Abstract
Objectives: To study the spectrum and classification of ATP7B variants in Iraqi children with Wilson disease by direct gene sequencing with clinical correlation.
Methods: Fifty-five unrelated children with a clinical diagnosis of Wilson disease (WD) were recruited. Deoxyribonucleic acid was extracted from peripheral blood samples, and variants in the ATP7B gene were identified using next-generation sequencing.
Results: Seventy-six deleterious variants were detected in 97 out of 110 alleles of the ATP7B gene. Thirty (54.5%) patients had 2 disease-causing variants (15 homozygous and 15 compound heterozygous). Twelve (21.8%) patients had one disease-causing variant and one variant of uncertain significance (VUS) with potential pathogenicity. Thirteen (23.6%) patients were carriers of a single disease-causing variant. The most frequent variants, c.3305T>C and c.956delC, were detected in 4 alleles each, followed by c.3741-3742dupCA and c.3694A>C, which were detected in 3 alleles each. Among the 76 variants, 42 were missense, 13 were stop-gain, 9 were frameshift, 1 was an in-frame deletion, and 11 were intronic variants. Notably, the globally common variant H1069Q was not detected in this study.
Conclusion: The mutational spectrum of ATP7B in the Iraqi population is diverse, despite the high rates of consanguinity. It differs from that of neighboring countries. We provided evidence for ten VUS to be reclassified as deleterious, raising questions about the diagnostic criteria for patients with higher Leipzig scores and a single deleterious variant.
Wilson disease (WD) (hepatolenticular degeneration) (MIM #277900) is a rare autosomal recessive disease caused by a defect in copper metabolism, which primarily accumulates in the liver and brain.1 Wilson disease is a well-established single-gene disease caused by biallelic mutations of the ATP7B gene (MIM number 606882), located at 13q14.3, which encodes a copper transporter responsible for biliary excretion of excess copper and its incorporation into ceruloplasmin.2 The ATP7B gene spans 80 kb. The longest transcript (NM_000053), detected in the liver, ranges from 7.5–8.5 kb and comprises 21 exons (encoding an approximately 7.5 kb transcript) and 20 introns.3
Wilson disease is a multisystem disorder with variable symptoms characterized by the accumulation of intracellular hepatic copper, which then spills into the blood and begins to accumulate in other organs and tissues, such as the putamen of the brain, subthalamus, kidneys, and cornea.4 The clinical manifestations depend on the organs in which copper accumulates during the pathological process. Therefore, liver and neurological involvement predominates. Wilson disease can have a pure hepatic, neurological, or a mixed presentation. Most pediatric patients present with liver disease, whereas patients diagnosed in adulthood predominantly have mixed presentations. Diagnosis is typically made in childhood, adolescence, or early adulthood (between 5 and 35 years of age); however, late presentation has also been described.5
An estimated disease prevalence is 1:30,000–1:50,000 in Europe, USA, and Asia. It is surprisingly close to the 1984 proposals of Scheinberg and Sternlieb.6 Other populations have a higher prevalence, such as the Middle East, Pakistan, and India.6
There is significant variability in WD in terms of the age at onset, impact on the liver or brain, severity, and response to treatment. Analysis of hundreds of different deleterious variants in WD has failed to find convincing evidence of genotype-phenotype correlations.7
The diagnosis of WD is currently based on a scoring system developed at the 8th International Meeting on WD in Leipzig,8 which includes clinical signs, histopathological studies, biochemical tests, and genetic analyses. A score of 4 or more confirms WD. This is achievable if 2 deleterious mutations in ATP7B are detected segregating with the disease. With a score of three, the diagnosis is possible, and more tests are indicated; while a score of 2 or less, the diagnosis is unlikely.2
In the modified Leipzig score, other points are assigned for example a family history of WD and a serum ceruloplasmin value of <5 mg/dL versus a value of serum ceruloplasmin ≥5.9
Molecular analysis of ATP7B is an essential step for diagnosing WD to initiate proper treatment and prevent or delay the development of fulminant hepatic failure, liver cirrhosis, or neurological manifestations, as neither clinical nor laboratory results are sufficient to confirm the diagnosis in many patients.
The study aims to detect variants of the ATP7B gene by long-read next-generation sequencing (NGS) among a group of clinically diagnosed or highly suspected children with WD in Iraq and to analyze the detected variants to determine the disease-causing variants in the ATP7B gene.
Methods
This cross-sectional study was carried out between October 2022 and October 2023. The patients were recruited from the Children’s Welfare Teaching Hospital, Medical City, Baghdad, Iraq. In total, 55 unrelated children with a diagnosis or high clinical suspicion of WD were recruited to this study. A thorough history was obtained, and physical examination and appropriate investigations were performed. Patients aged <18 years with clinical features suggestive of Wilson disease supported by laboratory evidence at presentation (such as: elevated 24-hour urinary copper level, reduced serum ceruloplasmin, and elevated liver enzymes), the presence of Kayser-Fleischer (KF) ring, and a family history of Wilson disease were included in this study. The data obtained were converted into numbers using a modified Leipzig scoring system.10 As a rule, any patient with a modified Leipzig score of ≥2 was recruited into this study. Patients with chronic viral hepatitis, autoimmune hepatitis, or other metabolic liver diseases were also excluded.
Two milliliter of peripheral blood samples were withdrawn from each patient, collected in K2EDTA tubes, and kept at 4°C for deoxyribonucleic acid (DNA) extraction and downstream molecular workup. DNA was extracted using a Reliaprep Blood gDNA Miniprep System (Promega, USA). DNA concentration was measured by the Qubit™ dsDNA HS Assay Kit from Invitrogen-Thermo Fisher®, USA.
Polymerase chain reaction (PCR), performed using the KAPA HiFi PCR Kit from Roche®, Switzerland, was used to amplify the whole ATP7B gene using 10 specific primers from Macrogen®, Korea in 14 different combinations (forward and reverse), which were put in 2 separate tubes for each combination. Polymerase chain reaction products were purified using Agencourt AMPure XP PCR Purification (Beckman Coulter, USA). Sequencing was performed using MinION mk1C Oxford Nanopore (Oxford, UK). Sequencing results were analyzed using MinKNOW, which acquires raw signals from the device and sends them to the analysis pipeline (basecaller) in chunks of defined size, according to the manufacturer’s instructions.
The entire coding, non-coding, promoter, 3’-UTR, and 5’-UTR regions of the ATP7B gene were sequenced using long-read NGS. This test can detect SNPs as well as small Integrative Genomics Viewer (IGV), Mutation Taster, and other software were used for data analysis. Bidirectional sequence reads were assembled and aligned to reference sequences based on the NCBI Reference Sequence Transcripts and the human genome build GRCh37/UCSC hg19.
Following gene-specific filtering, the data were analyzed to identify sequence variants as well as most deletions and duplications involving the entire gene. Alternative sequencing or copy number detection methods were used to analyze regions with inadequate sequences or copy number data. Reportable variants included pathogenic variants, likely pathogenic variants, and variants of uncertain significance.In silico analysis of all the variants was performed to assess their predicted effects on protein function.
This study was performed according to the Declaration of Helsinki and approved by the Research Ethics Committee in the Department of Pathology & Forensic Medicine, College of Medicine, University of Baghdad (issue no. 18 dated January 19th, 2023). Informed consent for the publication of medical information was obtained from parents/legal guardians of the enrolled patients.
Statistical analysis
Statistical data description and analysis were performed by Microsoft Excel version 2016 and by the IBM SPSS Statistics for Windows version 21 (IBMCorp, Armonk, NY, USA). Qualitative data were described by frequency and percentage. Fisher Exact Test was used to determine non-random associations between 2 categorical variables. A p-value <0.05 was considered statistically significant.
Results
For this study, 55 unrelated children from Iraq were recruited. There were 34 (61.8%) males and 21 (38.2%) females, with a male-to-female ratio of 1.62:1. Their ages ranged between 6 and 17 years (12.07±3.36).The detailed demographic and clinical data are presented in Table 1.
- Demographic and clinical characteristics with age of onset of the enrolled children
The asymptomatic patient showed elevated urinary copper levels. The patient was recruited because he had a deceased sister with WD and a cousin with liver cirrhosis.
Results of genetic analysis
Direct ATP7B gene sequencing using long-read NGS detected a wide range of disease-causing variants and a group of VUS with conflicting interpretations of their probable deleterious or damaging effects on protein function.
Overall findings
Molecular confirmation of the diagnosis of WD was obtained in 33 of the 55 patients, yielding a WD diagnostic rate of 60% among the recruited children. Thirty-four patients had a score of >4 prior to NGS, and the diagnosis was molecularly confirmed in 30 patients; the remaining four were heterozygous carriers (have one deleterious variant). Thirteen patients had a score of 3 prior to NGS, and the diagnosis was confirmed in 6 patients (4/13 were heterozygous carriers). The remaining 3 had one variant of uncertain significance (VUS) in addition to the disease-causing variant. Eight patients had a score of 2 prior to NGS, and the diagnosis was confirmed in 3 patients; while 5 patients were heterozygous.
Variants’ characteristics
Seventy-six distinct variants were identified in 97 out of 110 alleles of the ATP7B gene, with a detection rate of 88.18%; detailed data are presented in Table 2. Of the 76 variants, 42 (55.3%) were missense variants, 13 (17.1%) were stop-gains, 9 (11.8%) were frameshifts, 11 (14.5%) were intronic variants, and 1 (1.3%) was an in-frame deletion. The common worldwide variant (p.H1069Q) was not detected in any of the enrolled patients. Of the 76 detected variants, 2 were detected in four alleles each, 2 were detected in three alleles each, 16 were detected in 2 alleles each, and 56 were detected in one allele.
- Variants identified by targeted ATP7B gene sequencing by next-generation sequencing arranged by frequency.
Exon 2 harbors 7 (9.2%); exon 13 harbors 6 (7.9%); exons 8, 10, and 16 harbor 5 (6.6%) each; exons 18 and 20 harbor 4 (5.3%) each; exons 6, 11, 12, 15, 17, and 19 harbor 3 (3.95%) each; exons 4, 7, and 14 harbor 2 (2.6%) each; exons 3, 5, 9, and 21 harbor one (1.3%) each, while introns harbor 11 (14.5%) of the detected variants. No variants were detected in exon 1.
The molecular data of those patients are summarized as follows
Group 1: 30 patients with definitive diagnosis of WD: a) 15 Homozygous: 3 of them had additional deleterious variant; 13 variants were detected in those 15 patients, example were 2 variants were detected in 2 patients each; Table 3. b) 15 compound heterozygous: 1 of them had additional intronic pathogenic variant; Table 4.
- Variants detected in homozygous state in fifteen patients with Wilson disease.
- Variants detected in compound heterozygous state in 15 patients with Wilson disease.
Group 2: 12 heterozygous – 6 had additional VUS that affect protein function by SIFT; 4 had additional variant with conflicting interpretation by ClinVar and shown to affect protein function by SIFT; and 2 had additional VUS that is shown to be tolerated by SIFT; Table 5. Group 3: 13 heterozygous; 12 symptomatic, and 1 was asymptomatic carrier; Table 6.
- Variants characteristics detected in 12 children with 2 variants: a disease-causing variant plus a VUS or a variant with conflicting pathogenicity.
- Variant characteristics, clinical presentation, and MLS scores of 13 highly suspected children to have Wilson disease with a single heterozygous deleterious variant.
In this study, 12 (21%) patients had 2 heterozygous variants, one of which was disease-causing, whereas the second was either a VUS or had a conflicting interpretation. Six of them were VUS but showed affected protein function evidenced by Sorting Tolerant From Intolerant (SIFT) tool. Four of them had conflicting interpretations but showed evidence of affecting protein function by SIFT. The remaining 2 were VUS, which showed no evidence of an effect on protein function (tolerated) by SIFT (Table 5).
The 13 (23.63%) patients were carriers of a single pathogenic or likely pathogenic variant. One of them was asymptomatic and was included because of a positive family history and elevated urinary copper excretion (Table 6).
Additional findings
Two patients had neuropsychiatric manifestations. One with pure neurological manifestations (ID: 47) was found to be compound heterozygote for a missense pathogenic variants (p.Arg778Leu/p.Ile1148Thr), whereas the other patient (ID: 27) had neuropsychiatric manifestations and a single pathogenic stop-gain variant (p.Leu1088Ter).
Three patients harbored more than one disease-causing variant in the same allele, with a homozygous variant and one additional pathogenic variant.
Discussion
In this study, the target patients were children aged <18 years. The number of males was slightly higher than females, giving an M:F ratio of 1.62:1. This finding is similar to other local and regional studies.10-12 The vast majority (84%) of patients were offsprings of consanguineous unions. The overall consanguinity rate reported earlier in Iraq ranged from 47–60% in various studies.13 A high rate of consanguinity has also been reported in other clinical studies from Iraq10,14 and other regional countries, such as Turkey12 and the Arab Gulf.15 In this study, a family history of WD was found in 49.1% of patients, which is less than that reported by Hameed et al,16 who reported it in 64% of patients. Therefore, screening of first-degree relatives of the proband is highly recommended by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL).8,17
The age of onset and diagnosis has been reported to coincide with clinical presentation, where neurological symptoms are typically preceded by liver involvement.2 However, this was not observed in the present study.The clinical manifestations of WD may vary from asymptomatic patients with only biochemical abnormalities, to those with advanced disease such as liver cirrhosis, neurological deficits, and neuropsychiatric disorders. Hepatic manifestations are the predominant presenting features in pediatric patients.18 In the current study, the highest percentage of patients with WD presented with pure hepatic manifestations (72.7%), followed by mixed presentations (21.8%), similar to the pattern observed worldwide. The factors that determine whether a WD patient will develop hepatic or neurological diseases, or both, remains unclear.9
The diagnosis of WD, based on clinical and biochemical abnormalities alone, is often difficult. Similar non-specific chronic liver diseases with elevated urinary copper, reduced serum ceruloplasmin levels, and liver cirrhosis can be found in other inherited conditions, such as glycosylation disorders and manganese storage disease.2,19 Therefore, genetic testing using microarray-based methods or next-generation sequencing (NGS) is essential. Sequencing the entire ATP7B gene using NGS is an essential tool for diagnosing Wilson disease.9
In the present study, direct NGS analysis of the entire ATP7B gene in Iraqi patients with WD was performed. Previous Iraqi articles have studied the clinical and epidemiological characteristics of patients with WD.10,16 To date, 1,275 unique variants of the ATP7B gene have been identified.20 In this study, 76 variants were found in 97 of the 110 alleles; 56/76 were detected in only one allele, whereas only 2 variants were detected in four alleles of 2 homozygous patients. This demonstrated the tremendous heterogeneity of the detected variants, even in consanguineous marriages. This was also observed in other studies from India9 and China21 but not in Turkey.22 This heterogeneity among the Iraqi population has also been observed for other diseases.23
Single-nucleotide missense and nonsense variants are the most common types of the ATP7B gene worldwide, followed by insertions/deletions (indels) and splice-site mutations.25 Rare genetic mechanisms include whole-exon deletions, promoter region mutations, three concurrent pathogenic variants, and uniparental disomy.25,26 In the current study, the predominant variant type was missense, followed by nonsense, intronic, frameshift, and in-frame deletions, similar to what has been reported in other studies worldwide.20
The frequency of pathogenic variants associated with WD has regional differences. For example, the H1069Q variant is among the commonest mutations. It has a population allelic frequency of 10–40% (30–70% among Caucasians) and is detected most frequently in Central and Eastern Europe.27 However, its prevalence greatly varies based on geographic area. The most common variants identified in this study were p.Ile1102Thr and p.Pro319HisfsTer44, followed by p.Lys1248ThrfsTer83 and p.Thr1232Pro.
Genetically, there were 4 groups of patients in this study: homozygotes, compound heterozygotes, heterozygotes without additional variants, and heterozygotes with another variant (VUS).
Homozygous and compound heterozygous patients
The diagnosis was straightforward with no clinical problems in this group of patients, as the variants were either reported in other studies or classified according to the ACMG guidelines as disease-causing (pathogenic or likely pathogenic variants).
One patient (ID=47) in this study had pure neurological manifestations. The patient was a 16-year-old boy with a positive consanguinity and a negative family history; his MLS score was 8. A compound heterozygous state of the 2 pathogenic variants, p.Ile1148Thr and p.Arg778Leu, was observed.
The (p.Arg778Leu) variant is the most prevalent in East Asia and is considered the first hotspot mutation in the Chinese population.22 Recently published articles have reported that patients with WD with the p.Arg778Leu variant present with earlier disease onset and predominantly hepatic symptoms.28,29
Heterozygous patients with no additional variant
Twelve symptomatic patients (and one asymptomatic child) carried only one disease-causing variant without any additional variants on ATP7B. ATP7B is the only gene known to be responsible for WD.30 The frequency of heterozygote ATP7B mutations was considerably higher than the previously reported.31,32 The findings of heterozygous manifesting WD patients were also found in published articles, though they were not epidemiological studies, such as Paris33 and China.34
Early evidence suggests that among carriers of autosomal recessive conditions, intermediate phenotypes are plausible.35 The presence of symptoms suggestive of WD in patients with one deleterious variant can be explained by the inability of the test to detect a second variant. However, this was not the case in this study, as long-read NGS covered all regions of the ATP7B gene and all possible variant types, including gross deletions.
Another possibility is the presence of a modifier gene that can either alter gene function (mutation of a regulatory region) or presence of a coexisting gene mutation (in heterozygous state) causing hepatic manifestation. Such genes are ATP7A, PNPLA3, MTHFR, ESD, INO80, HTT, and others.7 These genetic modifiers can alter disease onset and phenotype, and some can even result in similar biochemical changes. In fact, 3 of the reported patients in this study had a clinical diagnosis of Gilbert disease, Alagille syndrome, and beta thalassemia major. They were found to have a single variant in ATP7B gene. This cannot be excluded as we tested only for ATP7B, whole genome sequencing can detect such possibility.
A third possibility is uniparental segmental isodisomy, which was documented as a novel disease-causing mechanism of WD in one study.25 This mechanism cannot be excluded from the present study because parental testing was not performed. A search for other causes of the WD phenotype for possible misdiagnosis, as it occurs in approximately 1% of cases, is necessary. The list of differential diagnosis is long, where similar clinical and/or biochemical changes are observed. This includes progressive familial intrahepatic cholestasis, congenital disorders of glycosylation, and manganese storage disease (referred to as the new Wilson disease).19
Urinary copper levels following a D-penicillamine challenge and symptomatic responses to chelation therapy can aid clinicians to manage these patients. Knowing that 7/12 of the carriers had an MLS score of 4 or more favored the diagnosis of WD. For patients with a score of 2, WD is unlikely.
Heterozygous patients with one additional variant
In this study, 12 patients carried a VUS in combination with a pathogenic or likely pathogenic variant. According to the ACMG, the most important criteria for establishing the causality of putative disease-causing mutations are minor allele frequency, co-segregation, and in silico pathogenicity scores (36).
In this study, an in-silico analysis of the variants was performed using the SIFT tool. According to the SIFT scores, 10 variants (p.Met668Val, p.Glu905Asp, p.Val966Gly, c.2576-44G>T, p.Val1298Ile, p.Ile116Thr, p.Asp118Asn, p.Ser440Gly, p.Pro539Leu, and c.4125-1G>A) were predicted to affect protein function, and 2 (p.Gly809Asp, p.Ala208Val) were predicted to be tolerated (Table 5).
Being detected in symptomatic patients with biochemical changes suggesting WD, their MLS score of ≥4, and the presence of an additional single deleterious variant, these 10 VUS that show an effect on protein function can be reclassified as disease-causing.
Only one case of an intronic VUS affecting a splice site has been reported. All other VUS occurred in the coding regions. Given that the in-silico analysis of intronic variants can reveal their impact on the splicing process, the consequences of a given substitution are generally unpredictable. Functional in-vitro analysis of the effects of potential splicing mutations can confirm the putative pathogenicity of non-coding mutations and thus help guide the patient’s clinical management and improve genetic counseling in affected families.36
The following cases showed evidence on the rationale for reclassification: i) An 8-year-old girl (ID=38) died from liver failure. Her MLS score was 4. She was born to consanguineous parents and had no relevant family history. The patient presented with hepatic manifestations at 5 years. She had 2 variants, p.Thr1232Pro (a likely pathogenic missense variant, also detected in patient 51 in the homozygous state and previously reported in Morocco.37 The second variant was p.Asp118Asn, a missense variant classified as VUS according to the ACMG guidelines. In silico analysis using SIFT revealed that it was deleterious. This variant has been identified on 79/280,864 chromosomes in the general population, using the Genome Aggregation Database (gnomAD). The disease was severe enough to cause liver cirrhosis and death, with one LP variant and one VUS shown by SIFT to be deleterious, making it more likely to be the second deleterious variant.
ii) The patient (ID=34) was a 10-year-old Kurdish female with a negative family history of WD and positive consanguinity who presented with hepatic manifestations at 6 years of age, leading to liver cirrhosis, for which liver transplantation was performed. Her MLS score was 6. Two variants were detected in the heterozygous state: a missense pathogenic variant (p.Thr766Arg) and a missense VUS variant (p.Val966Gly), according to ACMG. In silico analysis using SIFT revealed a damaging effect.
iii) The patient (ID=39) was an 8-year-old male with a positive consanguinity and a negative family history of WD. The patient presented with liver cirrhosis for which liver transplantation was performed. His MLS score was 6. Two heterozygous variants were detected: a frameshift pathogenic variant (p.Ala1183TyrfsTer2) (which was also detected in a heterozygous state in another patient) and a missense variant classified according to the ACMG guidelines as VUS (p.Ser440Gly). This variant has not been reported in individuals with WD in the literature and has been identified on 2/248,776 chromosomes in the general population with gnomAD.
iv) A 10-year-old boy (ID=35), who was a child of a consanguineous union with a negative family history, presented with hepatic manifestations and a score of 5. The patient also had a congenital heart defect and clubfoot. Brain MRI revealed abnormal signal intensity in white matter and the basal ganglia with mild brain atrophy. A pathogenic (p.Gln544Ter) stop-gain variant was detected along with another intronic variant (c.2576-44G>T) located at 13:52524342, intron 10, approximately 66 bp from exon 10 and 43 bp from exon 11, which was classified as VUS. In silico analysis revealed that it affects proteins. A previous analysis by Invitrogen Laboratories indicated that it is related to disease. The presence of congenital heart defects and clubfoot raised the suspicion of concomitant Alagille syndrome. Amson et al38 reported 2 siblings presented with liver disease and were carriers of 2 deleterious genetic variants.
v) A 17-year-old male (ID=36), who was a child of a consanguineous union and negative family history, presented with acute abdominal pain, shrunken lobulated liver, and signs of portal hypertension. His MLS was 4. Two variants were detected: a rare missense (p.Gly836Glu) likely pathogenic variant [also reported in 3 cases: one in a Moroccan patient,37 a second in an Italic patient of Moroccan origin,39 and a third in a French patient40]. The second variant was a missense VUS (p.Val1298Ile). Computational prediction in ClinVar suggests it may have a deleterious impact on protein structure and function. In-silico study by SIFT showed a score of 0.03, indicating that it affects protein function.
In summary, in a clinically relevant scenario, when a biallelic mutation is detected, no additional work is required. However, when a heterozygous variant is detected, with or without an additional VUS, a search for other variants in regulatory regions or in other related genes may be necessary. In addition, a comprehensive functional study is required to provide evidence of pathogenicity of the detected VUS with regular checkups for any update or reclassification.
Study limitation
Further molecular workup is needed for patients having disease manifestations and one deleterious variant, with or without additional VUS (variant functional study). This was not possible for this group of patients.
In conclusion, the ATP7B mutational spectrum in the Iraqi population is diverse, despite high rates of consanguinity. Evidence for ten VUS to be reclassified as deleterious was provided. Whether patients with a Leipzig score greater than 4 who carry a single deleterious variant can be definitively diagnosed as having WD or classified as manifesting carriers remains uncertain. Whole-exome or genome sequencing is recommended for patients with only one detected variant. Additionally, functional in-vitro or in-vivo studies are advised to ascertain the pathogenicity of the detected VUS.
Acknowledgment
The authors would like to thank Prof. Dr. Rabab Farhan Thejeal, Department of Pediatrics, College of Medicine, University of Baghdad, and Children Welfare Teaching Hospital, Medical City, Baghdad for assisting in clinical diagnosis of enrolled children.We would also like to thank editage (https://www.editage.com) for English language editing of the manuscript.
Footnotes
Disclosure. Authors have no conflict of interests, and the work was not supported or funded by any drug company.
- Received November 29, 2024.
- Accepted January 14, 2025.
- Copyright: © Saudi Medical Journal
This is an Open Access journal and articles published are distributed under the terms of the Creative Commons Attribution-NonCommercial License (CC BY-NC). Readers may copy, distribute, and display the work for non-commercial purposes with the proper citation of the original work.
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