Multiplex PCR-based Sequencing of ATP7B Gene in Wilson's Disease - A Preliminary Study.
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SUBMITTER: Aaron R
PROVIDER: S-EPMC9077194 | biostudies-literature | 2022 Mar-Apr
REPOSITORIES: biostudies-literature
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Multiplex PCR-based Sequencing of <i>ATP7B</i> Gene in Wilson's Disease - A Preliminary Study.
Aaron Rekha R Chapla Aaron A Danda Sumita S Zachariah Uday U Eapen Chundamannil E CE Goel Ashish A
Journal of clinical and experimental hepatology 20210806 2
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Project description:BACKGROUND:Wilson disease is a rare disorder of copper metabolism due to mutation in ATP7B gene. Proper counseling of patients with Wilson disease, and their families necessitates finding mutation in ATP7B gene. Finding mutations in ATP7B gene with 21 exons, and more than 500 mutations is expensive and time-consuming. OBJECTIVES:The aim of this study was to provide a simple multiplex amplification refractory mutation system PCR (M-ARMS-PCR) for screening eight common mutations in ATP7B gene. PATIENTS AND METHODS:Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2. The Multiplex ARMS assay was then subsequently tested in 65 patients with Wilson disease with known and unknown ATP7B mutations. RESULTS:Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients. CONCLUSIONS:The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.
| S-EPMC3753551 | biostudies-literature
Project description:BackgroundWilson's disease (WD) is an autosomal recessive genetic disease caused by mutations in ATP7B and characterized by copper metabolism disorders.MethodsDirect sequencing of the ATP7B gene is the most sensitive and widely used confirmatory testing method. Fourteen probands with WD and 12 family members participated in this study. The ATP7B gene was analyzed by direct sequencing.ResultsTwenty-nine different variants (27 substitutions, 1 duplication, 1 deletion) were found. Of the 23 reported variants, nine nondisease variants, 11 disease variants, one silent variant, and two variants with uncertain functions were identified. The six novel variants included c.1875T>A, c.2306T>C, c.3028A>G, c.3243G>A, c.3437_3438 delTG, and c.3903+5G>A.ConclusionThese findings will assist in the diagnosis of WD. The novel variants have enriched the WD database.
| S-EPMC6503029 | biostudies-literature
Project description:Background and aimsWilson's disease (WD) is a rare hereditary disorder due to ATP7B gene mutation, causing pathologic copper storage mainly in the liver and neurological systems. Hepatocyte transplantation showed therapeutic potential; however, this strategy is often hindered by a shortage of quality donor cells and by allogeneic immune rejection. In this study, we aimed to evaluate the function and efficacy of autologous reprogrammed, ATP7B gene-restored hepatocytes using a mouse model of WD.Approach and resultsSufficient liver progenitor cells (LPCs) were harvested by reprogramming hepatocytes from ATP7B-/- mice with small molecules, which exhibited strong proliferation and hepatic differentiation capacity in vitro. After lentivirus-mediated mini ATP7B gene transfection and redifferentiation, functional LPC-ATP7B-derived hepatocytes (LPC-ATP7B-Heps) were developed. RNA sequencing data showed that, compared with LPC-green fluorescent protein-Heps (LPC-GFP-Heps) with enrichment of genes that were mainly in pathways of oxidative stress and cell apoptosis, in LPC-ATP7B-Heps under high copper stress, copper ion binding and cell proliferation pathways were enriched. LPC-ATP7B-Heps transplantation into ATP7B-/- mice alleviated deposition of excess liver copper with its associated inflammation and fibrosis, comparable with those observed using normal primary hepatocytes at 4 months after transplantation.ConclusionsWe established a system of autologous reprogrammed WD hepatocytes and achieved ATP7B gene therapy in vitro. LPC-ATP7B-Heps transplantation demonstrated therapeutic efficacy on copper homeostasis in a mouse model of WD.
| S-EPMC9790736 | biostudies-literature
Project description:Wilson's disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver, brain, and other organs. The disease is caused by pathogenic variants in the ATP7B gene, which encodes a P-type copper transport ATPase. Diagnosing WD is associated with numerous difficulties due to the wide range of clinical manifestations and its unknown dependence on the physiological characteristics of the patient. This leads to a delay in the start of therapy and the subsequent deterioration of the patient's condition. However, in recent years, molecular genetic testing of patients using next generation sequencing (NGS) has been gaining popularity. This immediately affected the detection speed of WD. If, previously, the frequency of this disease was estimated at 1:35,000-45,000 people, now, when conducting large molecular genetic studies, the frequency is calculated as 1:7026 people. This certainly points to the problem of identifying WD patients. This review provides an update on the performance of epidemiological studies of WD and describes normal physiological functions of the protein and diversified disfunctions depending on pathogenic variants of the ATP7B gene. Future prospects in the development of WD genetic diagnostics are also discussed.
| S-EPMC10889319 | biostudies-literature
Project description:Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b -/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b -/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b -/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b -/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b -/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b -/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b -/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b -/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b -/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b -/- mice.
| S-EPMC6994990 | biostudies-literature
Project description:Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B resulting in copper overload in the liver and brain. Direct sequencing is routinely used to confirm WD diagnosis; however, partial and whole gene deletions in the heterozygous state cannot be detected by exon amplification since the normal allele will mask its presence. The aim of the present work was to search for unusual mutational events in the unexplained WD cases and to provide insight into the mechanisms. Out of 1420 clinically and biochemically confirmed WD samples received between 2000 and 2014 for routine mutation analysis, we were unable to detect mutant alleles in 142 samples, after extensive sequencing analysis. We used selective amplification and MLPA to identify the partial gene deletions and identified three different partial gene deletions in seven different families. All three deletions were fully characterized at the DNA sequence level. We report the first hemizygous case with WD due to intragenic deletion in the ATP7B (c.3134_3556+689del). This novel deletion resulted from an excision event mediated by consensus sequences in an AluSq2 repeat element and could be traced to micro homologous end joining (MMEJ). Finally, we determined the prevalence of the three deletions in DNA samples from a multinational group of WD patients. Our results emphasize the need for searching mutant alleles beyond routine methods and highlight that large ATP7B deletions are rare, but account for a detectable proportion in some WD patients. Screening for gene aberrations will further improve mutation detection in patients with unidentified ATP7B mutations presenting with clinical manifestations of WD.
| S-EPMC5167361 | biostudies-literature
Project description:Wilson's disease (WD) is an inherited disorder of copper metabolism characterized by liver disease and/or neurologic and psychiatric pathology. The disease is a result of mutation in ATP7B, which encodes the ATP7B copper transporting ATPase. Loss of copper transport function by ATP7B results in copper accumulation primarily in the liver, but also in other organs including the brain. Studies in the Atp7b(-/-) mouse model of WD revealed specific transcript and metabolic changes that precede development of liver pathology, most notably downregulation of transcripts in the cholesterol biosynthetic pathway. In order to gain insight into the molecular mechanisms of transcriptomic and metabolic changes, we used a systems approach analysing the pre-symptomatic hepatic nuclear proteome and liver metabolites. We found that ligand-activated nuclear receptors FXR/NR1H4 and GR/NR3C1 and nuclear receptor interacting partners are less abundant in Atp7b(-/-) hepatocyte nuclei, while DNA repair machinery and the nucleus-localized glutathione peroxidase, SelH, are more abundant. Analysis of metabolites revealed an increase in polyol sugar alcohols, indicating a change in osmotic potential that precedes hepatocyte swelling observed later in disease. This work is the first application of quantitative Multidimensional Protein Identification Technology (MuDPIT) to a model of WD to investigate protein-level mechanisms of WD pathology. The systems approach using "shotgun" proteomics and metabolomics in the context of previous transcriptomic data reveals molecular-level mechanisms of WD development and facilitates targeted analysis of hepatocellular copper toxicity.
| S-EPMC3695828 | biostudies-literature
Project description:BackgroundHigh resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.Material & methodsHere, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling.ResultsHigh-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry.ConclusionIn summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis.
| S-EPMC4459702 | biostudies-literature
Project description:BackgroundWilson's disease (WD) is a rare autosomal-recessive, inherited disorder caused by a mutation in the copper-transporting gene ATP7B affecting the liver and nervous system. About 30% of patients with WD may initially present with psychiatric symptoms, and diagnosis can be difficult to establish. The objectives of the present preliminary study were [1] to evaluate the relevance of serum copper (Cu) and ceruloplasmin (Cp) measures in hospitalized patients with psychiatric disorders; and [2] to identify possible mutations in the ATP7B gene in patients with abnormal biological copper profile.MethodsAll psychiatric patients who participated in this study were hospitalized in Saint-Jean de Dieu Hospital (Lyon, France). Cp was measured by immunoturbidimetry and serum Cu by inductively coupled plasma-optical emission spectrometry. When Cp and serum Cu levels were inferior to, respectively, 0.18 g/L and 0.88 mg/L in combination with atypical psychiatric presentations, complete clinical examinations were performed by multidisciplinary physicians specialized in WD. In addition, mutation detection in the ATP7B gene was performed.ResultsA total of 269 patients completed the study. (1) 51 cases (19%) showed both decreased Cp and Cu concentrations. (2) Molecular genetic tests were performed in 29 patients, and one ATP7B mutation (heterozygous state) was found in four patients. We identified three different missense mutations: p.His1069Gln, c.3207C>A (exon 14), p.Pro1379Ser, c.4135C>T (exon 21) and p.Thr1434Met, c.4301C>T (exon 21). No pathogenic mutation on either ATP7B allele was detected.ConclusionResults of Cp and/or serum Cu concentrations below the normal limits are common in patients with psychiatric disorders and nonrelevant and/or informative for the WD diagnosis. WD diagnosis is based on a combination of clinical and biological arguments. Psychiatric patients with suspicion of WD should be evaluated in a reference center. Trial registration CPP Lyon Sud-Est IVNo 10/044, CNIL No DR-2011-470, Afssaps No B100832-40 and CCTIRS No 10.612 bis, registered 8 June 2010.
| S-EPMC5379609 | biostudies-literature
Project description:Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.
| S-EPMC4188281 | biostudies-literature