Dual genetic diagnoses: AIRE and FOXRED1-Related Syndromes in two brothers

Dual genetic diagnoses: AIRE and FOXRED1-Related Syndromes in two brothers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Dual genetic diagnoses: AIRE and FOXRED1-Related Syndromes in two brothers Reheman Palati, Jirong Feng, Lei He, Maimaitiyiming Mireayi, Aimaiti Aerziguli, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8551715/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disorder caused by mutations in the AIRE gene. While the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency defines the syndrome, its phenotypic spectrum can be broad and complex. While a single genetic etiology is typically assumed, the co-occurrence of a second monogenic disorder can complicate the clinical picture. Here, we report two brothers from a non-consanguineous family who presented with a complex multisystem phenotype including hypocalcemia, seizures, and neurodevelopmental impairments. Whole-exome sequencing revealed a homozygous AIRE deletion in both siblings, confirming APS-1. Notably, the elder brother was also found to carry a homozygous variant in the FOXRED1 gene, which explained his severe neurological features, including cerebral atrophy and lactic acidosis, consistent with a dual diagnosis of APS-1 and FOXRED1-related mitochondrial encephalopathy. This case highlights that atypical or compounded clinical presentations should raise suspicion for dual genetic disorders, underscoring the critical role of comprehensive genetic analysis in reaching a precise diagnosis. Autoimmune polyglandular syndrome type 1 FXRED1 Dual genetic diagnosis Whole-exome sequencing Mitochondrial encephalopathy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The proband boy came to our department at the age of 7 after suffering a seizure. His first symptom was developmental delay, noted at age 4. No family history of genetic diseases was reported. The male patient was born via cesarean section at 32 weeks of gestation with a birth weight of 2,200g; Apgar scores were unrecorded. Following a three-day observation in the neonatal department, no abnormalities were initially noted. However, he exhibited global developmental delay: independent sitting at approximately 1year, independent walking at 2.6 years, and utterance of simple single words after age 4. He was first evaluated at age 4 for these delays, though no definitive diagnosis was established. At age 5, he presented with a limp in the left lower limb and weakness in the right upper limb, manifesting as an inability to lift it fully or hold heavy objects. Brain MRI and lumbar puncture (CSF fluid was normal), results led to a diagnosis of brainstem infarction（Figure 1）, for which physical rehabilitation was recommended. By age 6, his motor function had not improved, and he developed progressive pain in the left leg, alongside the onset of vitiligo. X-ray examinations subsequently revealed left hip dislocation and left femoral head necrosis（Figure 2）. The treatment plan included joint immobilization and activity restriction. At age 6.5, the patient's pain worsened, and he began experiencing episodes of blinking and staring. These episodes lasted 2-5 minutes and occurred once every 2-3 months. By age 7, he suffered a generalized tonic-clonic seizure (GTCS) and developed visual deterioration, along with cutaneous and mucosal candidiasis. Subsequent hospital evaluation led to diagnoses of epilepsy, hypocalcemia, hypoparathyroidism, and hypothyroidism. Treatment was initiated with oral vitamin D, calcium, levothyroxine, and valproic acid. Although Whole Exome Sequencing (WES) was recommended to identify a potential genetic etiology, the parents initially declined due to the absence of familial disease history. Despite further counseling on the necessity of genetic testing and its role in elucidating de novo mutations, they maintained their decision to refuse. By the age of 8, the boy's seizures had become more frequent. The clinical situation was further clarified when his younger brother also suffered a seizure and was diagnosed with hypocalcemia. This development led the parents to agree to genetic testing. The results demonstrated a homozygous deletion in the AIRE gene（Figure 3） in both brothers, with their parents being heterozygous carriers. A definitive diagnosis of Autoimmune Polyglandular Syndrome Type 1 (APS-1). The elder brother carries homozygous FOXRED1 Figure 4）variants. His clinical presentation—including vision loss, cerebral atrophy with partial encephalomalacia on MRI, microcephaly (head circumference 49cm, <-3SD), lactic acidosis (3.2mmol/L, high), developmental delay, and epilepsy—is consistent with FOXRED1 -related mitochondrial encephalopathy and supports the pathogenicity of these mutations. The ratio of the copy number of exons 2-4 of the AIRE gene in the proband to that of the normal control was approximately 0, indicating a homozygous deletion of exons 2-4 of the AIRE gene in the patient The ratio of the copy number of exons 2-4 of the AIRE gene in the patient's father and mother to that of the normal control was approximately 0.5, indicating a heterozygous deletion of exons 2-4 of the AIRE gene in the patient's parents. The ratio of the copy number of exons 2-4 of the AIRE gene in the patient's brother to that of the normal control was approximately 0, indicating a homozygous deletion of exons 2-4 of the AIRE gene in the patient's brother. ( Note : The normal reference range for the ratio is 0.8-1.2. A ratio greater than 1.25 but less than 1.75 suggests a suspected single-copy duplication; a ratio greater than 1.75 but less than 2.25 suggests a suspected two-copy duplication. A ratio greater than 0.35 but less than 0.75 suggests a suspected heterozygous deletion; a ratio of 0 indicates a homozygous deletion. Other values are considered ambiguous, and the specific result cannot be determined.) This chromatogram confirms the identified FOXRED1 variant (c.580C>T, p.Arg194Trp) in the family. Patient: Homozygous for the mutation (T/T), consistent with the autosomal recessive disease inheritance. Father & Mother: Both are heterozygous carriers (C/T), each having one mutant and one normal allele. Brother: Also a heterozygous carrier (C/T), confirming the familial segregation of the variant. Discuss Autoimmune polyglandular syndrome type 1 (APS-1) is a rare disorder, with a particularly high prevalence of 1 in 9,000 among Iranian or Persian Jews, whereas the global prevalence is estimated to be between 1 in 90,000 and 1 in 200,000 1 . Autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disorder caused by pathogenic variants in the AIRE gene. It typically presents in early childhood (2–5 years of age) and is characterized by the production of autoantibodies against type I interferons. The condition manifests with a spectrum of endocrine and non-endocrine features. Classic endocrine disorders include hypoparathyroidism, adrenal insufficiency, gonadal failure, type 1 diabetes, and autoimmune thyroiditis. Common non-endocrine manifestations encompass chronic mucocutaneous candidiasis, vitiligo, alopecia, as well as gastrointestinal, hepatic, and hematological involvements 2 , 3 , and in these cases, it’s included femoral head necrosis, brain infarction. Previous studies show that the onset of symptoms in APS type 1 corresponds to the age of early childhood, while the first clinical symptoms (candidiasis of the skin and mucous membranes) manifest themselves in early childhood up to 5 years, hypoparathyroidism up to 10 years, and Addison’s disease up to 15 years 4 . Diagnostic delay is a hallmark of APS-1 due to its variable symptomatology. Although our proband's age at first symptom (5 years) was typical, the presentation was complicated by a movement disorder, ultimately explained by a concurrent mitochondrial encephalopathy. The definitive diagnosis was achieved through WES. A significant factor in the delay was the initial parental decline of genetic testing, based on the absence of a family history. Parental consent was subsequently obtained following the onset of illness in the second child, which facilitated the identification of the underlying AIRE and FOXRED1 mutations in both siblings. A notable aspect of this case is the elder brother's severe neurological presentation, including global developmental delay, cerebral atrophy, partial encephalomalacia, microcephaly, and lactic acidosis—a constellation of features not typically associated with APS-1. The identification of a homozygous missense mutation in FOXRED1 via trio-WES provided a conclusive explanation, establishing a dual diagnosis of FOXRED1-related mitochondrial encephalopathy. This gene encodes a 486-amino acid, FAD-dependent oxidoreductase that functions as a mitochondria-targeted assembly factor for complex I of the oxidative phosphorylation system 5 , 6 . Pathogenic mutations in FOXRED1 have been established in association with severe neurological disorders, including Leigh syndrome and infantile-onset mitochondrial encephalopathy 7 . To date, 11 pathogenic variants in FOXRED1 have been reported, and all cases of associated mitochondrial encephalopathy are consistent with an autosomal recessive inheritance pattern 8 . Previous studies have established that FOXRED1 mutations are associated with a range of neurological features, including athetoid movements, hypotonia, irritability, acquired microcephaly, and cortical blindness, typically presenting in early childhood 9 . According to the ACMG criteria 2015 guidelines 10 , the homozygous variants of c.580(exon5)C > T in our report is classified as likely pathogenic (PM1, PM2, PP3, PP5), and FOXRED1 function has been confirming by animal experiment 11 . This case illustrates that in patients with multisystem involvement and atypical features, the possibility of dual genetic diagnoses should be actively considered, even in the absence of consanguinity. Our case underscores the critical importance of re-evaluating a diagnosis when a single genetic finding cannot fully explain the complete clinical phenotype. Although dual genetic diagnoses are increasingly recognized, conventional genetic tests may still miss such cases. For instance, D.D. Tian et al. reported one of the largest series of dual genetic diagnoses in recent years 12 ,conventional genetic tests. This report highlights that a deep understanding of clinical phenotypes is paramount, as it guides the interpretation of genetic results, not vice versa. Declarations Author Contributions Reheman Palati and Jing Yu contributed to conception. Reheman Palati contributed to literature review and drafted the manuscript. Jirong Feng, Lei He, Maimaitiyiming Mireayi, Aimaiti Aerziguli critically revised the manuscript. All authors gave final approval. Acknowledgments We are very grateful to the children, their families, and colleagues in the imaging department； Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Ethical Approval Verbal informed consent for publication of their clinical presentation and course was obtained from the patient and their parents. References Proust-Lemoine E, Saugier-Veber P, Wemeau JL. Polyglandular autoimmune syndrome type I. Presse Med. 2012;41:e651–62. Lankisch TO, Jaeckel E, Strassburg CP. The autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or autoimmune polyglandular syndrome type 1. Semin Liver Dis. 2009;29:307–14. Ahonen P, Myllarniemi S, Sipila I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med. 1990;322:1829–36. Kisand K, Peterson P. Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. J Clin Immunol. 2015;35:463–78. Formosa LE, Mimaki M, Frazier AE, McKenzie M, Stait TL, Thorburn DR, et al. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I. Hum Mol Genet. 2015;24:2952–65. Calvo SE, Tucker EJ, Compton AG, Kirby DM, Crawford G, Burtt NP, et al. High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency. Nat Genet. 2010;42:851–8. Apatean D, Rakic B, Brunel-Guitton C, Hendson G, Bai R, Sargent MA, et al. Congenital lactic acidosis, cerebral cysts and pulmonary hypertension in an infant with FOXRED1 related complex 1 deficiency. Mol Genet Metab Rep. 2019;19:100472. Hu C, Xu Q, Shen J, Wang Y. Clinical and Genetic Characteristics of Mitochondrial Encephalopathy Due to FOXRED1 Mutations: Two Chinese Case Reports and a Review of the Literature. Front Neurol. 2021;12:633397. Fassone E, Duncan AJ, Taanman JW, Pagnamenta AT, Sadowski MI, Holand T, et al. FOXRED1, encoding an FAD-dependent oxidoreductase complex-I-specific molecular chaperone, is mutated in infantile-onset mitochondrial encephalopathy. Hum Mol Genet. 2010;19:4837–47. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24. Salama M, El-Desouky S, Alsayed A, El-Hussiny M, Moustafa A, Taalab Y, et al. FOXRED1 silencing in mice: a possible animal model for Leigh syndrome. Metab Brain Dis. 2019;34:367–72. Tan DD, Liu YD, Fan YB, Wei CJ, Song DY, Yang HP, et al. [Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses]. Zhonghua Er Ke Za Zhi. 2023;61:345–50. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8551715","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":587924831,"identity":"95790025-eaf1-4d90-8480-58a55c2d0498","order_by":0,"name":"Reheman Palati","email":"","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Reheman","middleName":"","lastName":"Palati","suffix":""},{"id":587924832,"identity":"c540d814-5e07-4936-b8e3-b7790444fdb7","order_by":1,"name":"Jirong Feng","email":"","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Jirong","middleName":"","lastName":"Feng","suffix":""},{"id":587924833,"identity":"2898a1f5-7dcd-4509-877f-28e0bc48c53e","order_by":2,"name":"Lei He","email":"","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"He","suffix":""},{"id":587924834,"identity":"50ef5a1c-7b6b-46e3-b847-4256590b8bef","order_by":3,"name":"Maimaitiyiming Mireayi","email":"","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Maimaitiyiming","middleName":"","lastName":"Mireayi","suffix":""},{"id":587924835,"identity":"75c56db5-57d3-4eb7-a0f4-776942f30c65","order_by":4,"name":"Aimaiti Aerziguli","email":"","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Aimaiti","middleName":"","lastName":"Aerziguli","suffix":""},{"id":587924836,"identity":"17e75e47-1c9f-4772-b6cf-4045b2f6f8c0","order_by":5,"name":"Jing Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBAC+/bmA8Z//9jI8bM3EKnFgOdYQgFvQ5qxZM8BYrVI+Bh84G04nGhwI4FILeYSbIkbJHccTpCc+XjjDYYam2iCWixnNx82MDyTnscvnVZswXAsLbeBoJ47x9IMEtisiyVn55hJMDYcJkLLjRzzHwfYmBM33DxDpBaDGzkGho1tzokbbvAQqUWy51iCMcMZUCAD/ZJAjF/42YFRyVABisrDG298qLEhwi/IjpRIIEU5RAupOkbBKBgFo2BkAAAc9kQRerWKogAAAABJRU5ErkJggg==","orcid":"","institution":"Children’s Hospital of Xinjiang Uygur Autonomous Region，Xinjiang Hospital of Beijing Children’s Hospital，The Seventh People's Hospital of Xinjiang Uygur Autonomous Region，Department of Neurology","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-01-08 13:58:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8551715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8551715/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102745519,"identity":"91955e66-5a7f-47e7-933f-e3fa2abdba7d","added_by":"auto","created_at":"2026-02-16 08:51:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":732777,"visible":true,"origin":"","legend":"\u003cp\u003eMRI Brainstem Infarction\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8551715/v1/e34031d3a89cdafa2340fcfc.jpeg"},{"id":102440123,"identity":"f071e893-b559-48bd-afad-0b64cd35de74","added_by":"auto","created_at":"2026-02-11 16:45:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":437905,"visible":true,"origin":"","legend":"\u003cp\u003eLeft hip dislocation and femoral head necrosis\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8551715/v1/9d05a9f0d272bc0fa07f625b.jpeg"},{"id":102745635,"identity":"5a033032-5ccf-447a-8d7c-977adb1be6df","added_by":"auto","created_at":"2026-02-16 08:52:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63380,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of \u003cem\u003eAIRE\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8551715/v1/80cc822573b043b09856b95c.png"},{"id":102440120,"identity":"c4939e4d-7859-4cdf-aa31-3c6cd409a0ac","added_by":"auto","created_at":"2026-02-11 16:45:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264935,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of \u003cem\u003eFOXRED1\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8551715/v1/d3f9ddef7b017d28edf7cb7b.png"},{"id":107705121,"identity":"518a6193-6869-4f03-bdd2-44c68b2a3ace","added_by":"auto","created_at":"2026-04-24 09:08:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1548505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8551715/v1/6765ce72-cea1-4671-a266-291f6a812ca4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual genetic diagnoses: AIRE and FOXRED1-Related Syndromes in two brothers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe proband boy came to our department at the age of 7 after suffering a seizure. His first symptom was developmental delay, noted at age 4. No family history of genetic diseases was reported.\u003c/p\u003e\n\u003cp\u003eThe male patient was born via cesarean section at 32 weeks of gestation with a birth weight of 2,200g; Apgar scores were unrecorded. Following a three-day observation in the neonatal department, no abnormalities were initially noted. However, he exhibited global developmental delay: independent sitting at approximately 1year, independent walking at 2.6 years, and utterance of simple single words after age 4.\u003c/p\u003e\n\u003cp\u003eHe was first evaluated at age 4 for these delays, though no definitive diagnosis was established. At age 5, he presented with a limp in the left lower limb and weakness in the right upper limb, manifesting as an inability to lift it fully or hold heavy objects. Brain MRI and lumbar puncture (CSF fluid was normal), results led to a diagnosis of brainstem infarction（Figure 1）, for which physical rehabilitation was recommended.\u003c/p\u003e\n\u003cp\u003eBy age 6, his motor function had not improved, and he developed progressive pain in the left leg, alongside the onset of vitiligo. X-ray examinations subsequently revealed left hip dislocation and left femoral head necrosis（Figure 2）. The treatment plan included joint immobilization and activity restriction.\u003c/p\u003e\n\u003cp\u003eAt age 6.5, the patient\u0026apos;s pain worsened, and he began experiencing episodes of blinking and staring. These episodes lasted 2-5 minutes and occurred once every 2-3 months. By age 7, he suffered a generalized tonic-clonic seizure (GTCS) and developed visual deterioration, along with cutaneous and mucosal candidiasis. Subsequent hospital evaluation led to diagnoses of epilepsy, hypocalcemia, hypoparathyroidism, and hypothyroidism. Treatment was initiated with oral vitamin D, calcium, levothyroxine, and valproic acid.\u003c/p\u003e\n\u003cp\u003eAlthough Whole Exome Sequencing (WES) was recommended to identify a potential genetic etiology, the parents initially declined due to the absence of familial disease history. Despite further counseling on the necessity of genetic testing and its role in elucidating de novo mutations, they maintained their decision to refuse.\u003c/p\u003e\n\u003cp\u003eBy the age of 8, the boy\u0026apos;s seizures had become more frequent. The clinical situation was further clarified when his younger brother also suffered a seizure and was diagnosed with hypocalcemia. This development led the parents to agree to genetic testing. The results demonstrated a homozygous deletion in the \u003cem\u003eAIRE\u003c/em\u003e gene（Figure 3） in both brothers, with their parents being heterozygous carriers. A definitive diagnosis of Autoimmune Polyglandular Syndrome Type 1 (APS-1). The elder brother carries homozygous\u003cem\u003e\u0026nbsp;FOXRED1\u003c/em\u003e Figure 4）variants. His clinical presentation\u0026mdash;including vision loss, cerebral atrophy with partial encephalomalacia on MRI, microcephaly (head circumference 49cm, \u0026lt;-3SD), lactic acidosis (3.2mmol/L, high), developmental delay, and epilepsy\u0026mdash;is consistent with \u003cem\u003eFOXRED1\u003c/em\u003e-related mitochondrial encephalopathy and supports the pathogenicity of these mutations.\u003c/p\u003e\n\u003cp\u003eThe ratio of the copy number of exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the proband to that of the normal control was approximately 0, indicating a \u003cstrong\u003ehomozygous deletion\u0026nbsp;\u003c/strong\u003eof exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the patient The ratio of the copy number of exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the patient\u0026apos;s father and mother to that of the normal control was approximately 0.5, indicating a \u003cstrong\u003eheterozygous deletion\u003c/strong\u003e of exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the patient\u0026apos;s parents. The ratio of the copy number of exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the patient\u0026apos;s brother to that of the normal control was approximately 0, indicating a \u003cstrong\u003ehomozygous deletion\u003c/strong\u003e of exons 2-4 of the \u003cem\u003eAIRE\u003c/em\u003e gene in the patient\u0026apos;s brother.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eNote\u003c/strong\u003e: The normal reference range for the ratio is 0.8-1.2. A ratio greater than 1.25 but less than 1.75 suggests a suspected single-copy duplication; a ratio greater than 1.75 but less than 2.25 suggests a suspected two-copy duplication. A ratio greater than 0.35 but less than 0.75 suggests a suspected heterozygous deletion; a ratio of 0 indicates a homozygous deletion. Other values are considered ambiguous, and the specific result cannot be determined.)\u003c/p\u003e\n\u003cp\u003eThis chromatogram confirms the identified \u003cem\u003eFOXRED1\u0026nbsp;\u003c/em\u003evariant (c.580C\u0026gt;T, p.Arg194Trp) in the family.\u003c/p\u003e\n\u003cp\u003ePatient: Homozygous for the mutation (T/T), consistent with the autosomal recessive disease inheritance. Father \u0026amp; Mother: Both are heterozygous carriers (C/T), each having one mutant and one normal allele. Brother: Also a heterozygous carrier (C/T), confirming the familial segregation of the variant.\u003c/p\u003e"},{"header":"Discuss","content":"\u003cp\u003eAutoimmune polyglandular syndrome type 1 (APS-1) is a rare disorder, with a particularly high prevalence of 1 in 9,000 among Iranian or Persian Jews, whereas the global prevalence is estimated to be between 1 in 90,000 and 1 in 200,000\u003csup\u003e1\u003c/sup\u003e. Autoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disorder caused by pathogenic variants in the \u003cem\u003eAIRE\u003c/em\u003e gene. It typically presents in early childhood (2\u0026ndash;5 years of age) and is characterized by the production of autoantibodies against type I interferons. The condition manifests with a spectrum of endocrine and non-endocrine features. Classic endocrine disorders include hypoparathyroidism, adrenal insufficiency, gonadal failure, type 1 diabetes, and autoimmune thyroiditis. Common non-endocrine manifestations encompass chronic mucocutaneous candidiasis, vitiligo, alopecia, as well as gastrointestinal, hepatic, and hematological involvements\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and in these cases, it\u0026rsquo;s included femoral head necrosis, brain infarction.\u003c/p\u003e \u003cp\u003ePrevious studies show that the onset of symptoms in APS type 1 corresponds to the age of early childhood, while the first clinical symptoms (candidiasis of the skin and mucous membranes) manifest themselves in early childhood up to 5 years, hypoparathyroidism up to 10 years, and Addison\u0026rsquo;s disease up to 15 years\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Diagnostic delay is a hallmark of APS-1 due to its variable symptomatology. Although our proband's age at first symptom (5 years) was typical, the presentation was complicated by a movement disorder, ultimately explained by a concurrent mitochondrial encephalopathy. The definitive diagnosis was achieved through WES. A significant factor in the delay was the initial parental decline of genetic testing, based on the absence of a family history. Parental consent was subsequently obtained following the onset of illness in the second child, which facilitated the identification of the underlying \u003cem\u003eAIRE\u003c/em\u003e and \u003cem\u003eFOXRED1\u003c/em\u003e mutations in both siblings.\u003c/p\u003e \u003cp\u003eA notable aspect of this case is the elder brother's severe neurological presentation, including global developmental delay, cerebral atrophy, partial encephalomalacia, microcephaly, and lactic acidosis\u0026mdash;a constellation of features not typically associated with APS-1. The identification of a homozygous missense mutation in \u003cem\u003eFOXRED1\u003c/em\u003e via trio-WES provided a conclusive explanation, establishing a dual diagnosis of FOXRED1-related mitochondrial encephalopathy. This gene encodes a 486-amino acid, FAD-dependent oxidoreductase that functions as a mitochondria-targeted assembly factor for complex I of the oxidative phosphorylation system\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Pathogenic mutations in \u003cem\u003eFOXRED1\u003c/em\u003e have been established in association with severe neurological disorders, including Leigh syndrome and infantile-onset mitochondrial encephalopathy \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. To date, 11 pathogenic variants in \u003cem\u003eFOXRED1\u003c/em\u003e have been reported, and all cases of associated mitochondrial encephalopathy are consistent with an autosomal recessive inheritance pattern \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Previous studies have established that \u003cem\u003eFOXRED1\u003c/em\u003e mutations are associated with a range of neurological features, including athetoid movements, hypotonia, irritability, acquired microcephaly, and cortical blindness, typically presenting in early childhood \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. According to the ACMG criteria 2015 guidelines\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, the homozygous variants of c.580(exon5)C\u0026thinsp;\u0026gt;\u0026thinsp;T in our report is classified as likely pathogenic (PM1, PM2, PP3, PP5), and FOXRED1 function has been confirming by animal experiment\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This case illustrates that in patients with multisystem involvement and atypical features, the possibility of dual genetic diagnoses should be actively considered, even in the absence of consanguinity.\u003c/p\u003e \u003cp\u003eOur case underscores the critical importance of re-evaluating a diagnosis when a single genetic finding cannot fully explain the complete clinical phenotype. Although dual genetic diagnoses are increasingly recognized, conventional genetic tests may still miss such cases. For instance, D.D. Tian et al. reported one of the largest series of dual genetic diagnoses in recent years\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e,conventional genetic tests. This report highlights that a deep understanding of clinical phenotypes is paramount, as it guides the interpretation of genetic results, not vice versa.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReheman Palati\u0026nbsp;and\u0026nbsp;Jing Yu\u0026nbsp;contributed to conception.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReheman Palati\u0026nbsp;contributed to literature review and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eJirong Feng, Lei He, Maimaitiyiming Mireayi, Aimaiti Aerziguli\u0026nbsp;critically revised the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors\u0026nbsp;gave final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to the children, their families, and colleagues in the imaging department；\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVerbal informed consent for publication of their clinical presentation and course was obtained from the patient and their parents.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eProust-Lemoine E, Saugier-Veber P, Wemeau JL. Polyglandular autoimmune syndrome type I. Presse Med. 2012;41:e651\u0026ndash;62.\u003c/li\u003e\n\u003cli\u003eLankisch TO, Jaeckel E, Strassburg CP. The autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or autoimmune polyglandular syndrome type 1. Semin Liver Dis. 2009;29:307\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eAhonen P, Myllarniemi S, Sipila I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med. 1990;322:1829\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eKisand K, Peterson P. Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. J Clin Immunol. 2015;35:463\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eFormosa LE, Mimaki M, Frazier AE, McKenzie M, Stait TL, Thorburn DR, et al. Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I. Hum Mol Genet. 2015;24:2952\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eCalvo SE, Tucker EJ, Compton AG, Kirby DM, Crawford G, Burtt NP, et al. High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency. Nat Genet. 2010;42:851\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eApatean D, Rakic B, Brunel-Guitton C, Hendson G, Bai R, Sargent MA, et al. Congenital lactic acidosis, cerebral cysts and pulmonary hypertension in an infant with FOXRED1 related complex 1 deficiency. Mol Genet Metab Rep. 2019;19:100472.\u003c/li\u003e\n\u003cli\u003eHu C, Xu Q, Shen J, Wang Y. Clinical and Genetic Characteristics of Mitochondrial Encephalopathy Due to FOXRED1 Mutations: Two Chinese Case Reports and a Review of the Literature. Front Neurol. 2021;12:633397.\u003c/li\u003e\n\u003cli\u003eFassone E, Duncan AJ, Taanman JW, Pagnamenta AT, Sadowski MI, Holand T, et al. FOXRED1, encoding an FAD-dependent oxidoreductase complex-I-specific molecular chaperone, is mutated in infantile-onset mitochondrial encephalopathy. Hum Mol Genet. 2010;19:4837\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eSalama M, El-Desouky S, Alsayed A, El-Hussiny M, Moustafa A, Taalab Y, et al. FOXRED1 silencing in mice: a possible animal model for Leigh syndrome. Metab Brain Dis. 2019;34:367\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eTan DD, Liu YD, Fan YB, Wei CJ, Song DY, Yang HP, et al. [Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses]. Zhonghua Er Ke Za Zhi. 2023;61:345\u0026ndash;50. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autoimmune polyglandular syndrome type 1, FXRED1, Dual genetic diagnosis, Whole-exome sequencing, Mitochondrial encephalopathy","lastPublishedDoi":"10.21203/rs.3.rs-8551715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8551715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutoimmune polyglandular syndrome type 1 (APS-1) is a rare autosomal recessive disorder caused by mutations in the \u003cem\u003eAIRE\u003c/em\u003e gene. While the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency defines the syndrome, its phenotypic spectrum can be broad and complex. While a single genetic etiology is typically assumed, the co-occurrence of a second monogenic disorder can complicate the clinical picture. Here, we report two brothers from a non-consanguineous family who presented with a complex multisystem phenotype including hypocalcemia, seizures, and neurodevelopmental impairments. Whole-exome sequencing revealed a homozygous \u003cem\u003eAIRE\u003c/em\u003e deletion in both siblings, confirming APS-1. Notably, the elder brother was also found to carry a homozygous variant in the \u003cem\u003eFOXRED1\u003c/em\u003e gene, which explained his severe neurological features, including cerebral atrophy and lactic acidosis, consistent with a dual diagnosis of APS-1 and FOXRED1-related mitochondrial encephalopathy. This case highlights that atypical or compounded clinical presentations should raise suspicion for dual genetic disorders, underscoring the critical role of comprehensive genetic analysis in reaching a precise diagnosis.\u003c/p\u003e","manuscriptTitle":"Dual genetic diagnoses: AIRE and FOXRED1-Related Syndromes in two brothers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 16:45:44","doi":"10.21203/rs.3.rs-8551715/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c86eb62b-9359-4e04-b9db-af99159c7cb7","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-17T17:10:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 16:45:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8551715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8551715","identity":"rs-8551715","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}