Title: Homozygous Loss-of-Function Mutations in AP1B1, Encoding Beta-1 Subunit of Adaptor-Related Protein Complex 1, Cause MEDNIK-like Syndrome
Abstract: MEDNIK syndrome (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma) is an autosomal-recessive disorder caused by bi-allelic mutations in AP1S1, encoding the small σ subunit of the AP-1 complex. Central to the pathogenesis of MEDNIK syndrome is abnormal AP-1-mediated trafficking of copper transporters; this abnormal trafficking results in a hybrid phenotype combining the copper-deficiency-related characteristics of Menkes disease and the copper-toxicity-related characteristics of Wilson disease. We describe three individuals from two unrelated families in whom a MEDNIK-like phenotype segregates with two homozygous null variants in AP1B1, encoding the large β subunit of the AP-1 complex. Similar to individuals with MEDNIK syndrome, the affected individuals we report display abnormal copper metabolism, evidenced by low plasma copper and ceruloplasmin, but lack evidence of copper toxicity in the liver. Functional characterization of fibroblasts derived from affected individuals closely resembles the abnormal ATP7A trafficking described in MEDNIK syndrome both at baseline and in response to copper treatment. Taken together, our results expand the list of inborn errors of copper metabolism. MEDNIK syndrome (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma) is an autosomal-recessive disorder caused by bi-allelic mutations in AP1S1, encoding the small σ subunit of the AP-1 complex. Central to the pathogenesis of MEDNIK syndrome is abnormal AP-1-mediated trafficking of copper transporters; this abnormal trafficking results in a hybrid phenotype combining the copper-deficiency-related characteristics of Menkes disease and the copper-toxicity-related characteristics of Wilson disease. We describe three individuals from two unrelated families in whom a MEDNIK-like phenotype segregates with two homozygous null variants in AP1B1, encoding the large β subunit of the AP-1 complex. Similar to individuals with MEDNIK syndrome, the affected individuals we report display abnormal copper metabolism, evidenced by low plasma copper and ceruloplasmin, but lack evidence of copper toxicity in the liver. Functional characterization of fibroblasts derived from affected individuals closely resembles the abnormal ATP7A trafficking described in MEDNIK syndrome both at baseline and in response to copper treatment. Taken together, our results expand the list of inborn errors of copper metabolism. Copper is an essential metal in the human body, which contains 1.2-2.2 mg of copper per kg of total weight.1Olivares M. Uauy R. Limits of metabolic tolerance to copper and biological basis for present recommendations and regulations.Am. J. Clin. Nutr. 1996; 63: 846S-852SCrossref PubMed Scopus (65) Google Scholar Exogeneous copper is absorbed by enterocytes via the human copper transport protein 1 (hCTR1), secreted into the circulation via a copper membrane-transporter (ATP7A), transported in blood in both ceruloplasmin-bound (in plasma) and superoxide-dismutase-bound (in red blood cells) forms, and is avidly taken up by hepatocytes via hCTR1.2Latorre M. Troncoso R. Uauy R. Biological Aspects of Copper.in: Kerkar N. Roberts E.A. Clinical and Translational Perspectives on Wilson Disease. Elsevier, 2019: 25-31Crossref Scopus (21) Google Scholar In the liver, another copper membrane transporter (ATP7B) transports copper into the secretory pathway for incorporation into apoceruloplasmin as well as biliary excretion.3Ferenci P. Pathophysiology and clinical features of Wilson disease.Metab. Brain Dis. 2004; 19: 229-239Crossref PubMed Scopus (108) Google Scholar Also important for copper metabolism are intracellular proteins known as metallothioneins, which bind metals, including copper, to protect against their toxicity, and metallochaperones, which mediate the transfer of copper from metallothioneins to physiologically active copper-containing proteins.4Palmiter R.D. The elusive function of metallothioneins.Proc. Natl. Acad. Sci. USA. 1998; 95: 8428-8430Crossref PubMed Scopus (602) Google Scholar, 5Huffman D.L. O'Halloran T.V. Function, structure, and mechanism of intracellular copper trafficking proteins.Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (420) Google Scholar, 6Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase.Science. 1999; 284: 805-808Crossref PubMed Scopus (1366) Google Scholar In addition to the liver, the brain and kidney represent the other major sites of copper concentration. Copper influences numerous cellular processes with important physiological consequences. As the central metal ion of cytochrome c oxidase, it mediates energy production by the electron transport chain. It also serves as anti-oxidant as part of Cu/Zn superoxide dismutase, helps dopamine synthesis as a co-factor for dopamine β-hydroxylase, and maintains extracellular matrix integrity by promoting Cu-dependent lysyl oxidase-mediated collagen crosslinking.7Kaplan J.H. Maryon E.B. How mammalian cells acquire copper: An essential but potentially toxic metal.Biophys. J. 2016; 110: 7-13Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Copper has also been demonstrated to modulate the activity of several signaling pathways, including those involving Akt and MAPK, and to be involved in inflammation, fibrosis, and lipogenesis, among other physiological roles.8Grubman A. White A.R. Copper as a key regulator of cell signalling pathways.Expert Rev. Mol. Med. 2014; 16: e11Crossref PubMed Scopus (104) Google Scholar, 9Tallino S. Duffy M. Ralle M. Cortés M.P. Latorre M. Burkhead J.L. Nutrigenomics analysis reveals that copper deficiency and dietary sucrose up-regulate inflammation, fibrosis and lipogenic pathways in a mature rat model of nonalcoholic fatty liver disease.J. Nutr. Biochem. 2015; 26: 996-1006Crossref PubMed Scopus (34) Google Scholar, 10Quiroz N. Rivas N. del Pozo T. Burkhead J. Suazo M. González M. Latorre M. Transcriptional activation of glutathione pathways and role of glucose homeostasis during copper imbalance.Biometals. 2015; 28: 321-328Crossref PubMed Scopus (7) Google Scholar The two classic inborn errors of copper metabolism are Wilson disease [MIM: 277900] and Menkes disease [MIM: 309400]. Wilson disease is caused by recessive mutations in ATP7B [MIM: 606882]; these mutations result in progressive hepatotoxicity due to failure of biliary excretion of copper. Eventually, the growing hepatic stores of copper escape into the circulation, largely in a ceruloplasmin-free form because the synthesis of ceruloplasmin requires active ATP7B, and cause widespread copper-induced tissue damage, especially in the brain.3Ferenci P. Pathophysiology and clinical features of Wilson disease.Metab. Brain Dis. 2004; 19: 229-239Crossref PubMed Scopus (108) Google Scholar Menkes disease, on the other hand, represents a true copper-deficiency state caused by an inability to utilize dietary copper, which becomes locked inside enterocytes as a result of hemizygous mutations in the X chromosome gene ATP7A [MIM: 300011]; active ATP7A is required for the efflux of copper from enterocytes into the circulation. The phenotype of Menkes disease, therefore, is very different from that of Wilson disease and is characterized by severe encephalopathy, growth retardation, severe connective-tissue manifestations, and death in early childhood.11Kaler S.G. ATP7A-related copper transport diseases-emerging concepts and future trends.Nat. Rev. Neurol. 2011; 7: 15-29Crossref PubMed Scopus (414) Google Scholar More recently, inborn errors of copper metabolism have been expanded to include less familiar multisystem disorders. Huppke-Brendel syndrome (HBS) was originally proposed in 2005 and later confirmed in 2012 by the identification of additional individuals with the same phenotype (severe encephalopathy, cataract, progressive hearing loss, and very low serum copper and ceruloplasmin levels) and was found to be caused by recessive mutations in SLC33A1 [MIM: 603690], encoding the acetyl-CoA transporter AT-1.12Horváth R. Freisinger P. Rubio R. Merl T. Bax R. Mayr J.A. Shawan Müller-Höcker J. Pongratz D. Moller L.B. et al.Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.J. Inherit. Metab. Dis. 2005; 28: 479-492Crossref PubMed Scopus (21) Google Scholar, 13Huppke P. Brendel C. Kalscheuer V. Korenke G.C. Marquardt I. Freisinger P. Christodoulou J. Hillebrand M. Pitelet G. Wilson C. et al.Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin.Am. J. Hum. Genet. 2012; 90: 61-68Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar The pathophysiology is thought to be related to impaired post-translational acetylation of ceruloplasmin and, potentially, of ATP7A and ATP7B.14Bandmann O. Weiss K.H. Kaler S.G. Wilson's disease and other neurological copper disorders.Lancet Neurol. 2015; 14: 103-113Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar Another multisystem disorder characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratoderma (MEDNIK [MIM: 609313]) was originally described in 2008 but was only shown to be related to abnormal copper metabolism in 2013.15Montpetit A. Côté S. Brustein E. Drouin C.A. Lapointe L. Boudreau M. Meloche C. Drouin R. Hudson T.J. Drapeau P. Cossette P. Disruption of AP1S1, causing a novel neurocutaneous syndrome, perturbs development of the skin and spinal cord.PLoS Genet. 2008; 4: e1000296Crossref PubMed Scopus (109) Google Scholar, 16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar MEDNIK is caused by recessive mutations in AP1S1 [MIM: 603531], encoding the σ1A small subunit of the adaptor-related protein complex-1 (AP-1), which is involved in intracellular trafficking of transmembrane proteins, including ATP7A.16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar The resulting hypocupremia, hypoceruloplasminemia, and liver copper accumulation lead to a hybrid phenotype combining the copper-deficiency-related characteristics of Menkes disease and the copper-toxicity-related characteristics of Wilson disease.16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar Here, we describe two unrelated families where loss-of-function variants in a gene encoding another subunit of AP-1 segregate with a phenotype that overlaps considerably with that of MEDNIK; this overlap includes evidence of copper metabolic derangement and abnormal ATP7A trafficking. Family 1 is from the UK and of Pakistani origin (Figure 1). The index individual (family 1_II:1, individual 1) is the first child to healthy parents, who are half second cousins. She has congenital ichthyosis, enteropathy, and mild persisting hepatopathy. Failure to thrive, global developmental delay, and bilateral severe to profound sensorineural hearing loss ensued later. Clinical examination revealed mild facial dysmorphism and generalized ichthyosis with a fine, whitish scale associated with erythroderma and sparse hair. Her body tone is normal, but all of her deep tendon reflexes are brisk. Liver biopsy at the age of 10 months showed normal histology with no evidence of copper accumulation. Skin biopsy at the age of 14 months was consistent with ichthyosis, but precise subtyping was not possible. The parents' second pregnancy resulted in the birth of a son (family 1_II:2, individual 2) who had ichthyosis with erythroderma and diarrhea in the neonatal period. Subsequent problems included enteropathy, severe failure to thrive, global developmental delay, hearing loss, narrow and incomplete cleft of the soft palate, anemia, and respiratory infections. Clinical examination revealed frontal bossing, sparse scalp hair, generalized ichthyosis and erythroderma, and hepatomegaly, and a hypoplastic scrotum with undescended testes was also noted. He has biochemical abnormalities similar to those of his sister, i.e., these include variably elevated ALT levels, normal GGT levels, persistently low plasma copper and caeruloplasmin levels, and mildly elevated very-long-chain fatty acids (Figure 1, Table 1, and Supplemental Clinical Data).Table 1Summary of Clinical Characteristics of MEDNIK-like PhenotypeFamily112Individual ID123Pedigree IDII-1II-2II-2GenderFMMAge4 years, 4 months1 year, 5 months4 years, 6 monthsGenotypegross deletion, chr22: 29758984–29815476gross deletion, chr22: 29758984–29815476NM_001127: c.38-1G>AClinical FeaturesMEDNIKIntellectual disability+NA++Global dev delay++++Enteropathy+ (early onset)+ (later onset)-+Deafness++++Neuropathy---+Ichthyosis++++Palmoplantar keratoderma--++Erythroderma++++Hepatopathy++-+Hairsparse; wiry texturesparse; wiry texturesparsesparseLaboratory abnormalitiesAST and ALT (<48 U/L)ALT measured many times: from 29–376ALT measured many times: from 21–57AST 37.1 and ALT 16.2±↑Serum albumin (40–50 g/L)Measured many times: from 22–29NA40.3NGGT (8–61 U/L)measured many times: from 11–48measured many times: from 14–488NALP (142–335 U/L)measured many times: from 253–928measured many times: from 204–1,278236.3↑Plasma alanine (70–700 umol/L)NANA243↑Plasma lactate (0.5–2 mmol/L)0.9 and 1.8NA1.84↑Total bile acidsNAglyco-dihydroxycholanoate 2.6 μmol/mmol Cr (normal 0.02–0.47); glyco-trihydroxycholanoate 34.8 μmol/mmol Cr (normal 0.04–1.39); tauro-dihydroxycholanoate 2.20 μmol/L (normal 0.01–0.08); tauro-trihydroxycholanoate 20.1 μmol (normal 0.01–0.08)NA↑Plasma copper (12–25 μmol/L)measured many times: from 5–10measured many times: from 2–105.4↓Liver copper 8–40 mcg/g17NANA↑Ceruloplasmin (150–300 mg/L)measured many times: from 98–119measured many times: from 93–100131↓VLCFA: C26 (0.45–1.32 μmol/L) C24/C22 ratio (≤1.2) C26/C22 ratio (0.005–0.030)↑ C26 (1.63 μmol/L), C24/C22 ratio (0.83) and ↑ C26/C22 ratio (0.047)↑ C26 (2.12 μmol/L), C24/C22 ratio (1.08) and ↑ C26/C22 ratio (0.053)C26 (0.477 μmol/L), C24/C22 ratio (1.15) and C26/C22 ratio (0.021)↑Phytanate (0.2–19.3 μmol/L)1.40.50.82NPristanate (0–1.88 μmol/mmol Cr)0.1900.245NPlasma zinc (10.6–19 μmol/L)measured many times: from 8–22measured many times: from 9–1610±Brain MRICerebral atrophy-+NA+Basal ganglia abnormalities--NA+Nerve conduction studyNANAunremarkableperipheral neuropathyAbbreviations are as follows: AST, aspartate aminotransferase; ALT, alanine aminotransferase; and NA, not applicable. Open table in a new tab Abbreviations are as follows: AST, aspartate aminotransferase; ALT, alanine aminotransferase; and NA, not applicable. Chromosomal microarray with the Affymetrix 750K CytoScan chip revealed that both children have a homozygous loss of 75 Kb at chromosomal region 22q12.2; this loss spans AP1B1 (chromosomal coordinates 29,756,293–29,831,540 with probe positions mapped to NCBI Build 37). The healthy parents are both heterozygous for this microdeletion. The index individual in family 2 (family 2_II:2, individual 3) is a 4-year-, 6-month-old boy born to consanguineous healthy Saudi parents (Figure 1). He had been noted since birth to have scaly skin, which persisted and evolved into generalized ichthyosis with associated palmoplantar hyperkeratosis. He later developed developmental delay, bilateral profound sensorineural deafness, and failure to thrive. Clinical examination revealed mild frontal bossing, sparse eyebrows, and generalized ichthyosiform scaling with palmoplantar keratoderma. He has low serum copper and ceruloplasmin levels, and normal liver function (Figure 1, Table 1 and Supplemental Clinical Data). Clinical exome sequencing suggested a homozygous variant in AP1B1 (NM_001127:c.38-1G>A) as a potential candidate. Through personal communication between the teams involved in the care of the two families and in light of the remarkably similar phenotype, both families were recruited with informed consent under an IRB-approved protocol (KFSRHC RAC# 2121053). The deletion breakpoints in family 1 were delineated by PCR deletion mapping and Sanger sequencing (chr22: 29758984-29815476). Interestingly, this deletion is flanked by two homologous segments of DNA, suggesting a potential underlying mechanism (Figure S1). This deletion removes the tentative promoter as well as the first two exons of AP1B1 (exon 2 is the first coding exon), so it is predicted to be a null at the transcript level or protein level. Indeed, a skin biopsy was obtained from individual 2, and fibroblast-derived RNA was tested by RT-PCR, which confirmed abrogation of the AP1B1 transcript (Figure 2). Similarly, to investigate the effect of c.38-1G>A variant in family 2, we obtained a skin biopsy from individual 3 and confirmed by RT-PCR the deleterious splicing nature of this variant on the canonical splice acceptor. Specifically, this variant leads to replacement of the normal transcript by an aberrant transcript that harbors one base-pair deletion (r.40del), again predicting loss of function (p.Glu14Argfs∗5) (Figure 2). Thus, our data show that a highly consistent MEDNIK-like phenotype segregates with a different homozygous loss-of-function variant in each of the two affected families (Figure 2). Adaptins are ∼100 kDa proteins first identified as binding partners of clathrin in the study of clathrin-coated vesicles (CCVs) and later found to be subunits of heterotetrameric adaptor protein (AP) complexes.17Boehm M. Bonifacino J.S. Adaptins: the final recount.Mol. Biol. Cell. 2001; 12: 2907-2920Crossref PubMed Scopus (359) Google Scholar Each of the four known AP complexes (AP-1 to AP-4) comprises two large (one each of γ, α, δ, ε, and β1–β4, respectively), one medium (μ1– μ4), and one small adaptin (σ1–σ4).17Boehm M. Bonifacino J.S. Adaptins: the final recount.Mol. Biol. Cell. 2001; 12: 2907-2920Crossref PubMed Scopus (359) Google Scholar The AP-1 complex functions in conjunction with clathrin to mediate the trafficking of membrane proteins to the plasma membrane from the trans-Golgi network (TGN) and endosomes.18Fölsch H. Ohno H. Bonifacino J.S. Mellman I. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.Cell. 1999; 99: 189-198Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar Interestingly, it has been shown that two of the key copper transporters, ATP7A and ATP7B, depend on AP-1 for their trafficking.19Holloway Z.G. Velayos-Baeza A. Howell G.J. Levecque C. Ponnambalam S. Sztul E. Monaco A.P. Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2-, AP-1-, and Rab22-dependent steps.Mol. Biol. Cell. 2013; 24 (S1–S8): 1735-1748Crossref PubMed Scopus (44) Google Scholar, 20Hirst J. Borner G.H. Antrobus R. Peden A.A. Hodson N.A. Sahlender D.A. Robinson M.S. Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system.Curr. Biol. 2012; 22: 1711-1716Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Jain S. Farías G.G. Bonifacino J.S. Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine signal by AP-1.Mol. Biol. Cell. 2015; 26: 218-228Crossref PubMed Scopus (37) Google Scholar Indeed, in their pursuit to explain the copper metabolic defect observed in MEDNIK individuals, Martinelli and colleagues showed that AP1S1 regulates the correct intracellular localization of ATP7A.16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar Specifically, they showed that both the baseline TGN localization and copper-induced plasma membrane localization of ATP7A are impaired in fibroblasts derived from MEDNIK-affected individuals but readily rescued by the heterologous expression of normal AP1S1.16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar In order to test whether the strong, yet incomplete phenotypic overlap between the affected individuals reported here and MEDNIK (they did not have evidence of copper toxicity or neuropathy) is mirrored by an overlap in the pathophysiological perturbation, we sought to replicate the above experiment by Martinelli and colleagues (Supplemental Methods). As shown in Figure 3, we observed an even more dramatic perturbation of ATP7A trafficking than that caused by AP1S1 deficiency. Specifically, the colocalization of ATP7A with TGN at baseline was completely random in the affected individuals, in contrast to the nearly perfect colocalization observed in control (Figure 3). In addition, we quantified this trafficking abnormality by using 2D intensity histogram and Pearson's correlation coefficient, and the difference from control was highly significant (p value (2 tailed) = 7 × 10−10, Figure S4). This suggests that, similar to σ deficiency, deficiency of the β1 subunit of the AP-1 complex leads to abnormal trafficking of ATP7A and results in copper metabolic derangement very similar to that observed in MEDNIK. Our results are also consistent with a study noting abnormal AP-1-mediated trafficking resulting from an ap1b1 mutation in zebrafish, although in that case the studied protein was the Na+/K+-ATPase pump.22Clemens Grisham R. Kindt K. Finger-Baier K. Schmid B. Nicolson T. Mutations in ap1b1 cause mistargeting of the Na(+)/K(+)-ATPase pump in sensory hair cells.PLoS ONE. 2013; 8: e60866Crossref PubMed Scopus (20) Google Scholar Although Martinelli and colleagues could not study the trafficking of ATP7B in cells from affected individuals because it is a liver-specific copper transporter, they speculated that the liver copper toxicity in those individuals is related to abnormal ATP7B trafficking.16Martinelli D. Travaglini L. Drouin C.A. Ceballos-Picot I. Rizza T. Bertini E. Carrozzo R. Petrini S. de Lonlay P. El Hachem M. et al.MEDNIK syndrome: a novel defect of copper metabolism treatable by zinc acetate therapy.Brain. 2013; 136: 872-881Crossref PubMed Scopus (96) Google Scholar None of the three affected individuals we report in this study had evidence of copper toxicity. Remarkably, even individual 1, with the most significant hepatopathy, had no increased copper content on direct measurement in the liver, which indicates another mechanism for liver involvement. On the other hand, it appears likely that abnormal ATP7A trafficking and the resulting impaired utilization of dietary copper is not the sole pathomechanism becuase there are clear differences between the phenotype of the affected individuals we report here and the Menkes disease phenotype. For example, their elevated very-long-chain fatty acids (VLCFA), which was also observed in MEDNIK, suggests a potential peroxisomal biogenesis defect (PBD). However, liver involvement in PBD is different from that observed here, e.g., jaundice is typical. Furthermore, when we analyzed peroxisomal integrity by using the 70 kDa peroxisomal membrane protein (PMP70), results were inconsistent with a bona fide PBD (Figure S3). In order to investigate the possibility that AP1B1 deficiency might exert its pathogenesis through a generalized defect in CCVs, we pursued proteomic analysis of CCVs essentially as described before (Supplemental Methods).23Navarro Negredo P. Edgar J.R. Wrobel A.G. Zaccai N.R. Antrobus R. Owen D.J. Robinson M.S. Contribution of the clathrin adaptor AP-1 subunit μ1 to acidic cluster protein sorting.J. Cell Biol. 2017; 216: 2927-2943Crossref PubMed Scopus (24) Google Scholar As shown in Table S1, AP1B1 was the only AP1 or AP2 CCV component that was consistently reduced in both individual 2 and individual 3 beyond a log2 ratio cutoff of 0.5 for these individuals compared to controls. These results suggest that AP1B1 deficiency results in a rather specific defect in CCVs, at least to the extent this was evaluated in a single cell type, i.e., fibroblasts. However, they also make it clear that future work is needed to investigate the consequences of AP1B1-related defective trafficking on additional proteins that might play a role in the pathogenesis of this disease. In summary, we show that bi-allelic loss-of-function variants in AP1B1 largely phenocopy MEDNIK syndrome caused by AP1S1 mutation. Although part of the phenotypic discrepancy between the individuals we report here and those affected by MEDNIK (lack of neuropathy specifically) may be related to their young age, it seems likely that lack of copper toxicity reflects a fundamental difference in the pathogenesis of AP1B1 and AP1S1 deficiencies. Our findings not only expand the list of copper-related metabolic diseases in humans but also point to a potential therapeutic avenue in the form of copper replacement. Authors declare no conflict of interest. We thank the study families for their enthusiastic participation. We thank Mais Hashem, Firdous Abdulwahab, and Niema Ibrahim for their help as clinical coordinators, Eman Al-Obeid, Tarfa Alshidi, and Mona Alanazi for their help with the tissue culture, and the Genotyping and Sequencing Core Facilities at King Faisal Specialist Hospital and Research Center for their technical help. This work was supported in part by King Salman Center for Disability Research. Download .pdf (1.59 MB) Help with pdf files Document S1. Supplemental Clinical Data, Figures S1–S4, and Supplemental Methods Download .xlsx (.02 MB) Help with xlsx files Table S1. Proteomic Analysis of CCVs in Individuals and Controls Imagej, https://imagej.net/index.htmlOMIM, http://omim.org/ Recessive Mutations in AP1B1 Cause Ichthyosis, Deafness, and PhotophobiaBoyden et al.The American Journal of Human GeneticsOctober 17, 2019In BriefWe describe unrelated individuals with ichthyosis, failure to thrive, thrombocytopenia, photophobia, and progressive hearing loss. Each have bi-allelic mutations in AP1B1, the gene encoding the β subunit of heterotetrameric adaptor protein 1 (AP-1) complexes, which mediate endomembrane polarization, sorting, and transport. In affected keratinocytes the AP-1 β subunit is lost, and the γ subunit is greatly reduced, demonstrating destabilization of the AP-1 complex. Affected cells and tissue contain an abundance of abnormal vesicles and show hyperproliferation, abnormal epidermal differentiation, and derangement of intercellular junction proteins. Full-Text PDF Open Archive