Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis
2018; Springer Nature; Volume: 10; Issue: 4 Linguagem: Inglês
10.15252/emmm.201708483
ISSN1757-4684
AutoresMarianna Parlato, Fabienne Charbit‐Henrion, Jie Pan, Claudio Romano, Rémi Duclaux‐Loras, Marie‐Hélène Le Du, Neil Warner, Paola Francalanci, Julie Bruneau, Marc Bras, Mohammed Zarhrate, Bernadette Bègue, Nicolas Guégan, Sabine Rakotobé, Nathalie Kapel, Paola De Angelis, Anne M. Griffiths, Karoline Fiedler, Eileen Crowley, Frank M. Ruemmele, Aleixo M. Muise, Nadine Cerf–Bensussan,
Tópico(s)Galectins and Cancer Biology
ResumoResearch Article22 March 2018Open Access Source DataTransparent process Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis Marianna Parlato Marianna Parlato INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Fabienne Charbit-Henrion Fabienne Charbit-Henrion INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Jie Pan Jie Pan SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Claudio Romano Claudio Romano GENIUS group from ESPGHAN Unit of Pediatrics, Department of Human Pathology in Adulthood and Childhood "G. Barresi", University of Messina, Messina, Italy Search for more papers by this author Rémi Duclaux-Loras Rémi Duclaux-Loras INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Marie-Helene Le Du Marie-Helene Le Du Department of Biochemistry, Biophysics and Structural Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, UMR 9198 CNRS, Université Paris-Sud, Gif-sur-Yvette, France Search for more papers by this author Neil Warner Neil Warner SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Paola Francalanci Paola Francalanci Digestive Endoscopy and Surgery Unit and Pathology Unit Bambino Gesù Children Hospital, IRCCS, Rome, Italy Search for more papers by this author Julie Bruneau Julie Bruneau Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pathology, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France Search for more papers by this author Marc Bras Marc Bras Bioinformatics Platform, Université Paris-Descartes-Paris Sorbonne Centre and Institut Imagine, Paris, France Search for more papers by this author Mohammed Zarhrate Mohammed Zarhrate Genomic Platform, INSERM, UMR1163, Imagine Institute, Paris Descartes-Sorbonne Paris Cite University, Paris, France Search for more papers by this author Bernadette Bègue Bernadette Bègue INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Nicolas Guegan Nicolas Guegan INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Sabine Rakotobe Sabine Rakotobe INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Nathalie Kapel Nathalie Kapel Department of Functional Coprology, Pitié Salpêtrière Hospital, Assistance publique-Hôpitaux de Paris (AP-HP), Paris, France Search for more papers by this author Paola De Angelis Paola De Angelis Digestive Endoscopy and Surgery Unit and Pathology Unit Bambino Gesù Children Hospital, IRCCS, Rome, Italy Search for more papers by this author Anne M Griffiths Anne M Griffiths SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Karoline Fiedler Karoline Fiedler orcid.org/0000-0001-5627-1916 SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Eileen Crowley Eileen Crowley SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Frank Ruemmele Frank Ruemmele INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Aleixo M Muise Corresponding Author Aleixo M Muise [email protected] SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Department of Biochemistry, Institute of Medical Science, University of Toronto, Toronto, ON, Canada Search for more papers by this author Nadine Cerf-Bensussan Corresponding Author Nadine Cerf-Bensussan [email protected] orcid.org/0000-0003-0665-1245 INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Marianna Parlato Marianna Parlato INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Fabienne Charbit-Henrion Fabienne Charbit-Henrion INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Jie Pan Jie Pan SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Claudio Romano Claudio Romano GENIUS group from ESPGHAN Unit of Pediatrics, Department of Human Pathology in Adulthood and Childhood "G. Barresi", University of Messina, Messina, Italy Search for more papers by this author Rémi Duclaux-Loras Rémi Duclaux-Loras INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Marie-Helene Le Du Marie-Helene Le Du Department of Biochemistry, Biophysics and Structural Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, UMR 9198 CNRS, Université Paris-Sud, Gif-sur-Yvette, France Search for more papers by this author Neil Warner Neil Warner SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Paola Francalanci Paola Francalanci Digestive Endoscopy and Surgery Unit and Pathology Unit Bambino Gesù Children Hospital, IRCCS, Rome, Italy Search for more papers by this author Julie Bruneau Julie Bruneau Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pathology, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France Search for more papers by this author Marc Bras Marc Bras Bioinformatics Platform, Université Paris-Descartes-Paris Sorbonne Centre and Institut Imagine, Paris, France Search for more papers by this author Mohammed Zarhrate Mohammed Zarhrate Genomic Platform, INSERM, UMR1163, Imagine Institute, Paris Descartes-Sorbonne Paris Cite University, Paris, France Search for more papers by this author Bernadette Bègue Bernadette Bègue INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Nicolas Guegan Nicolas Guegan INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Sabine Rakotobe Sabine Rakotobe INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Search for more papers by this author Nathalie Kapel Nathalie Kapel Department of Functional Coprology, Pitié Salpêtrière Hospital, Assistance publique-Hôpitaux de Paris (AP-HP), Paris, France Search for more papers by this author Paola De Angelis Paola De Angelis Digestive Endoscopy and Surgery Unit and Pathology Unit Bambino Gesù Children Hospital, IRCCS, Rome, Italy Search for more papers by this author Anne M Griffiths Anne M Griffiths SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Karoline Fiedler Karoline Fiedler orcid.org/0000-0001-5627-1916 SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Eileen Crowley Eileen Crowley SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Search for more papers by this author Frank Ruemmele Frank Ruemmele INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Aleixo M Muise Corresponding Author Aleixo M Muise [email protected] SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Department of Biochemistry, Institute of Medical Science, University of Toronto, Toronto, ON, Canada Search for more papers by this author Nadine Cerf-Bensussan Corresponding Author Nadine Cerf-Bensussan [email protected] orcid.org/0000-0003-0665-1245 INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France GENIUS group from ESPGHAN Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Author Information Marianna Parlato1,2, Fabienne Charbit-Henrion1,2,3,4, Jie Pan5, Claudio Romano2,6, Rémi Duclaux-Loras1,2,3, Marie-Helene Le Du7, Neil Warner5, Paola Francalanci8, Julie Bruneau3,9, Marc Bras10, Mohammed Zarhrate11, Bernadette Bègue1,2, Nicolas Guegan1,3, Sabine Rakotobe1,2, Nathalie Kapel12, Paola De Angelis8, Anne M Griffiths5, Karoline Fiedler5, Eileen Crowley5, Frank Ruemmele1,2,3,4, Aleixo M Muise *,5,13,14,‡ and Nadine Cerf-Bensussan *,1,2,3,‡ 1INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France 2GENIUS group from ESPGHAN 3Université Paris Descartes-Sorbonne Paris Cité, Paris, France 4Department of Pediatric Gastroenterology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France 5SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada 6Unit of Pediatrics, Department of Human Pathology in Adulthood and Childhood "G. Barresi", University of Messina, Messina, Italy 7Department of Biochemistry, Biophysics and Structural Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, UMR 9198 CNRS, Université Paris-Sud, Gif-sur-Yvette, France 8Digestive Endoscopy and Surgery Unit and Pathology Unit Bambino Gesù Children Hospital, IRCCS, Rome, Italy 9Department of Pathology, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France 10Bioinformatics Platform, Université Paris-Descartes-Paris Sorbonne Centre and Institut Imagine, Paris, France 11Genomic Platform, INSERM, UMR1163, Imagine Institute, Paris Descartes-Sorbonne Paris Cite University, Paris, France 12Department of Functional Coprology, Pitié Salpêtrière Hospital, Assistance publique-Hôpitaux de Paris (AP-HP), Paris, France 13Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada 14Department of Biochemistry, Institute of Medical Science, University of Toronto, Toronto, ON, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 416 813 7735; E-mail: [email protected] *Corresponding author. Tel: +33 1 4275 4288; E-mail: [email protected] EMBO Mol Med (2018)10:e8483https://doi.org/10.15252/emmm.201708483 PDFDownload PDF of article text and main figures. 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 Herein, we report the first identification of biallelic-inherited mutations in ALPI as a Mendelian cause of inflammatory bowel disease in two unrelated patients. ALPI encodes for intestinal phosphatase alkaline, a brush border metalloenzyme that hydrolyses phosphate from the lipid A moiety of lipopolysaccharides and thereby drastically reduces Toll-like receptor 4 agonist activity. Prediction tools and structural modelling indicate that all mutations affect critical residues or inter-subunit interactions, and heterologous expression in HEK293T cells demonstrated that all ALPI mutations were loss of function. ALPI mutations impaired either stability or catalytic activity of ALPI and rendered it unable to detoxify lipopolysaccharide-dependent signalling. Furthermore, ALPI expression was reduced in patients' biopsies, and ALPI activity was undetectable in ALPI-deficient patient's stool. Our findings highlight the crucial role of ALPI in regulating host–microbiota interactions and restraining host inflammatory responses. These results indicate that ALPI mutations should be included in screening for monogenic causes of inflammatory bowel diseases and lay the groundwork for ALPI-based treatments in intestinal inflammatory disorders. Synopsis Whole-exome sequencing in two unrelated patients with severe intestinal inflammation identified loss-of-function mutations in ALPI, a brush border enzyme which detoxifies lipopolysaccharides (LPS) through dephosphorylation of its lipid A moiety, linking ALPI deficiency to inflammatory bowel diseases. Structural modelling of ALPI mutants indicated that affected residues are critical for inter-subunit interactions. Heterologous expression of ALPI mutants in HEK293T cells demonstrated that all mutations were loss of function and impaired either stability or catalytic activity of ALPI which could no more detoxify LPS. ALPI expression was strongly decreased in small intestinal biopsies from ALPI deficient patients. ALPI activity was undetectable in ALPI deficiency patient's stools. Introduction Inflammatory bowel diseases (IBDs) are complex, and severe disorders ascribed to alterations in the dialogue between the microbiota and the host immune system (Bouma & Strober, 2003; Maloy & Powrie, 2011). Insights into pathogenesis, first derived from animal studies, have highlighted how multiple mechanisms cooperate to build an efficacious and tightly regulated intestinal barrier that enables hosts to cope with the complex and diverse community of microbes inhabiting the bowel (Bouma & Strober, 2003; Maloy & Powrie, 2011). In humans, the common polygenic forms of IBDs have been thoroughly investigated using genomewide association studies (GWAS). These studies demonstrated that variants affecting expression or function of genes involved in innate defence or cell homeostasis may predispose to or protect against intestinal inflammation (Jostins et al, 2012). Recent advances in next-generation sequencing methods have enabled the identification of an expanding number of monogenic forms of IBDs generally characterized by very early onset and extreme severity (Uhlig, 2013; Uhlig & Muise, 2017). Genetic dissection of these rare disorders is indispensable to identify as early as possible the best therapeutic option and to improve long-term outcome and life quality. It also provides novel insight into the non-redundant mechanisms that are necessary to maintain mutualistic host–microbiota interactions in the gut. Sensing of commensal bacteria by Toll-like receptors (TLRs) is critical to maintain intestinal epithelial homeostasis and to protect against direct injury (Rakoff-Nahoum et al, 2004), while hyperactive TLR signalling can foster intestinal inflammation and autoimmunity in many experimental settings (Asquith et al, 2010). In the gut, TLR4 is expressed by both epithelial and immune cells and can be activated by lipopolysaccharides (LPS), a family of complex glycolipids that are major constituents of the outer membrane of Gram-negative bacteria. In order to cope with the huge load of LPS released in the intestinal lumen by the microbiota and to restrain TLR4-dependent inflammatory signals, hosts have evolved several regulatory mechanisms. Animal studies have shown that one crucial mechanism is intestinal alkaline phosphatase (ALPI), a brush border metalloenzyme that catalyses phosphate hydrolysis of the lipid moiety of LPS and thereby drastically reduces LPS pro-inflammatory activity (Schromm et al, 1998; Goldberg et al, 2008). Herein, using whole-exome sequencing (WES), we report the identification of ALPI mutations in two unrelated patients displaying severe intestinal inflammation and autoimmunity. Our data highlight how LPS detoxification by ALPI plays a crucial role in humans to restrain inflammatory responses to the microbiota. Moreover, our findings provide a genetic-based rationale for ALPI oral therapy in IBD. Results Clinical description of the patients Two patients, P1 and P2, from two unrelated kindreds were investigated. P1 was born at term to non-consanguineous parents living in Italy. He presented at 2 years of age with severe diarrhoea and weight loss. Histology showed increased duodenal intraepithelial lymphocytes with mild villous atrophy (Fig 1A). Since serum IgA and IgG anti-tissue transglutaminase 2 (anti-TG2) titres were over 100 U/ml and HLA typing revealed DR3-DQ2/DR7-DQ8 haplotypes, he was diagnosed with coeliac disease (Fig EV1). Gluten-free diet for 1 year resolved his clinical symptoms and lowered TG2 titres. He was again referred to the clinic at 3 years of age because of recurrent abdominal pain, rectal bleeding and severe diarrhoea. Repeated endoscopic evaluations showed pancolitis with continuous inflammation and ulcerations of severe intensity from transverse colon to rectum. Mucosa was normal in the terminal ileum. Histological lesions combined dense lymphoplasmacytic infiltrate, mucosal ulcerations and erosions, mucin depletion, Paneth cell metaplasia and diffuse thickening of the muscularis mucosae in all colonic biopsies. Of note, no epithelioid granulomas were identified. Immunological workup showed normal serum concentrations of IgG, IgA, IgE and IgM and negative Epstein–Barr virus and cytomegalovirus serology, but the presence of cytoplasmic anti-neutrophil cytoplasmic (cANCA) antibodies. Biological evaluation showed elevated faecal calprotectin (200 μg/g). He was initially treated with steroids and azathioprine. In view of continued disease activity and steroid dependence, infliximab was initiated with no significant improvement. Due to lack of response, he underwent colectomy with ileorectal anastomosis. No extra-gastrointestinal manifestation was reported. A first screen using a targeted sequencing of 66 genes known to be associated with intestinal disorders did not detect any possibly damaging rare (frequency < 1%) variants in public or in-house databases (Uhlig et al, 2014). Figure 1. Colonic inflammation in ALPI-deficient patients A, B. Haematoxylin–eosin staining of colonic biopsies from P1 (A) and P2 (B). Scale bars: 100 μm (A) and 50 μm (B). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Duodenal inflammation in ALPI-deficient patient P1Haematoxylin–eosin staining of duodenal biopsy from P1. Scale bar: 100 μm. Download figure Download PowerPoint P2, a boy born to non-consanguineous parents of Jewish Ashkenazi origin, initially presented at the age of 15.4 years with non-painful swelling of an ankle and hip. Family history revealed psoriatic arthritis in a maternal uncle and ulcerative colitis in the maternal grandmother. P2 was HLA-B27 negative but had anti-nuclear antibodies (titre 1:320). He was treated with non-steroidal anti-inflammatory drugs without improvement. Six months later, he became acutely sick with vomiting and abdominal pain, biological evidence of severe intestinal inflammation [white cell counts 16 (4–10 × 109/l), neutrophils 12 (2–7.5 × 109/l), platelets 891 (150–400 × 109/l), CRP 127.5 (0–8 mg/l), ESR 93 (1–10 mm/h)], thickening of small and large bowel wall at ultrasound examination, abnormal distal ileum at barium contrast imaging, endoscopic and histological evidence of mild inflammation in stomach and duodenum and of more extensive inflammation in the distal intestine with ulcerations of the ileocaecal valve and of the colon hepatic flexure, inflamed granulation tissue with a single granuloma in the ascending colon and one ulcer in sigmoid (Fig 1B). Serum IgM was normal, serum IgG and IgA increased (21.2 and 4.9 g/l, respectively), and anti-TG antibodies were negative. P2 was diagnosed with ileocolonic Crohn's disease and was first treated by exclusive enteral nutrition. Due to iterative clinical symptoms of partial small bowel obstructions, he received corticosteroids and anti-TNF therapy, and finally underwent ileocaecal resection, which showed moderate-to-severe chronic active ileitis with transmural chronic inflammation, fissure-type ulcerations, serosal fibrosis but no evidence of granulomas. After a few months on metronidazole, treatment could be discontinued without digestive relapse. His joint symptoms have resolved post-ileocaecal resection, and he is no longer on any rheumatological medications. Identification of heterozygous compound mutations in ALPI by WES Whole-exome sequencing was performed on trios including affected individuals and their unaffected parents. Annotated data were analysed with in-house software. WES of patient 1 (and parents) resulted in > 144× the mean depth of coverage of the exonic targeted bases covered by a minimum of 30 independent reads and identified 101,319 variants. For P2, exome sequencing coverage was 20× or greater for > 85% of the bases targeted and identified 106,869 variants. Polymorphisms reported in public databases with minor allele frequency (MAF) > 1%, and synonymous variants were filtered out. Potential pathogenicity of each protein-coding variant was evaluated using OMIM (Online Mendelian Inheritance in Man), evolutionary conservation and prediction tools (SIFT, PolyPhen-2.2.2, Mutation Taster). Mutations were next ranked by combined annotation-dependent depletion (CADD) and compared with the mutation significance cut-off (MSC), a gene-level-specific cut-off for CADD scores (Itan et al, 2016). This pipeline did not identify any causal variants in known disease-relevant genes (Appendix Tables S1 and S2). Instead, it led to the identification of compound heterozygous mutations in the ALPI gene, encoding intestinal alkaline phosphatase, in both patients (Fig 2A and B, Table 1). Each variant was confirmed by Sanger sequencing (Figs 2C and EV2) with the segregation within each family being consistent with autosomal recessive inheritance. ALPI has eleven exons spanning 4.6 kb (UCSC Genome Browser hg37, chr2:233, 320, 822–233, 325, 455) and encodes for a 528 amino acid (aa) precursor protein featuring a signal peptide at its N-terminus (NT; 1–19 aa) and a C-terminal (CT) phosphatidyl-inositol glycan (GPI) anchor signal (504–528 aa). Active ALPI consists of two identically processed subunits (each lacking the first 19 and last 24 aa) bound to the cell surface via a post-translationally added GPI anchor (Fig 2D). All mutations were designated as disease causing by the majority of in silico predictions tools, with three variants resulting in the substitution of residues highly conserved across species (A97T, A350V and A360; Fig EV2) and one variant (Q439X) introducing a premature stop codon. Moreover, the combined annotation-dependent depletion (CADD) scores of each variant were well above the mutation significance cut-off (MSC) of 3.13 defined for ALPI (Table 1). All mutations were either absent or present in only a heterozygous state at a frequency of less than 0.001% in the Exome Aggregation Consortium (ExAC) database (Table 1; Lek et al, 2016). Overall, in silico analyses suggested that the four variants identified in the two patients were not irrelevant polymorphisms but rare pathogenic mutations. Figure 2. WES identification of heterozygous compound mutations in ALPI Variant identification pipeline for WES in P1 and P2 (see also Appendix Tables S1 and S2). Unrelated familial trees showing affected children in black and healthy individuals in white. Sanger sequencing of the region corresponding to mutations in ALPI in both families. Location of ALPI on chromosome 2 and diagrams featuring ALPI gene with its nine exons and ALPI protein with its N-terminal signal peptide, phosphatase domain and C-terminal recognition signal for the transamidase complex (GPI-anchor attachment site), which removes the GPI signal sequence and replaces it by a preformed GPI precursor glycolipid. Arrows point to mutations identified in P1 and P2. Numbers indicate amino acid position. Download figure Download PowerPoint Table 1. Features of variants identified in P1 and P2 Patient Exon Position MAF Prediction Genomic Chr2(GRCh37) cDNA NM_001631.4 Proteins CADD MSC:3.13 PoliPhen-2 SIFT Mutation Taster 1 3 g.233321394G>A c.289G>A p.Ala97Thr 0.0004362 27.1 Probably damaging score: 1.0 Deleterious score: 0.03 Disease causing P-value: 0.997 9 g.233322984C>T c.1049C>T p.Ala350Val No Freq 19.95 Possibly damaging score: 0.532 Tolerated score: 0.35 Disease causing P-value: 0.644 2 9 g.233323014C>T c.1079C>T p.Ala360Val 0.0006363 27.8 Probably damaging score: 0.990 Tolerated score: 0.36 Disease causing P-value: 1 11 g.233323584C>T c.1315C>T p.Gln439X 0.0001295 19.3 – – – MAF, minor allelic frequency based on 60,706 individuals genotyped as part of the Exome Aggregation Consortium (ExAC: http://exac.broadinstitute.org). Variant predictions are based on PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), SIFT (http://sift.jcvi.org), Mutation Taster (http://www.mutationtaster.org). Mutation significance cut-off (MSC), a gene-level-specific cut-off for CADD scores, was generated for ALPI (http://pec630.rockefeller.edu:8080/MSC/). Click here to expand this figure. Figure EV2. Autosomal recessive ALPI deficiency Confirmatory Sanger sequencing for siblings in Family 2. Multiple alignments of ALPI orthologs from different species using the Clustal Omega software. Residues altered by mutations in P1 and P2 are boxed in red. Conserved residues are indicated as follow: full identity (*), similar characteristics (:) (> 0.5 in the Gonnet PAM 250 matrix), weak similarities (.) (< 0.5 in the Gonnet PAM 250 matrix). Download figure Download PowerPoint Impaired expression of ALPI A97T and Q439X mutants To assess the impact of the mutations on protein stability of ALPI, HEK293T cells were stably transduced with lentiviral particles encoding WT and mutant proteins. In parallel, and in order to define structure–function correlation, we generated a homology model of human ALPI based on the crystal structure of human placental alkaline phosphatase (ALPP), which shares 87% identity with ALPI (Le Du et al, 2001; Appendix Fig S1). We then analysed how mutations might affect the 3D structure of the ALPI homodimer using ConSurf server (Ashkenazy et al, 2016). Immunoblotting (Fig 3A) and cell surface flow cytometry (Fig 3B) revealed significantly less ALPI protein in HEK293T cells stably transduced with A97T mutant than with the WT construct despite comparable mRNA expression (Fig EV3). Since the highly conserved alanine 97 is located at the β-strand in the dimer interface with each alanine facing the other (Fig 3C), we posited that substitution by threonine with its significantly larger, polar projecting side chain might impair protein dimerization and therefore stability. As expected, given the lack of a GPI anchoring site at the CT domain, the Q439X truncated protein was undetectable at the cell surface by flow cytometry (Fig 3B). However, a weak band with the predicted size of the truncated Q439X mutant was detected by immunoblot (Fig 3A). Reduced abundance of the corresponding transcript (Fig EV3) suggested nonsense-mediated decay. Since the 3D model predicts that each CT region is deeply buried at the interface between the two monomers and engaged in non-covalent interactions, the lack of CT might also impair proper folding and dimerization of the truncated Q439X mutant
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