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• W2898095583 abstract "Ca2+ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, and dysregulation of ER Ca2+ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca2+ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca2+ storage, impaired Ca2+ signaling mediated by the IP3R Ca2+ release channel, and reduced ER Ca2+ refilling via store-operated Ca2+ entry. These results, together with the previously described role of CCDC47 in Ca2+ signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder. Ca2+ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, and dysregulation of ER Ca2+ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca2+ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca2+ storage, impaired Ca2+ signaling mediated by the IP3R Ca2+ release channel, and reduced ER Ca2+ refilling via store-operated Ca2+ entry. These results, together with the previously described role of CCDC47 in Ca2+ signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder. Ca2+ signaling is a multipurpose intracellular signaling system that regulates a number of cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, transcription, and cellular proliferation.1Berridge M.J. Bootman M.D. Roderick H.L. Calcium signalling: dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4201) Google Scholar The endoplasmic reticulum (ER), or the sarcoplasmic reticulum (SR) in muscle cells, is the largest store of intracellular Ca2+.2Somlyo A.P. Bond M. Somlyo A.V. Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in vivo.Nature. 1985; 314: 622-625Crossref PubMed Scopus (221) Google Scholar ER Ca2+ depletion is also observed in a number of genetic disorders due to variants in Ca2+ channels and sensors. For example, Brody myopathy (MIM: 601003) is caused by recessive variants in ATP2A1 (MIM: 611974), which encodes the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA1),3Odermatt A. Taschner P.E. Khanna V.K. Busch H.F. Karpati G. Jablecki C.K. Breuning M.H. MacLennan D.H. Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease.Nat. Genet. 1996; 14: 191-194Crossref PubMed Scopus (184) Google Scholar while Darier disease (MIM: 124200) occurs due to variants in ATP2A2 (MIM: 108740), which encodes another sarcoplasmic reticulum Ca2+ ATPase, SERCA2.4Sakuntabhai A. Ruiz-Perez V. Carter S. Jacobsen N. Burge S. Monk S. Smith M. Munro C.S. O’Donovan M. Craddock N. et al.Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease.Nat. Genet. 1999; 21: 271-277Crossref PubMed Scopus (616) Google Scholar Minicore myopathy (MIM: 255320) and central core disease (MIM: 117000) result from variants in RYR1 (MIM: 180901), which encodes a major Ca2+ release channel,5Zhang Y. Chen H.S. Khanna V.K. De Leon S. Phillips M.S. Schappert K. Britt B.A. Browell A.K. MacLennan D.H. A mutation in the human ryanodine receptor gene associated with central core disease.Nat. Genet. 1993; 5: 46-50Crossref PubMed Scopus (291) Google Scholar and autosomal centronuclear myopathy (MIM: 160150) is associated with variants in MTMR14 (MIM: 611089), which encodes a muscle-specific inositol phosphatase.6Shen J. Yu W.M. Brotto M. Scherman J.A. Guo C. Stoddard C. Nosek T.M. Valdivia H.H. Qu C.K. Deficiency of MIP/MTMR14 phosphatase induces a muscle disorder by disrupting Ca(2+) homeostasis.Nat. Cell Biol. 2009; 11: 769-776Crossref PubMed Scopus (78) Google Scholar Stormorken syndrome (MIM: 185070), tubular aggregate myopathy 1 (MIM: 160565), and immunodeficiency 10 (MIM: 612783) are caused by variants in STIM1 (MIM: 605921),7Misceo D. Holmgren A. Louch W.E. Holme P.A. Mizobuchi M. Morales R.J. De Paula A.M. Stray-Pedersen A. Lyle R. Dalhus B. et al.A dominant STIM1 mutation causes Stormorken syndrome.Hum. Mutat. 2014; 35: 556-564Crossref PubMed Scopus (123) Google Scholar, 8Böhm J. Chevessier F. Maues De Paula A. Koch C. Attarian S. Feger C. Hantaï D. Laforêt P. Ghorab K. Vallat J.M. et al.Constitutive activation of the calcium sensor STIM1 causes tubular-aggregate myopathy.Am. J. Hum. Genet. 2013; 92: 271-278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Picard C. McCarl C.A. Papolos A. Khalil S. Lüthy K. Hivroz C. LeDeist F. Rieux-Laucat F. Rechavi G. Rao A. et al.STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity.N. Engl. J. Med. 2009; 360: 1971-1980Crossref PubMed Scopus (401) Google Scholar which encodes a Ca2+ sensor. Tubular aggregate myopathy 2 (MIM: 615883) and immunodeficiency 9 (MIM: 612782) are caused by variants in ORAI1 (MIM: 610277),10Endo Y. Noguchi S. Hara Y. Hayashi Y.K. Motomura K. Miyatake S. Murakami N. Tanaka S. Yamashita S. Kizu R. et al.Dominant mutations in ORAI1 cause tubular aggregate myopathy with hypocalcemia via constitutive activation of store-operated Ca2+ channels.Hum. Mol. Genet. 2015; 24: 637-648Crossref PubMed Scopus (108) Google Scholar, 11Feske S. Gwack Y. Prakriya M. Srikanth S. Puppel S.H. Tanasa B. Hogan P.G. Lewis R.S. Daly M. Rao A. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.Nature. 2006; 441: 179-185Crossref PubMed Scopus (1849) Google Scholar which encodes a Ca2+ channel that coordinates ER Ca2+ refilling via store-operated Ca2+ entry (SOCE). Additionally, disruption of ER Ca2+ homeostasis contributes to the pathogenesis of several common diseases including diabetes mellitus, neurological diseases, and cancer.12Mekahli D. Bultynck G. Parys J.B. De Smedt H. Missiaen L. Endoplasmic-reticulum calcium depletion and disease.Cold Spring Harb. Perspect. Biol. 2011; 3: 3Crossref Scopus (305) Google Scholar CCDC47, also known as calumin, is present in several tissues including brain, lung, heart, stomach, liver, spleen, kidney, muscle, and testis.13Zhang M. Yamazaki T. Yazawa M. Treves S. Nishi M. Murai M. Shibata E. Zorzato F. Takeshima H. Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum.Cell Calcium. 2007; 42: 83-90Crossref PubMed Scopus (20) Google Scholar CCDC47 is an ER transmembrane Ca2+-binding protein involved in embryogenesis and development.13Zhang M. Yamazaki T. Yazawa M. Treves S. Nishi M. Murai M. Shibata E. Zorzato F. Takeshima H. Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum.Cell Calcium. 2007; 42: 83-90Crossref PubMed Scopus (20) Google Scholar, 14Yamamoto S. Yamazaki T. Komazaki S. Yamashita T. Osaki M. Matsubayashi M. Kidoya H. Takakura N. Yamazaki D. Kakizawa S. Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.Dev. Biol. 2014; 393: 33-43Crossref PubMed Scopus (11) Google Scholar A reported Ccdc47-knockout mouse model exhibited delayed development, atrophic neural tubes, heart abnormalities, a paucity of blood cells in the dorsal aorta, and embryonic lethality.14Yamamoto S. Yamazaki T. Komazaki S. Yamashita T. Osaki M. Matsubayashi M. Kidoya H. Takakura N. Yamazaki D. Kakizawa S. Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.Dev. Biol. 2014; 393: 33-43Crossref PubMed Scopus (11) Google Scholar Further, mouse embryonic fibroblasts (MEFs) from these mice exhibited impaired Ca2+ signaling.13Zhang M. Yamazaki T. Yazawa M. Treves S. Nishi M. Murai M. Shibata E. Zorzato F. Takeshima H. Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum.Cell Calcium. 2007; 42: 83-90Crossref PubMed Scopus (20) Google Scholar These data suggest that CCDC47 is critical for Ca2+ signaling and normal development. In this study, we report four unrelated individuals presenting with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay; the clinical features of the probands are summarized in Table 1. We performed molecular analyses on probands who were referred to one of the collaborating centers for diagnostic evaluation of an undiagnosed genetic disorder and for whom prior genetic testing had been unrevealing. The parents of probands 1, 3, and 4 provided informed consent for sample collection and molecular analyses under protocol 76-HG-0238 approved by the NHGRI Institutional Review Board. The family of proband 2 gave consent for research studies through the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) initiative under protocol #H-29697 approved by the Institutional Review Board at Baylor College of Medicine. The families of probands 3 and 4 were recruited for research studies through the Clinic for Special Children under a Lancaster General Hospital Institutional Review Board-approved protocol. Blood samples were collected from the probands and their unaffected parents and, when available, their unaffected siblings for whole-exome sequencing. Skin biopsies or peripheral blood leukocytes were obtained from the proband when possible for further molecular analyses. Using whole-exome sequencing, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. Further details on the methodologies used in this study are available in the Supplemental Data.Table 1Summary of Clinical Features of Individuals with Bi-allelic Loss-of-Function CCDC47 VariantsClinical FeaturesProband 1Proband 2Proband 3Proband 4Prenatal and Perinatal HistoryDeliveryC-sectionC-sectionNSVDC-sectionPremature birth+termtermtermPolyhydramnios+–––Respiratory distress–NA++Decreased fetal movementsNA++NABradycardia++–+Birth weight (%ile)75thNA<3rd10thGrowth ParametersDecreased body weight++++Microcephaly++++Physical FindingsCoarse facies++++Midface hypoplasia+++–Hypertelorism+++–Almond-shaped palpebral fissure++––Epicanthal folds––+–Ptosis++++Long eyelashes++––Synophrys+–++Ectropion++––Unusual nose++++Downturned mouth++++Macrostomia–+wide mouth–Macroglossia–+++Full or thick lips++++Dental abnormalities+–++High arched palate++++Ear abnormalities++++Bilateral otitis media++++Bitemporal narrowing–+++Brachycephaly++++Plagiocephaly+++–Pruritus++++Unusual hair++++Thoracic hypertrichosis++++Fifth digit hypoplasia and/or clinodactyly++++Dystrophic nails––+–Overlapping toes++++Distal arthrogryposis / joint laxity++++Hypoplastic nipples++++Genital anomaly++––Musculoskeletal FindingsHypotonia++++Bilateral hip dislocation++ND–Hip dysplasia++ND+Bilateral coxa valga+–ND+Abnormal bone density++NDNDNarrow chest++––Fibular bowing++––Genu valgum––+–Bilateral clubfoot++–+Small feet++++Pectus excavatum+––+Scoliosis–++–Ocular FindingsHyperopia+NA–+Astigmatism+NA––Cortical visual impairment+NA++Immunological FindingsRecurrent infections–++–Immunodeficiency–++–Endocrine FindingsHypothyroidism–NA+–Rickets–++–Respiratory FindingsObstructive sleep apnea+++–Central sleep apnea+NA+–Heart FindingsVentricular septal defect–+––Patent ductus arteriosus++––Gastrointestinal FindingsHepatosplenomegaly+–+–Liver dysfunction+ND++Recurrent pancreatitis+NA––Exocrine pancreatic insufficiency+NA––Gastresophageal reflux+NA++Steatorrhea++––Chronic diarrhea–+–+Gallstones+––+Gastrostomy tube+–++Elevated bile acids+NA++Renal FindingsRenal abnormalities++––Neurological FindingsSevere global developmental delay++++Hyperreflexia––++Reduced tendon reflexes++––Absent Achilles reflex++––Behavioral issues––++Seizures–NA+–EEG abnormalities+NA++Neuroimaging FindingsAbnormal ventricle morphology++–+Abnormal corpus callosum+––+Cerebral atrophy++++White matter abnormalities–––+Cerebellar hypoplasia–––+Abbreviations: +, present; – absent; C-section, Caesarean section; EEG, electroencephalogram; NA, not available; ND, not done; NSVD, normal spontaneous vaginal delivery. Open table in a new tab Abbreviations: +, present; – absent; C-section, Caesarean section; EEG, electroencephalogram; NA, not available; ND, not done; NSVD, normal spontaneous vaginal delivery. Proband 1 (1: II-1) was a 5-year-old female at the time she was evaluated through the National Institutes of Health Undiagnosed Diseases Program.15Gahl W.A. Tifft C.J. The NIH Undiagnosed Diseases Program: lessons learned.JAMA. 2011; 305: 1904-1905Crossref PubMed Scopus (89) Google Scholar, 16Gahl W.A. Markello T.C. Toro C. Fajardo K.F. Sincan M. Gill F. Carlson-Donohoe H. Gropman A. Pierson T.M. Golas G. et al.The National Institutes of Health Undiagnosed Diseases Program: insights into rare diseases.Genet. Med. 2012; 14: 51-59Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 17Gahl W.A. Mulvihill J.J. Toro C. Markello T.C. Wise A.L. Ramoni R.B. Adams D.R. Tifft C.J. UDNThe NIH Undiagnosed Diseases Program and Network: Applications to modern medicine.Mol. Genet. Metab. 2016; 117: 393-400Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar She was born to non-consanguineous parents of mixed Northern European and Native American descent. There were three miscarriages subsequent to the birth of proband 1. The pregnancy was complicated by premature rupture of membranes at 30 weeks of gestation, requiring preterm delivery by Caesarean section. At birth, she exhibited microcephaly, hypotonia, bilateral club foot deformities, and a patent ductus arteriosus (PDA). Complete blood counts identified anemia during infancy, which resolved by 5 years of age. She was unable to breast or bottle feed and was admitted to the NICU, where she was diagnosed with oral motor dyspraxia and severe gastresophageal reflux (GERD). At approximately 3 years of age, she experienced an episode of pancreatitis with liver inflammation. She had recurrent steatorrhea and low fecal elastase levels, and multiple gray-black cholesterol stones; cholecystectomy failed to resolve the problem and she underwent recurrent hospitalizations for similar episodes of pancreatitis. Evaluations for primary biliary cholangitis and autoimmune hepatitis were negative, and she was diagnosed with exocrine pancreatic insufficiency. She had dysmorphic facial features including coarse and woolly hair, midface hypoplasia, hypertelorism, ptosis, a downturned mouth, full lips, dental abnormalities, a high arched palate, low-set ears, brachycephaly, and plagiocephaly (Figure 1A and Table 1). Further clinical examination revealed distal arthrogryposis, fifth digit hypoplasia, a narrow chest, hypoplastic nipples, hip dysplasia, clitoral hyperplasia, fibular bowing, and overlapping toes (Figure 1A and Table 1). Ophthalmologic evaluation showed astigmatism, hyperopia, and cortical visual impairment. Skeletal survey was consistent with osteopenia. She exhibited both truncal and appendicular hypotonia with poor head control and severe global developmental delay. She had frog leg posturing when supine. She could not hold objects, bear weight, or sit up without support. A brain MRI showed abnormalities of the corpus callosum as well as mild prominence of the third ventricle (Figure S1A). Additional clinical data are available in the Supplemental Note. Proband 2 (2: II-3) was a male first seen by the Department of Medical Genetics at Dr. Sami Ulus Research and Training Hospital of Women’s and Children’s Health and Diseases at age 2 years 7 months. He was born at term via Caesarean section with a birth weight of 3,000 g. The parents were first-degree cousins of Turkish origin and they had two healthy living children and reported four previous miscarriages as well as two miscarriages subsequent to the birth of proband 2. The parents first noticed decreased spontaneous movements and hypotonia at 2 months of life. The infant had no head control and no single words. He was below the 3rd percentile for all anthropometric measurements, with severe malnutrition. Dysmorphic features included woolly and thin blonde hair, macroglossia, macrostomia, and simple large ears (Figure 1B and Table 1). The proband also exhibited bilateral cryptorchidism. Ophthalmologic evaluation was unremarkable. Laboratory studies including chemistry, blood count, metabolic testing (urine organic acid, ammonia, plasma amino acid, lactate, and pyruvate), congenital disorders of glycosylation testing, karyotype, and subtelomeric FISH were negative or inconclusive. Echocardiogram showed a ventricular septal defect (VSD) and PDA; abdominal ultrasound revealed nephrocalcinosis. Skeletal survey was consistent with osteoporosis (Figure S1B). Brain CT showed mild dilation of the lateral ventricles and cerebral atrophy. The boy was last evaluated at age 8 and the mother had two more miscarriages in the interim. He was referred to Baylor-Hopkins Center for Mendelian Genomics (BHCMG) to identify the molecular etiology. Proband 3 (3: II-8) was an Old Order Amish female first seen at The Community Health Clinic (Topeka, IN) at age 7 years 7 months. She was born at 38 weeks of gestation via normal spontaneous vaginal delivery at home with a birth weight of 2,070 g and considered to be small for gestational age. The mother noticed slower and less frequent movements compared to her previous pregnancies. Due to respiratory distress, the newborn was transported to Wright Memorial Hospital (Trenton, MO) where she was placed on oxygen for 12–24 hr. She was frequently ill and diagnosed with failure to thrive (FTT); at 3 months of age a gastrostomy tube (G-tube) was placed. Dysmorphic features included microcephaly, dark and curly hair, epicanthal folds, hypertelorism, a bulbous nasal tip, and a wide and downturned mouth (Figure 1C and Table 1). She also had small hands and feet, dystrophic nails, and abnormal chubby toes that overlapped (Figure 1C and Table 1). The proband had a history of feeding issues, FTT, GERD, and liver dysfunction with mild splenomegaly and a prominent left hepatic lobe; itching improved on cholestyramine. She had recurrent infections due to a Toll-like receptor signaling defect, which was treated with IVIG, as well as central hypothyroidism and vitamin D-deficient rickets. Proband 3 also had chronic respiratory insufficiency and a history of apnea and sleep disturbances. Developmentally, she was severely delayed, non-verbal, and had generalized hypotonia. She could not grasp objects or sit up but was able to roll onto her side. Behaviorally, she displayed bruxism and self-mutilation; treatment with Risperdal resolved these behaviors. A brain MRI revealed mild prominence of the CSF space (Figure S1A). The parents had five healthy living children, two miscarriages, and two males who passed away at 3.5 months (3: II-3) due to aspiration pneumonia and at 7 months of gestation (3: II-6) (Figure 2A). A maternal uncle passed away at 9 weeks of age due to kidney failure. All had dark curly hair similar to proband 3 (3: II-8). Additional clinical data are available in the Supplemental Note. Proband 4 (4: II-1) was first seen at The Community Health Clinic (Topeka, IN) at age 6 years 6 months. At birth, she was transferred to the Memorial Hospital NICU (South Bend, IN) for 5 days due to episodes of oxygen desaturation and poor feeding. At 1 year 6 months, she had a G-tube placed due to FTT. Her dysmorphic features included microcephaly, red curly hair, synophrys, full lips, and a downturned mouth (Figure 1D and Table 1). A skeletal survey showed that she had bilateral talipes equinovarus, coxa valga, bilateral overlapping toes, pectus excavatum, and hypermobile joints (Figure 1D and Table 1). Ophthalmologic evaluation showed she had hyperopia and cortical visual impairment. Proband 4 had abnormal liver function tests, elevated serum bile acids, and pruritus; itching improved on cholestyramine. She also had cholelithiasis without secondary evidence of acute cholecystitis; partial visualization of the pancreas was unremarkable. Neurologically, she had severe global developmental delay, hyperreflexia, hypotonia, and poor head control; she was non-verbal, although she sometimes answered “yah.” She displayed bruxism and self-mutilation and also clapped or hit herself when excited. A brain MRI showed minimal prominence of the cerebral sulci and ventricular enlargement, global white matter paucity, and a thin corpus callosum (Figure S1A). The parents had three healthy living children subsequent to the birth of the proband (Figure 2A). Additional clinical data are available in the Supplemental Note. Whole-exome sequencing was performed on these four probands at three different research centers to identify pathogenic variants underlying their disease (Supplemental Subjects and Methods).18Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (830) Google Scholar Variant interpretation and prioritization was based on the clinical relevance of the gene and the pathogenicity of the variants using ACMG-AMP guidelines.19Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. et al.ACMG Laboratory Quality Assurance CommitteeStandards 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: 405-424Abstract Full Text Full Text PDF PubMed Scopus (14827) Google Scholar Further variant prioritization was based on Mendelian consistency and segregation, observed frequency of the variants in public and internal population databases, conservation, and predicted deleteriousness coalesced with published biological and functional data of the candidate genes. Each center independently identified compound heterozygous or homozygous variants in CCDC47 (GenBank: NM_020198.2) segregating according to Mendelian expectations for an autosomal-recessive disease trait (Figures 2A, 2B, and S2; Table 2). All of the CCDC47 variants identified were either nonsense or frameshift variants that are predicted to lead to nonsense-mediated mRNA decay or premature truncation of the protein (Figure 2C and Table 2). The allele frequencies of the identified CCDC47 variants in population databases, such as the Genome Aggregation Database (gnomAD), were very low ranging from 0.000% to 0.010% (Table 2) with no homozygotes recorded. In addition, these variants are predicted to be pathogenic by multiple bioinformatic algorithms (Table 2).Table 2Summary of Bi-allelic Loss-of-Function Variants Identified in CCDC47 (GenBank: NM_020198.2)ProbandAncestryReported ConsanguinityNucleotide Change (hg19 genomic coordinates)Coding Sequence ChangeAmino Acid ChangeInheritanceParent of OrigingnomAD AllCADD Phred ScoreAOH Region Containing Candidate (Mb)Genomewide AOH (Mb)1Northern European/Native AmericannoChr17:g.61829694G>Ac.1189C>Tp.Arg397∗compound heterozygousP0.010%40NANAChr17:g.61829718delc.1165delTp.Ser389Leufs∗25M0.000%34NANA2TurkishyesChr17:g.61833855G>Ac.811C>Tp.Arg271∗homozygousboth0.001%391.3268.43AmishnoChr17:g.61829738delc.1145delTp.Leu382Argfs∗2homozygousboth0.002%3519.681.24AmishnoChr17:g.61829738delc.1145delTp.Leu382Argfs∗2homozygousboth0.002%3515.765.1Abbreviations: AOH, absence of heterozygosity; CADD, Combined Annotation Dependent Depletion; gnomAD, Genome Aggregation Database; M, maternal; NA, not applicable; P, paternal. Open table in a new tab Abbreviations: AOH, absence of heterozygosity; CADD, Combined Annotation Dependent Depletion; gnomAD, Genome Aggregation Database; M, maternal; NA, not applicable; P, paternal. To experimentally assess the functional consequences of the variants identified, we performed TaqMan gene expression analysis to quantify CCDC47 mRNA, western blot to assess the levels of CCDC47, and indirect immunofluorescence microscopy to assess the localization of the protein (Supplemental Subjects and Methods and Tables S1–S3). Gene expression analyses showed that the relative CCDC47 mRNA was decreased in the primary dermal fibroblasts of probands 1 (1: II-1), 3 (3: II-8), and 4 (4: II-1) compared to two unaffected sex-matched pediatric controls (Figure 2D, upper panel) and in the lymphoblastoid cells of proband 2 (2: II-3) compared to his father (2: I-1) and unaffected sibling (2: II-2) (Figure 2D, lower panel). Consistent with the predicted loss-of-function effect of the identified variants, CCDC47 levels were nearly undetectable in the cells from all four probands, as assessed using an antibody that recognizes the C terminus of CCDC47 (Figure 2E). These results were consistent using an antibody that recognizes the N terminus of CCDC47 (data not shown). Cell studies showed that CCDC47 was localized in an ER-like pattern in unaffected control cells and that the signal for CCDC47 was undetectable by immunofluorescence using primary dermal fibroblasts from proband 1 (Figure 2F), consistent with the observation from western blot experiments. Altogether, our experiments support the hypothesis that the variants in CCDC47 lead to nonsense-mediated decay of the prematurely truncated transcripts and result in the absence of protein and a functional loss of CCDC47. To further explore the functional effects of loss of CCDC47, we performed in vitro experiments to interrogate ER Ca2+ storage, Ca2+ release, and store-operated Ca2+ entry (SOCE). CCDC47 has been previously shown to bind Ca2+,13Zhang M. Yamazaki T. Yazawa M. Treves S. Nishi M. Murai M. Shibata E. Zorzato F. Takeshima H. Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum.Cell Calcium. 2007; 42: 83-90Crossref PubMed Scopus (20) Google Scholar so we hypothesized that the loss of CCDC47 expression would lead to impaired ER Ca2+ storage, signaling, and refilling. We performed live-cell imaging using the cell-permeable Ca2+ indicator Fura-2-acetoxymethyl ester (Fura-2AM) to monitor the elevation of cytoplasmic Ca2+ following the addition of either the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin at a high concentration to completely deplete ER Ca2+ levels and assess total ER Ca2+. In addition, IP3 was used to induce Ca2+ release via the inositol 1,4,5-trisphosphate receptor (IP3R), ryanodine to induce Ca2+ release via the ryanodine receptor (RyR), thapsigargin at a low concentration to determine ER Ca2+ leak, and thapsigargin at a high concentration to deplete ER Ca2+ levels followed by CaCl2 to assess refilling via SOCE (Supplemental Subjects and Methods). Complete inhibition of SERCA, which transports Ca2+ from the cytoplasm into the ER, was achieved by the addition of 2 μM thapsigargin, which leads to rapid depletion of ER Ca2+ stores and reflects total ER Ca2+ levels.20Thastrup O. Cullen P.J. Drøbak B.K. Hanley M.R. Dawson A.P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.Proc. Natl. Acad. Sci. USA. 1990; 87: 2466-2470Crossref PubMed Scopus (2999) Google Scholar Our results show that total ER Ca2+ was decreased in the primary dermal fibroblasts of all three probands tested compared to unaffected control cells (Figure 3A). Ca2+ release via IP3R was decreased in the primary dermal fibroblasts of all three probands tested (Figure 3B), while Ca2+ release via RyR was decreased only in proband 1 compared to that of unaffected control subjects (Figure 3C). Although proband 4 has generally lower Ca2+ released after addition of ryanodine, the difference from control subjects was not statistically significant. Partial inhibition of SERCA by the addition of 0.2 μM thapsigargin unmasks ER Ca2+ leak, a constitutive process mediated via ion channels such as presenilin 1 and bax inhibitor 1.21Nelson O. Tu H. Lei T." @default.
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• W2898095583 date "2018-11-01" @default.
• W2898095583 modified "2023-10-18" @default.
• W2898095583 title "Bi-allelic CCDC47 Variants Cause a Disorder Characterized by Woolly Hair, Liver Dysfunction, Dysmorphic Features, and Global Developmental Delay" @default.
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• W2898095583 doi "https://doi.org/10.1016/j.ajhg.2018.09.014" @default.
• W2898095583 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6218603" @default.
• W2898095583 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30401460" @default.
• W2898095583 hasPublicationYear "2018" @default.
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