Title: <i>S1P</i> defects cause a new entity of cataract, alopecia, oral mucosal disorder, and psoriasis‐like syndrome
Abstract: Article1 April 2022Open Access Source DataTransparent process S1P defects cause a new entity of cataract, alopecia, oral mucosal disorder, and psoriasis-like syndrome Fuying Chen Fuying Chen orcid.org/0000-0002-7439-4845 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Cheng Ni Cheng Ni Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Data curation, Formal analysis, Writing - original draft, Writing - review & editing Search for more papers by this author Xiaoxiao Wang Xiaoxiao Wang orcid.org/0000-0003-3490-6735 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Ruhong Cheng Ruhong Cheng Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Resources, Supervision, Investigation, Visualization Search for more papers by this author Chaolan Pan Chaolan Pan orcid.org/0000-0003-3378-4676 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Methodology, Writing - review & editing Search for more papers by this author Yumeng Wang Yumeng Wang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Visualization, Writing - review & editing Search for more papers by this author Jianying Liang Jianying Liang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Jia Zhang Jia Zhang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Visualization, Writing - review & editing Search for more papers by this author Jinke Cheng Jinke Cheng orcid.org/0000-0002-4344-5363 Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China Contribution: Investigation, Writing - review & editing Search for more papers by this author Y Eugene Chin Y Eugene Chin orcid.org/0000-0003-4962-1396 Instituteof Health Sciences, Chinese Academy of Sciences, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Resources, Writing - review & editing Search for more papers by this author Yi Zhou Yi Zhou orcid.org/0000-0002-7785-7112 Department of gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China Contribution: Writing - review & editing Search for more papers by this author Zhen Wang Zhen Wang Department of Dermatology, Children's Hospital of Shanghai Jiaotong University, Shanghai, China Contribution: Methodology, Project administration, Writing - review & editing Search for more papers by this author Yiran Guo Yiran Guo Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, PA, USA Contribution: Writing - review & editing Search for more papers by this author She Chen She Chen NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China Contribution: Methodology, Writing - review & editing Search for more papers by this author Stephanie Htun Stephanie Htun Division of Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Erin F Mathes Erin F Mathes Departments of Dermatology and Pediatrics, University California, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Alejandra G de Alba Campomanes Alejandra G de Alba Campomanes Department of Ophthalmology, University of California, San Francisco, CA, USA Search for more papers by this author Anne M Slavotinek Anne M Slavotinek Division of Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Si Zhang Corresponding Author Si Zhang [email protected] orcid.org/0000-0002-5682-4995 NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Ming Li Corresponding Author Ming Li [email protected] orcid.org/0000-0003-3053-2756 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China 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 Zhirong Yao Corresponding Author Zhirong Yao [email protected] orcid.org/0000-0003-0785-8985 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Visualization, Writing - review & editing Search for more papers by this author Fuying Chen Fuying Chen orcid.org/0000-0002-7439-4845 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Cheng Ni Cheng Ni Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Data curation, Formal analysis, Writing - original draft, Writing - review & editing Search for more papers by this author Xiaoxiao Wang Xiaoxiao Wang orcid.org/0000-0003-3490-6735 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Ruhong Cheng Ruhong Cheng Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Resources, Supervision, Investigation, Visualization Search for more papers by this author Chaolan Pan Chaolan Pan orcid.org/0000-0003-3378-4676 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Methodology, Writing - review & editing Search for more papers by this author Yumeng Wang Yumeng Wang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Visualization, Writing - review & editing Search for more papers by this author Jianying Liang Jianying Liang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Jia Zhang Jia Zhang Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Visualization, Writing - review & editing Search for more papers by this author Jinke Cheng Jinke Cheng orcid.org/0000-0002-4344-5363 Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China Contribution: Investigation, Writing - review & editing Search for more papers by this author Y Eugene Chin Y Eugene Chin orcid.org/0000-0003-4962-1396 Instituteof Health Sciences, Chinese Academy of Sciences, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Resources, Writing - review & editing Search for more papers by this author Yi Zhou Yi Zhou orcid.org/0000-0002-7785-7112 Department of gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China Contribution: Writing - review & editing Search for more papers by this author Zhen Wang Zhen Wang Department of Dermatology, Children's Hospital of Shanghai Jiaotong University, Shanghai, China Contribution: Methodology, Project administration, Writing - review & editing Search for more papers by this author Yiran Guo Yiran Guo Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, PA, USA Contribution: Writing - review & editing Search for more papers by this author She Chen She Chen NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China Contribution: Methodology, Writing - review & editing Search for more papers by this author Stephanie Htun Stephanie Htun Division of Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Erin F Mathes Erin F Mathes Departments of Dermatology and Pediatrics, University California, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Alejandra G de Alba Campomanes Alejandra G de Alba Campomanes Department of Ophthalmology, University of California, San Francisco, CA, USA Search for more papers by this author Anne M Slavotinek Anne M Slavotinek Division of Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA Contribution: Resources, Writing - review & editing Search for more papers by this author Si Zhang Corresponding Author Si Zhang [email protected] orcid.org/0000-0002-5682-4995 NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China Contribution: Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Ming Li Corresponding Author Ming Li [email protected] orcid.org/0000-0003-3053-2756 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China 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 Zhirong Yao Corresponding Author Zhirong Yao [email protected] orcid.org/0000-0003-0785-8985 Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Visualization, Writing - review & editing Search for more papers by this author Author Information Fuying Chen1,2,†, Cheng Ni1,2,†, Xiaoxiao Wang1,2,†, Ruhong Cheng1,2,†, Chaolan Pan1,2,†, Yumeng Wang1,2, Jianying Liang1, Jia Zhang1, Jinke Cheng3, Y Eugene Chin4, Yi Zhou5, Zhen Wang6, Yiran Guo7, She Chen8, Stephanie Htun9, Erin F Mathes10, Alejandra G de Alba Campomanes11, Anne M Slavotinek9, Si Zhang *,8, Ming Li *,1,2 and Zhirong Yao *,1,2 1Department of Dermatology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2Institute of Dermatology, Shanghai Jiaotong University School of Medicine, Shanghai, China 3Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China 4Instituteof Health Sciences, Chinese Academy of Sciences, Shanghai Jiaotong University School of Medicine, Shanghai, China 5Department of gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China 6Department of Dermatology, Children's Hospital of Shanghai Jiaotong University, Shanghai, China 7Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia, PA, USA 8NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China 9Division of Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA 10Departments of Dermatology and Pediatrics, University California, San Francisco, CA, USA 11Department of Ophthalmology, University of California, San Francisco, CA, USA † These authors contributed equally to this work *Corresponding author. Tel: +86-21-54237624; E-mail: [email protected] *Corresponding author. Tel: +86-21-25078571; E-mail: [email protected] *Corresponding author. Tel: +86-21-25076428; E-mail: [email protected] EMBO Mol Med (2022)14:e14904https://doi.org/10.15252/emmm.202114904 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 In this report, we discovered a new entity named cataract, alopecia, oral mucosal disorder, and psoriasis-like (CAOP) syndrome in two unrelated and ethnically diverse patients. Furthermore, patient 1 failed to respond to regular treatment. We found that CAOP syndrome was caused by an autosomal recessive defect in the mitochondrial membrane-bound transcription factor peptidase/site-1 protease (MBTPS1, S1P). Mitochondrial abnormalities were observed in patient 1 with CAOP syndrome. Furthermore, we found that S1P is a novel mitochondrial protein that forms a trimeric complex with ETFA/ETFB. S1P enhances ETFA/ETFB flavination and maintains its stability. Patient S1P variants destabilize ETFA/ETFB, impair mitochondrial respiration, decrease fatty acid β-oxidation activity, and shift mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Mitochondrial dysfunction and inflammatory lesions in patient 1 were significantly ameliorated by riboflavin supplementation, which restored the stability of ETFA/ETFB. Our study discovered that mutations in MBTPS1 resulted in a new entity of CAOP syndrome and elucidated the mechanism of the mutations in the new disease. Synopsis Autosomal recessive variants in MBTPS1 gene cause a new entity of cataract, alopecia, oral mucosal disorder and psoriasis-like (CAOP) syndrome. Further in vitro and in vivo analyses confirmed the pathogenicity of MBTPS1 variants. Our study also demonstrates that the CAOP syndrome patient was responsive to riboflavin treatment. S1P interacts with and flavinates the electron transferring flavoprotein (ETF). Inefficient import of mutant S1P into mitochondria impairs mitochondrial respiration and shifts mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Riboflavin supplementation increases ETF stability and restores mitochondrial dysfunction and inflammatory lesions in patient. The paper explained Problem CAOP syndrome is characterized by ocular abnormalities, noncicatricial generalized alopecia, oral mucosal disorder, and psoriasiform skin lesions. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of riboflavin supplementation. Results In this report, we discovered a new entity named CAOP syndrome in a 13-year-old Chinese boy. Furthermore, the patient 1 failed to respond to the regular treatment. We found that the CAOP syndrome is caused by autosomal recessive defects in mitochondrial MBTPS1. MBTPS1 interacts with and activates ETFB. As a result, dysfunction of MBTPS1 in the patient disrupted the electron transport chain, leading to decreased ATP production, and increased reactive oxygen species. Consistent with these findings, abnormalities in lipid metabolism and in mitochondria were observed. Finally, the patient 1 was responsive to riboflavin treatment, which is a cofactor of ETFB. Impact To our knowledge, this is the first study to report MBTPS1 as a disease-causing gene of CAOP syndrome. We showed that MBTPS1 regulates lipid metabolism and mitochondrial function by activating ETFB. Our study is also the first to demonstrate that the MBTPS1 variant patient was responsive to riboflavin treatment, which is the cofactor of ETFB. Introduction Two sporadic cases, a 14-year-old Chinese male patient (patient 1) and a 5-year-old Hispanic female patient (patient 2), were first diagnosed with cataract, alopecia, oral mucosal disorder, and psoriasis-like (CAOP) syndrome, which was characterized by early-onset bilateral lens cataract, generalized nonscarring alopecia, oral mucosal disorder, and severe psoriasiform skin lesions affecting the scalp, facial, inguinal region, buttocks, and lower extremities. Hematoxylin–eosin (HE)-stained sections of the skin of patient 1 showed psoriasiform perivasculitis. To the best of our knowledge, the patients’ clinical symptoms were different from those of all the known dermatoses, and patient 1 was unresponsive to regular treatment, including oral acitretin, zinc sulfate, and topical steroids. Whole-exome sequencing and Sanger sequencing of both patients identified compound heterozygous variants in the membrane-embedded zinc metalloprotease/site-1 protease (MBTPS1/S1P) gene in these patients. S1P is essential for the regulation of cholesterol homeostasis and endoplasmic reticulum (ER) stress responses. S1P catalyzes the first step in the proteolytic activation of transcription factor sterol regulatory element-binding proteins (SREBPs) and the first step in the proteolytic activation of cyclic AMP-dependent activating transcription factor 6 (ATF6), which is confirmed by the variants identified in patients 1 and 2. S1P has also been proven to be a regulator of lysosome biogenesis via proteolytic activation of the hexametric GlcNAc-1-phosphotransferase complex, which is needed for the modification of newly synthesized lysosomal enzymes (Yang et al, 2001; Marschner et al, 2011). The role of S1P in mitochondrial function has not yet been reported. The mitochondrial electron transport chain is essential for ATP production and provides intermediates to maintain metabolic homeostasis. Deficiency or dysfunction in the mitochondrial electron transport chain causes metabolic disorders (Missaglia et al, 2021). Electron transfer flavoprotein (ETF), which is composed of two different subunits, ETFA and ETFB, is the third major electron provider in the mitochondrial electron transport chain after complex I and complex II (Nolfi-Donegan et al, 2020). Mutations in the ETFA or ETFB genes cause metabolic disorders such as multiple acyl-CoA dehydrogenase deficiency (MADD) (Yotsumoto et al, 2008). ETF functions as a hub that takes up electrons from at least 14 dehydrogenases and feeds them into the mitochondrial respiratory chain. As a result, ETF accepts electrons from dehydrogenases, transfers the electrons to ETF-ubiquinone oxidoreductase (ETF-QO) through its cofactors flavin adenine dinucleotide (FAD), and then transports the electrons to the ubiquinone (UQ) pool, and UQ ultimately transfers the electrons to complex III (Nolfi-Donegan et al, 2020). Treatment with riboflavin, a precursor of FAD, could increase ETF stability and thereby partially or fully restore protein function (Zhang et al, 2006). However, the mechanism underlying the posttranscriptional regulation of the ETF protein remains unclear. Here, we found that patient 1 with CAOP syndrome displayed mitochondrial abnormalities. We further discovered that the S1P variants severely impair flavination and subsequently destabilize the ETF complex, which consequently disrupts the mitochondrial respiration chain reaction. These mitochondrial abnormalities and the induced phenotypes of CAOP syndrome were significantly improved by supplementation with riboflavin. Results Clinical features and variants of individuals with CAOP syndrome We identified two sporadic cases with CAOP syndrome, a 14-year-old Chinese male patient (patient 1) and a 5-year-old Hispanic female patient (patient 2) (Table 1). Both patients exhibited mild follicular keratosis, ichthyosis, generalized alopecia, photophobia, red and swollen gums, psoriasis-like lesions, paronychia, and bilateral cataracts (Fig 1A). Histopathological examination of the disorder also revealed psoriasiform perivasculitis (Fig 1B). Patient 2 showed developmental delay (Appendix Table S1). Table 1. Clinical features in our two patients. Clinical features Patient 1 Patient 2 Reference Skeletal dysplasia + − Kondo et al (2018) Elevated blood lysosomal enzymes − ND Kondo et al (2018) Neurological − ND Kondo et al (2018) Focal myoedema − ND Schweitzer et al (2019) Myalgias − ND Schweitzer et al (2019) hyperCKemia − ND Schweitzer et al (2019) Cutaneous lesions + + Schweitzer et al (2019) Ocular involvement + + NA Mucosal lesions + ND NA ND, not detected; NA, not available. Figure 1. CAOP syndrome patients carry MBTPS1 variants and inflammatory disorders A. Representative clinical picture of two patients with CAOP syndrome (14-year-old patient 1 and 5-year-old patient 2). B. Hematoxylin and eosin (H&E) staining of skin biopsies from patient 1 and healthy controls. Scale bars: 200 µm. C, D. Gene sequencing revealed heterozygous MBTPS1 p.Val355Gly (c.1064T>G) and p.Ter1053Arg (c.3157T>C) variants in patient 1 and heterozygous MBTPS1 p.Ter1053Cys (c.3159A>T) and c.2072-2A>T variants in patient 2. The arrows indicate the variants. E. Schematic diagram of the S1P domain structure. The p.Val355Gly variant is localized in the peptidase S8 domain, the c.2072-2A>T variant is located in the ABC transp-aux domain, and p.Ter1053Arg and p.Ter1053Cys are found in the cytoplasmic domain. Download figure Download PowerPoint Due to a lower blood zinc level (9.9 µM, normal range: 11–22 µM) combined with alopecia, angular cheilitis, and rash that involved the extremities, perineum, and buttocks, the Chinese patient was previously diagnosed with acrodermatitis enteropathica. He was treated with oral zinc sulfate at 250 mg/day for 6 months at the age of 10 years but failed to respond to oral zinc sulfate, and no SLC39A4 mutation was detected (Küry et al, 2002). We then administered oral acitretin at 20 mg/day for 6 months when patient 1 was 14 years of age with the aim of alleviating psoriasiform lesions, but he again exhibited a poor response. Nonscarring generalized alopecia, psoriasiform lesions, follicular keratosis, ichthyosis vulgaris phenotype, and photophobia can also be found in X-linked genodermatosis ichthyosis follicularis, atrichia and photophobia (IFAP) syndrome, and keratosis follicularis spinulosa decalvans (KFSD) (Mégarbané & Mégarbané, 2011), and these symptoms have been proven to be caused by mutations in membrane-embedded zinc metalloprotease/site-2 protease (MBTPS2, S2P) and sterol regulatory element-binding protein 1 (SREBP1). However, no mutations in MBTPS2 or SREBP1 were detected in our case. Whole-exome sequencing (Fig EV1A) and Sanger sequencing (Fig 1C) identified compound heterozygous variants comprising p. Val355Gly (c.1064T>G) and p. Ter1053Arg (c.3157T>C) in the MBTPS1 gene (NM_003791.4) in the Chinese patient. The sequencing analyses revealed that both of his biological parents were heterozygous carriers of one MBTPS1 gene variant and that his healthy brother was negative for both variants (Fig 1D). Similarly, compound heterozygous variants in MBTPS1 were found in patient 2, and these included p. Ter1053Cys (c.3159A>T) at the same amino acid site as the Chinese patient and c.2072-2A>T (Fig 1C–E). Click here to expand this figure. Figure EV1. Variants of MBTPS1 in CAOP syndrome Whole-exome sequencing analysis of patient 1. No meaningful new variants were detected based on the autosomal dominant pattern, X-linked hemizygous genetic model, or autosomal recessive inheritance pattern. The analysis according to an autosomal recessive genetic mode detected compound heterozygous variants of the MBTPS1 gene. Protein expression of S1P in skin biopsies of patient 1 and healthy controls. The heterozygous variants in the MBTPS1 gene resulted in two bands of S1P protein: one band was similar to the S1P band of the healthy control, and the other band was markedly larger than that of the healthy control. Ethidium bromide-stained agarose gel of the cDNA products resulting from the splicing assay of the wild-type (WT) and variant (c.2072-2A>T) pSPL3-MBTPS1 minigenes. The 491-base-pair (bp) product represents the mutant RNA transcript lacking MBTPS1 exon 16, and the 648-bp band corresponds to a wild-type transcript that includes MBTPS1 exon 16. The identity of the PCR fragments was confirmed by sequencing. Source data are available online for this figure. Download figure Download PowerPoint We confirmed that the variants identified in patients 1 and 2 were pathogenic by screening a panel of 1200 healthy alleles from public databases and 1,500 additional exomes from internal data. All identified variants were absent from public databases (Data ref: 1,000 genomes, ClinVar, Ensemble, and gnomAD) and healthy controls. The p. Val355Gly (c.1064T>G) variant (patient 1) in the MBTPS1 gene resulted in a missense mutation of 355 amino acids. The p. Ter1053Arg (c.3157T>C) (patient 1) and p. Ter1053Cys (c.3159A>T) (patient 2) variants in the MBTPS1 gene led to destruction of the stop codon and production of a protein markedly larger than wild-type S1P (Fig EV1B). To investigate the role of the c.2072-2A>T variant (patient 2), we performed a reverse transcription PCR study. Minigene spliced products demonstrated that the presence of the c.2072-2A>T variant partially abolished the expression of the normal transcript (648-bp band) and increased the skipping of exon 16 (491-bp band) (Fig EV1C). Sequencing of these PCR products revealed the presence of two different transcripts. S1P deficiency causes abnormal mitochondrial morphology and defective cholesterol metabolism A significant increase in the number of mitochondria (161.5% increase) and morphological abnormalities in the mitochondria (100.7% increase in the length and 10.3% decrease in the length-to-width ratio) were observed in the skin lesions of patient 1 by ultrastructural analysis (Fig 2A and C). Moreover, the mitochondrial lamellar cristae structures were disorganized, indicating severe damage to the mitochondria (Fig 2A). However, no obvious ER abnormality was found in the skin lesions of patient 1 (Fig EV2A, upper panel), which indicated that mitochondrial abnormalities are not induced by whole-cell disease. Figure 2. S1P dysfunction impairs mitochondrial impor