ATP7A Transcript Research Articles

Heterogeneous nuclear ribonucleoprotein hnRNPA2/B1 regulates the abundance of the copper-transporter ATP7A in an isoform-dependent manner.

Copper (Cu) is an essential micronutrient with a critical role in mammalian growth and development. Imbalance of Cu causes severe diseases in humans; therefore, cellular Cu levels are tightly regulated. Major Cu-transport proteins and their cellular behavior have been characterized in detail, whereas their regulation at the mRNA level and associated factors are not well-understood. We show that the heterogeneous nuclear ribonucleoprotein hnRNPA2/B1 regulates Cu homeostasis by modulating the abundance of Cu(I)-transporter ATP7A. Downregulation of hnRNPA2/B1 in HeLa cells increases the ATP7A mRNA and protein levels and significantly decreases cellular Cu; this regulation involves the 3' UTR of ATP7A transcript. Downregulation of B1 and B1b isoforms of hnRNPA2/B1 is sufficient to elevate ATP7A, whereas overexpression of either hnRNPA2 or hnRNPB1 isoforms decreases the ATP7A mRNA levels. Concurrent decrease in hnRNPA2/B1, increase in ATP7A, and a decrease in Cu levels was observed in neuroblastoma SH-SY5Y cells during retinoic acid-induced differentiation; this effect was reversed by overexpression of B1/B1b isoforms. We conclude that hnRNPA2/B1 is a new isoform-specific negative regulator of ATP7A abundance.

Occipital Horn Syndrome as a Result of Splice Site Mutations in ATP7A. No Activity of ATP7A Splice Variants Missing Exon 10 or Exon 15.

Disease-causing variants in ATP7A lead to two different phenotypes associated with copper deficiency; a lethal form called Menkes disease (MD), leading to early death, and a much milder form called occipital horn syndrome (OHS). Some investigators have proposed that an ATP7A transcript missing exon 10 leads to a partly active protein product resulting in the OHS phenotype. Here, we describe an individual with OHS, a biology professor, who survived until age 62 despite a splice site mutation, leading to skipping of exon 15. ATP7A transcripts missing exon 10, or exon 15 preserve the reading frame, but it is unknown if either of these alternative transcripts encode functional protein variants. We have investigated the molecular consequence of splice site mutations leading to skipping of exon 10 or exon 15 which have been identified in individuals with OHS, or MD. By comparing ATP7A expression in fibroblasts from three individuals with OHS (OHS-fibroblasts) to ATP7A expression in fibroblasts from two individuals with MD (MD-fibroblasts), we demonstrate that transcripts missing either exon 10 or exon 15 were present in similar amounts in OHS-fibroblasts and MD-fibroblasts. No ATP7A protein encoded from these transcripts could be detected in the OHS and MD fibroblast. These results, combined with the observation that constructs encoding ATP7A cDNA sequences missing either exon 10, or exon 15 were unable to complement the high iron requirement of the ccc2Δ yeast strain, provide evidence that neither a transcript missing exon 10 nor a transcript missing exon 15 results in functional ATP7A protein. In contrast, higher amounts of wild-type ATP7A transcript were present in the OHS-fibroblasts compared with the MD-fibroblasts. We found that the MD-fibroblasts contained between 0 and 0.5% of wild-type ATP7A transcript, whereas the OHS-fibroblasts contained between 3 and 5% wild-type transcripts compared with the control fibroblasts. In summary these results indicate that protein variants encoded by ATP7A transcripts missing either exon 10 or exon 15 are not functional and not responsible for the OHS phenotype. In contrast, expression of only 3-5% of wild-type transcript compared with the controls permits the OHS phenotype.

Estimated birth prevalence of Menkes disease and ATP7A-related disorders based on the Genome Aggregation Database (gnomAD)

BackgroundPrevious estimates of the prevalence of Menkes disease, a lethal X-linked recessive disorder of copper metabolism, were based on confirmed clinical cases ascertained from specific populations and varied from 1 in 40,000 to 1 in 354,507. With newly available population-based allelic frequencies of DNA sequence variants, the expected birth prevalence of Menkes disease and other ATP7A-related phenotypes can be reconsidered using Hardy-Weinberg theoretical principles. MethodsWe reviewed the canonical ATP7A transcript in the current version of gnomAD (v2.1.1) to evaluate frequency of complete loss-of-function alleles in a diverse normal control population. As a comparator, we used the DMD locus, associated with Duchenne and Becker Muscular Dystrophy, another X-linked recessive trait. We applied Hardy-Weinberg theory and PolyPhen-2 in silico plus REVEL and CADD ensemble analyses to calculate estimated frequencies of normal and predicted deleterious ATP7A alleles. ResultsWe identified 1106 total ATP7A variants out of 205,523 alleles in gnomAD, with missense variants most common (43.4%). Complete loss-of-function variants were found in four ATP7A alleles (frequency = 0.0000194), including three frameshift/nonsense mutations and one canonical splice donor site defect. Assuming Hardy-Weinberg equilibrium, this frequency of pathogenic alleles predicts 1 in 34,810 live male births with Menkes disease or other ATP7A-related disorders each year in the US. The same analysis for DMD loss-of-function variants predicted 1 in 7246 newborn males with Duchenne (or Becker) muscular dystrophy. We also identified nine ATP7A missense variants in gnomAD predicted as deleterious by PolyPhen-2 and stringent REVEL/CADD criteria, comprising 12 more disease-causing alleles and raising the estimated birth prevalence to 1 in 8664 and predicting 225 newborns with Menkes disease or other ATP7A-related disorders per year in the US alone. ConclusionsAssuming Hardy-Weinberg equilibrium, the allelic frequency of deleterious ATP7A variants in a genomic database from a large diverse population predicts a birth prevalence of Menkes disease or ATP7A-related disorders as high as 1 in 8664 live male births. This genome-driven ascertainment of deleterious ATP7A alleles in the population implies a higher birth prevalence of Menkes disease and ATP7A-related conditions than previously appreciated. A population-based newborn screening pilot study for Menkes disease will be instrumental in confirming the prediction.

Evidence of common cadmium and copper uptake routes in zebrafish Danio rerio.

Cadmium and copper accumulations in gills of zebrafish were measured during a 48 h exposure to 0.025 μM 106Cd and 0.05 or 0.5 μM 65Cu as a single metal or their mixtures. The gill transcript levels of genes involved in the transport of Cu (CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1), and Zn (ZIP8) were also compared between treatments at 24 and 48 h. Cd uptake was significantly suppressed in the presence of Cu, indicating interaction between Cu and Cd at uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation rates in the presence of Cu was associated with an increase in transcript abundance of ECaC at 24 h and DMT1 at 48 h and a decrease in Zip8 transcript levels, all known as routes for Cd uptake. Fish exposed to 0.5 μM 65Cu show an increase in gill ATP7a transcript abundance, suggesting that Cu is removed from the gill and is transferred to other organs for detoxification. A reduction in gill CTR1 transcript abundance was observed during the Cu-Cd exposure; this may be a regulatory mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes, such as ECaC and DMT1.

Small amounts of functional ATP7A protein permit mild phenotype

Mutations in ATP7A lead to at least three allelic disorders: Menkes disease (MD), Occipital horn syndrome and X-linked distal motor neuropathy. These disorders are mainly seen in male individuals, but a few affected females have been described. More than 400 different mutations have been identified in the ATP7A gene. We have conducted several studies in the hope of uncovering the relationship between genotype and phenotype. We have examined the X-inactivation pattern in affected females, the effect of exon-deletions and--duplications, and splice-site mutations on the composition and amount of ATP7A transcript, and we have examined the structural location of missense mutations. The X-inactivation pattern did not fully explain the manifestation of MD in a small fraction of carriers. Most of the affected females had preferential inactivation of the X-chromosome with the normal ATP7A gene, but a few individuals exhibited preferential inactivation of the X-chromosome with the mutated ATP7A gene. The observed mild phenotype in some patients with mutations that effect the composition of the ATP7A transcript, seems to be explained by the presence of a small amount of normal ATP7A transcript. The location of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with copper, whereas at higher concentrations, ATP7A shifts to the post-Golgi compartments or to the plasma membrane to export copper out of the cell. Impaired copper-regulation trafficking has been observed for ATP7A mutants, but its impact on the clinical outcome is not clear. The major problem in patients with MD seems to be insufficient amounts of copper in the brain. In fact, prenatal treatment of mottled mice as a model for human MD with a combination of chelator and copper, produces a slight increase in copper levels in the brain which perhaps leads to longer survival and more active behavior. In conclusion, small amounts of copper at the right location seem to relieve the symptoms.

Prenatal diagnostic conundrum involving a novel ATP7A duplication

Prenatal diagnostic conundrum involving a novel <i><scp>ATP7A</scp></i> duplication

Multiple Menkes copper ATPase (Atp7a) transcript and protein variants are induced by iron deficiency in rat duodenal enterocytes

The Menkes copper ATPase (Atp7a) pumps copper into the trans-Golgi for cuproenzyme synthesis, and translocates to the basolateral membrane of enterocytes for copper export. Recent studies demonstrated that three 5′ end splice variants of the Atp7a transcript exist in rat duodenum, all of which are strongly induced during iron deprivation. To explore a possible role for Atp7a (and copper) in intestinal iron absorption, the current studies were undertaken to test the hypothesis that multiple Atp7a transcript and protein variants exist in intestinal epithelial cells. Northern blot analyses using probes generated from the full-length Atp7a cDNA revealed several specific hybridization bands, all of which were more intense in RNA samples extracted from duodenal enterocytes isolated from iron-deficient rats. A PCR-based approach, using forward primers specific for the alternative 5′ end splice variants and a reverse primer in exon 23, demonstrated that 3 full-length transcripts exist in rat IEC-6 cells. To identify possible Atp7a protein variants, three distinct polyclonal antisera were utilized. The specificity of the antisera was first established by western blotting and immunoprecipitation studies using samples derived from isolated rat enterocytes and Atp7a knockdown IEC-6 cells. Several specific immunoreactive bands were documented, and a unique Atp7a protein distribution in cytosolic vesicle-like structures was noted. In conclusion, multiple Atp7a transcript and protein variants exist in rodent intestinal epithelial cells and are induced by dietary iron deprivation. Further studies will be designed to determine the subcellular distribution of Atp7a protein variants and possible unique functions of each.

Twenty-five novel mutations including duplications in the ATP7A gene

Twenty-five novel mutations including duplications in the ATP7A gene. Menkes disease (MD) and occipital horn syndrome (OHS) are allelic X-linked recessive copper deficiency disorders resulting from ATP7A gene mutations. MD is a severe condition leading to progressive neurological degeneration and death in early childhood, whereas OHS has a milder phenotype with mainly connective tissue abnormalities. Until now, molecular analyses have revealed only deletions and point mutations in both diseases. This study reports new molecular data in a series of 40 patients referred for either MD or OHS. We describe 23 point mutations (9 missense mutations, 7 splice site variants, 4 nonsense mutations, and 3 small insertions or deletions) and 7 intragenic deletions. Of these, 18 point mutations and 3 deletions are novel. Furthermore, our finding of four whole exon duplications enlarges the mutation spectrum in the ATP7A gene. ATP7A alterations were found in 85% of cases. Of these alterations, two thirds were point mutations and the remaining one third consisted of large rearrangements. We found that 66.6% of point mutations resulted in impaired ATP7A transcript splicing, a phenomenon more frequent than expected. This finding enabled us to confirm the pathogenic role of ATP7A mutations, particularly in missense and splice site variants.

Differences in ATP7A gene expression underlie intrafamilial variability in Menkes disease/occipital horn syndrome

Background: Pronounced intrafamilial variability is unusual in Menkes disease and its variants. We report two unrelated families featuring affected members with unusually disparate clinical and biochemical phenotypes and explore the...

Atp7a determines a hierarchy of copper metabolism essential for notochord development

The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.

Similar Splice-Site Mutations of the ATP7A Gene Lead to Different Phenotypes: Classical Menkes Disease or Occipital Horn Syndrome

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

The mottled mouse as a model for human Menkes disease: identification of mutations in the Atp7a gene.

Mutations in the Atp7a gene, the mouse homologue of the MNK (ATP7A) gene, have been suggested to be responsible for the mottled phenotype. To date, despite considerable effort, changes associated with the mottled mutations have been detected in only two such mutants. In this study, we identify changes in the level of Atp7a transcript and mutations which could explain the mottled phenotype in nine out of the 10 mutants analysed. The fluorescence-assisted mismatch analysis method used here has proved particularly well suited for mRNA scanning of heterozygous carrier animals, because of its ability to detect mutations even in the presence of an excess of wild-type mRNA. The three new underlying mutations identified at the Atp7a locus include a splice mutation and two missense mutations. While the spectrum of mutations detected in the Atp7a murine gene provides an explanation for at least part of the wide phenotypic variation observed in mottled mutant mice, there is a singular absence of deletions which are associated with a sizeable fraction of human Menkes syndrome cases.