead, plagiocephaly, depressed nasal bridge, nasal septum deviation, low set ears, ear deformities, micrognathia, and occult cleft palate (Figs 1C–E and EV1). The patients also suffered from microphthalmia, cataract, and bilateral esotropia. They had translucent, wrinkled skin with patent acrogeria and sparse hair with premature depigmentation (Figs 1C–E and EV1). They also displayed pectus excavatum and brachydactyly of both hands and feet (Figs 1C–E and EV1). X-ray and MRI (magnetic resonance imaging) tests were performed for patient V.1 (F1). X-ray images showed extensive deformity of the bones, bone dysplasia with bowing, and evidence of previous multiple fractures (Fig 1F). This proband had spared joints, a flattened epiphysis of the humeral bone, irregular growth of arm bones resulting in small deformed radius bone, and a bowed ulnar bone. She also presented a deformed clavicular bone with displacement of both claviculosternal and acromioclavicular joints, deformed shoulders, irregular development of the scapula, bilateral shallow acetabulum, abnormal contour of bilateral femoral head, and absent femoral diaphysis. X-rays also revealed severe calcification defects involving premature atherosclerotic vascular calcification, periarticular soft tissue calcification, and irregular calcification of carpal bones (Fig 1F). Brain abnormalities were also reported with cranial MRI showing defects in the white matter of the frontal and occipital lobes with pachygyria, possibly representing some form of leukodystrophy. The probands V.1 (F1) and V.5 (F1) died of severe respiratory infection and inflammation at the age of 10 and 4.5 years, respectively. The history of a similar disease was remarkable in this extended kindred. Two affected girls (IV.7 (F1) and V.13 (F1)) born to the mother's aunts who showed similar clinical manifestation and died of severe respiratory distress at the age of 5 years. Another case (V.12 (F1)) of 2 years of age is alive and manifests similar clinical features. Figure 1. Patients from two distantly related families present with a recessively inherited syndrome characterized by osteogenesis imperfecta and neonatal progeria A, B. Pedigrees of two distantly related consanguineous families from Jordan, showing an autosomal recessive mode of inheritance of the disease. Black symbols and crossed symbols represent affected and deceased individuals, respectively. C–E. Pictures of investigated patients showing severe bone deformities and fractures, neonatal progeria, wrinkled skin, prominent forehead and pectus excavatum. F. Radiographs of affected V.1 (F1) showing several deficits in the bones including deformity, dysplasia, spared joints and evidence of previous fractures. Severe calcification defects can also be noticed, involving premature atherosclerotic vascular calcification, periarticular soft tissue calcification and irregular calcification of carpal bones. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Clinical pictures of the affected V.12 (F1) individualThe patient presented with multiple abnormalities including bone and joint deformities, pectus excavatum, plagiocephaly microphthalmia and bilateral hypotropia. Moreover, she had apparent dysmorphic facial features such as a depressed nasal bridge and low set of ears. Download figure Download PowerPoint A deep intronic TAPT1 variant segregates with the disease Although the two families were reported to be unrelated, both originated from the Jordan valley. Assuming a founder mutation, we carried out homozygosity mapping for a total of 15 individuals including 3 affected patients (V.1 and V.5 (F1), IV.1 (F2)), 3 pairs of parents from both families, and 6 unaffected siblings from F1 (IV.4, IV.5, IV.6, V.2, V.3 and V.4). Homozygosity mapping confirmed distant relatedness between the 2 families with a minimal shared locus on chromosome 4 (4p16.1-p15.31) (hg19). The length of this Identical-by-Descent (IBD) locus was 8.4 Mb spanning a total of 39 candidate genes (Figs 2A and EV2A). We first performed whole-exome sequencing (WES) for V.1 (F1) and IV.1 (F2), but no compelling recessive mutations were found. To expand our search, we turned to an unbiased RNA-seq approach using primary cutaneous fibroblasts from 2 affected individuals (V.1 and V.5 (F1)), and 2 unrelated wild-type (WT1 and WT2) controls. Of the 39 candidate genes in the IBD region, our differential expression analysis data disclosed that TAPT1 was the only significantly dysregulated transcript in the patient primary dermal fibroblasts (Log2 fold change = −2.5; Figs 2B and EV2B and D). Moreover, the alternative splicing analysis identified 63 genes with splicing defects in patient samples, TAPT1 being the top significant transcript with an exon 12 skipping event (Figs 2C and D, and EV2C and D). Interestingly, homozygous mutations in TAPT1 were previously reported as the genetic cause of complex osteochondrodysplasia (MIM616897) (Symoens et al, 2015). Although less severe, this disease bears strong clinical overlap with the syndrome reported in this study. The exon 12-skipping event prompted us to search for the presence of possible TAPT1 intronic mutations. Targeted Sanger sequencing for this exon and its neighboring nucleotides (~ 300 bps) revealed a deep intronic single nucleotide polymorphism (NM_153365.3, c.1237-52 G>A) within intron 11 that entirely segregated with the disease in all available family members (Figs 2D and 3A). This variant was not reported in the Genome Aggregation Database (gnomAD). While there were similar variants described to occur in its vicinity, none of them were homozygous. Together, these findings indicate that the c.1237-52 G>A mutation within TAPT1 intron 11 most likely caused the disease for the 6 affected children. Figure 2. Homozygosity mapping followed by RNA-seq uncovers a deep intronic recessive mutation in TAPT1 Schematic representation of the shared IBD region between both Jordanian families, located on Chromosome 4 (4p16.1–p15.31) with a size of ~ 12 cM. Although WES analysis did not reveal any mutations in the coding sequences located in the IBD region, RNA-seq analysis helped us to identify the disease causative gene from this locus. Volcano plot showing differentially expressed genes between WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the −log10 P-value, whereas the horizontal axis (x-axis) displays the log2 fold change value. The red dots represent the upregulated transcripts; the blue dots represent the downregulated transcripts. A total of 172 genes were found significantly dysregulated. TAPT1, a gene located in the IBD region, appeared among the most significantly downregulated genes in the patients. Plot showing the alternative splicing analysis results from WT (WT1 and WT2) and patient (V.1 (F1), V.5 (F1)) primary dermal fibroblasts. The vertical axis (y-axis) shows the −log10 FDR (False Discovery Rate), whereas the horizontal axis (x-axis) represents the exon inclusion level (value ranging from −1 to 1). The red dots represent transcripts with exon inclusion events; the blue dots represent transcripts affected by exon skipping. A total of 63 aberrantly spliced genes were found in the patient cells, being TAPT1 the most significant exon skipping event. (Left) Schematic representation showing the complete loss of exon 12 from TAPT1 transcript in patient cells, as defined by our splicing analysis data. (Right) Chromatogram showing the novel intronic mutation (c.1237-52 G>A) we found entirely segregating with the disease in all available family members. For display purposes, results from the targeted Sanger sequencing in WT, IV.3 (F1) and V.5 (F1) individuals are shown. The mutation is present in heterozygosis in IV.3 (F1) (unaffected mother) and in homozygosis in V.5 (F1) (affected patient). Download figure Download PowerPoint Figure 3. TAPT1 c.1237-52 G>A mutation triggers exon 12 skipping Schematic representation of TAPT1 and TAPT1-AS1, indicating the causative intronic mutation (c.1237-52 G>A). The transcription start sites and the direction of transcription are indicated by arrows. Scale bar represents 2 kb. Diagram showing the branchpoint scores for the target c.1237-52 position and flanking nucleotides in TAPT1 intron 11 in both WT (+/+) and patient cells (−/−), as obtained from the RNABPS (Nazari et al, 2018), LaBranchoR (Paggi & Bejerano, 2018) and BPP (Zhang et al, 2017a) softwares. High branchpoint scores were predicted for the G>A transition in the patient cells using the RNABPS and LaBranchoR methods. The x-axis represents the nucleotide distance to the 3′ splice site (3´SS). Schematic illustration of minigene constructs and RT–PCR analysis of splicing products. The pSPL3 vect