dataesr/scientific-paragraphs-categorization huggingface dataset:
A BS TRACT: Background: Wilson's disease (WD) is usually diagnosed in children and young adults; limited data exist on late-onset forms. Objective: The aim was to characterize the clinical and paraclinical presentations, therapeutic management, and outcomes in patients with late-onset WD. Methods: Patients diagnosed with WD after age 40 years were identified from the French Wilson's Disease Registry (FWDR). Clinical, laboratory, and imaging findings and treatment were reported at diagnosis and last follow-up. Results: Forty-five patients were identified (median age: 49, range: 40-64) and placed in three groups according to their clinical presentation: neurological (n = 20, median diagnostic delay: 20 months), hepatic (n = 13, diagnostic delay: 12 months), and family screening (n = 12), all confirmed genetically.
The analysis of the second patient found no tremor or other abnormal movement. The sixth patient had isolated dysarthria for almost 10 years, with progressive worsening over time, and then developed tremor of upper limbs and walking problems. Surprisingly brain MRI of all 6 patients showed no T2/FLAIR hyperintensities (Fig. S1 ); however, the brain MRI severity scale, according to the recent validated score by Dusek et al, revealed mild atrophy in 4 of them  24  (Table  S1 ). Only 1 of these 6 patients had a KFR. high intrahepatic copper. All these atypical neurological forms of late-onset WD were confirmed by molecular analysis (Table  S2 ). Three mutations were novel: their pathogenicity was assessed using the American College of Medical Genetics and Genomics classification,  26  based on general population databases, missense variant prediction algorithms/software, international databases of pathogenic variants, and the literature.
Screened Patients (n = 12) At diagnosis (Table  1 ). Eleven patients were diagnosed by family screening, and 1 was diagnosed fortuitously when being assessed for recurrent vasovagal malaise, for which copper tests were performed. She had a normal brain MRI, low serum copper and ceruloplasmin, and normal UCE at diagnosis. Three of 7 patients for whom full data were available were related to other patients with late-onset WD. When available (n = 3), brain MRI was normal. KFR was present in 2 of 10 patients. Liver assessments were abnormal in 3 of 7 patients, with minimal liver ultrasound abnormalities. Three patients with normal copper balance were not treated but followed a low-copper diet. After 7. 0 Years (IQR: 7. 0-22. 0) of Follow-Up. All screened patients remained free of neurological symptoms (Table  3 ).
Quantification of Vanillin Methanolic extracts of fruits were filtered through a 0. 45 µm pore-size syringe filter and the solvent of 10 mL fruit extract was completely evaporated using nitrogen gas. The concentrate was dissolved in methanol (70%, 1 mL) and used for the analysis. Vanillin was quantified using the method described by Waliszewski et al. [34]. A Shimadzu Nexera-XR HPLC consisting of a LC-20ADxr pump, SPD-20AV UV detector, a CBM-20A/CBM-20Alite system controller, Lab Solutions software, and an auto sample injector was used. Vanillin (99. 9% purity) stock solution (1 mg/mL) was prepared and stored in an amber-colored bottle at 4 • C. Water: methanol at 40:60 was used for isocratic elution to quantify vanillin in fruit extracts since this mixture showed the best resolving ability.
Statistical Analysis All reported data were expressed as means ± standard deviation (three repetitions). A two-way analysis of variance (ANOVA) with Duncan post hoc tests was performed using STATISTICA software package (StatSof, Tulsa, OK, USA). Differences between means at 5%, 1%, and 0. 1% levels (p < 0. 05, 0. 01, or 0. 001, respectively) were considered statistically significant.
Vertebrate pigmentation patterns are amongst the best characterised model systems for studying the genetic basis of adaptive evolution. The wealth of available data on the genetic basis for pigmentation evolution allows for meta-analysis of trends and quantitative testing of evolutionary hypotheses. We employed Gephebase, a database of genetic variants associated with natural and domesticated trait variation, to examine trends in how cisregulatory and coding mutations contribute to vertebrate pigmentation phenotypes, as well as factors that favour one mutation type over the other. We found that studies with lower ascertainment bias identified higher proportions of cis-regulatory mutations, and that cisregulatory mutations were more common amongst animals harboring a higher number of pigment cell classes.
Here, we first outline the basic biology of vertebrate pigmentation, as well as the key differences between different vertebrate clades. We then utilise a dataset of vertebrate pigmentation literature to analyse trends in the underlying genetics. For this purpose, we used Gephebase (https://www. gephebase. org/) -a knowledge base dedicated to the compilation of literature on genes and mutations underlying natural and domesticated organismal variation in Eukaryotes  (Courtier-Orgogozo et al. , 2020). Gephebase gathers published data on evolutionarily relevant mutations, each entry representing a causal association between an allelic change at a given locus and trait variation between individuals or species. Each entry includes information relating to the species/population, the type of trait, the gene, the nature of the mutation(s), whether they represent null alleles, and the study that identified it.
Using the vertebrate pigmentation entries in this database, we focused on the relative abundance of cis-regulatory and coding mutations contributing to vertebrate pigmentation evolution. Finally, we discuss the direction of future vertebrate pigmentation research and the role to be played by recent model systems and innovative approaches. Haplochromis sauvagei (bottom) showing differences in horizontal stripes and vertical bars  (Kratochwil et al. , 2018). Photographs courtesy of Claudius Kratochwil. D: Different species of Peromyscus mice showing differences in colour phenotypes -P. maniculatus nubiterrae (top left, image credit to Evan P Kingsley), P. polionotus phasma (top right, image credit to  JB Miller) , P. polionotus sumneri (bottom left, image credit to Nicole Bedford) and P. gossypinus (bottom right, image credit to  Nicole Bedford).
Genetic variants associated with pigmentation are asymmetrically distributed across vertebrate clades To examine the representation of different pigmentation systems across vertebrate evolutionary studies, we looked at the number of entries in Gephebase across five cladesteleost fishes, amphibians, squamates, birds and mammals. All of the 363 vertebrate pigmentation entries belonged to one of these clades. The significant majority (71. 1%, n = 258) of entries were mammals, whilst birds (20. 9%, n = 76) were also highly prevalent (Table  1 ). Teleosts (5. 5%, n = 20) and squamates (2. 2%, n = 8) were scarcely represented, and amphibians were associated with only one entry (0. 3%). As such, clades in which multiple distinct chromatophore classes are found represent less than 10% of our dataset, in comparison to those clades which harbour only melanocytes and no other pigment cell classes.
Variation in vertebrate pigmentation is associated with a majority of coding mutations We examined the relative prevalence of protein coding and cis-regulatory mutations across the dataset. Cis-regulatory mutations, and coding sequence mutations together account for 330 entries (90. 9%) out of the full dataset, with coding mutations being the majority (262 entries, 72. 2%). The remaining classes of genetic variation (9. 1%) were gene amplification, gene loss, intronic mutations, and unknown (when the gene has been identified but the exact mutation(s) have not) (Figure  2A ). The overall prevalence of coding mutations is in contrast to the hypothesis that cis-regulatory mutations have a greater likelihood of generating phenotypic change  (Carroll, 2008). This may reflect the relative ease of identifying coding mutations compared with cis-regulatory changes  (Stern and Orgogozo, 2008).
Study methodologies with less ascertainment bias exhibit slightly higher proportion of cis-regulatory mutations Three methodology categories are represented in the dataset --Candidate Gene, Linkage Mapping, and Association Mapping  (Courtier-Orgogozo et al. , 2020). Candidate gene studies exhibit the highest ascertainment bias and may be expected to identify a higher proportion of coding mutations. Indeed, many candidate gene studies have historically focused on identifying amino-acid changes within coding regions in new species, based on previous findings in other species. However, they also facilitate comparisons across broad taxonomic distances compared with other mapping approaches. In contrast, both linkage and association mapping studies start with a phenotypic difference and attempt to pin it to a sequence change, at least as a locus interval.
Linkage mapping involves crossing populations to generate recombinant hybrids, and thus can only map variation between closely related organisms which are interfertile. However, such studies are usually less biased than candidate gene approaches in their detection of causative loci, depending on the resolution and coverage of the mapping. Finally, association mapping approaches involve identification of significant association within large, heavily intermixed populations, and typically have minimal ascertainment bias as a result. To account for potential differences between methodologies we plotted the proportion of cis-regulatory mutations for each method (Figure  2B ). As expected, candidate gene approaches identified the lowest proportion of cis-regulatory mutations (12. 2%) and was the only category below the dataset average.
Changes in cis-regulatory reporting over time do not fully explain the disparity in mutation types The Gephebase dataset includes studies published between 1993 and 2020  (Courtier-Orgogozo et al. , 2020). Owing to the increasing availability of genomic resources facilitating linkage and association mapping in a broader range of model systems  (Kratochwil and Meyer, 2015) , we expected the cis-regulatory proportion of the dataset to increase over time. We therefore examined the cumulative number of cis-regulatory and coding mutations over the time period covered by the dataset, for each study methodology. We used this approach as the low numbers of entries for each individual year made non-cumulative comparison misleading -for instance, there were several years with no entries for a particular study methodology.
We found that cis-regulatory entries began to appear in the dataset later than coding entries  (Figure 2C and 2D). Prior to 2005 only a single cis-regulatory mutation was identified, compared with 65 coding. However, past this point cis-regulatory entries were added at approximately the same rate as coding, for each study methodology. Thus, although the overall cis-regulatory proportion does increase over the entire time period covered, this is largely due to coding mutation discovery beginning earlier. We also did not observe an appreciable increase in the relative prevalence of linkage mapping or association mapping studies. Taken together, this suggests that the overall trend is a slight increase in cis-regulatory mutation reporting, but that differences in the relative prevalence of different study methodologies are not sufficient to explain the disparity between cis-regulatory and coding mutations.
As such, it is possible that there is a discovery bias towards the identification of coding mutations in all methods, or alternatively, that evolutionary variations in vertebrate pigmentation involve a higher proportion of coding mutations. In the next sections, we further examine this proposition with particular attention to research biases and phenotypic categories. The relative frequency of cis-regulatory and coding mutations holds in the absence of the most represented genes in the dataset Historically, most evolutionary studies have centred on the melanic mammalian and bird pigmentation systems owing to the important knowledge in mice coat colour genetics. Therefore, we further explored if the prevalence of protein coding mutations was due to the overrepresentation of melanic pigmentation case studies.
Mammal and avian melanic pigment patterns are the result of dynamic switching between the synthesis of two types of melanin. The switch between black eumelanin and reddish-brown pheomelanin is controlled by the melanocortin 1 receptor, MC1R  (García-Borrón et al. , 2005). Mc1r is the most represented gene in our dataset, with 87 entries that account for 26. 4% of the dataset. The second-most represented gene in the dataset is kit, a cell-surface receptor that is essential for the expression of tyrosinase, a rate-limiting enzyme in melanin biosynthesis  (Hou et al. , 2000). 40 kit entries account for 12. 1% of entries. Finally, the third-most represented gene is agouti signalling protein, asip. ASIP acts as an antagonist to MC1R -in the absence of ASIP, MC1R initiates a signalling cascade resulting in eumelanin production, while ASIP antagonism reverts the melanocyte to pheomelanin synthesis  (García-Borrón et al.
, 2005). There are 33 asip entries, corresponding to 10% of the dataset. The high representation of these three genes in Gephebase reflects their relevance in mammalian and other vertebrate systems, and particularly their frequent selection as candidate genes for further study. To remove a potential bias of methodology resulting from a focus on melanic genes, we performed the same analysis with these three entries -mc1r, kit, and asip -removed. When removing mc1r, kit, and asip entries from the dataset, the overall proportion of cis-regulatory mutations increases from 20. 6% to 27. 6%, owing to the prevalence of coding mutations amongst mc1r entries in particular (Figure  2E ). The cis-regulatory proportion of all three methdology categories increases, and association mapping remains the category with the highest cis-regulatory proportion, followed by linkage mapping (Figure  2E ).
In addition, contexts of artificial selection and favourable breeding conditions by humans can overcome the cost of mutations that would normally be counter-selected in the wild  (Cieslak et al. , 2011; Courtier-Orgogozo and Martin, 2020; Hanly et al. , 2021). To test if the same is true for vertebrate pigmentation systems, we plotted the proportion of cis-regulatory mutations by taxonomic status. The dataset includes three categories of taxonomic status -Domesticated, Intraspecific, and Interspecific. Gephebase also includes 'Intergeneric or higher', but no vertebrate colouration entries were assigned this category. The domesticated category included cases of artificial selection by breeders and fanciers, with pigment trait variations being directly selected in most cases. The intraspecific category contained studies that investigated natural phenotypic differences between morphs of the same species.
Similarly, genes implicated in direct cellular interaction might be expected to suffer negative effects from coding sequence mutations, owing to the specificity of recognition that is typical of signalling molecules and their receptors. We therefore examined whether clades with distinct pigmentation systems lead to differences in the mutation proportion. For this purpose, we plotted the proportion of mutations by the five vertebrate clades (Table  1 , Figure  3B ). The only amphibian entry in the dataset was coding. Excluding amphibians, mammals had the lowest proportion of cis-regulatory changes (16. 2%). Birds had a similarly low cis-regulatory proportion (21. 7%). In contrast, squamates and teleosts both showed higher rates of cis-regulatory change -50%, and 55. 0%, respectively. Although the low number of entries associated with multiple chromatophore pigment systems does preclude drawing definitive conclusions, this trend fits with the hypothesis that variation in pigmentation systems utilising multiple pigment cell classes exhibit a higher proportion of cis-regulatory mutations.
Teleosts, amphibians, and squamates combined had a cisregulatory proportions of 55. 5%, compared with 17. 5% for birds and mammals combined. Notably, teleosts exhibited a much higher proportion of interspecific studies than the dataset average -6 of the 18 teleost entries were interspecific (Figure  S2 ). The strong association between teleost entries and interspecific comparison makes it difficult to conclude which (if either) is the more important factor in yielding higher cis-regulatory proportions. Although, when taking into account only the intraspecific cases, the proportion of cis-regulatory mutations is still higher in teleosts, amphibians, and squamates (42. 1%, 8 out of 19) than in birds and mammals (23. 6%, 17 out of 72) (Figure  S2 ). A larger sample of studies conducted in model systems harbouring multiple pigment cell classes, together with more interspecific studies in other clades, would provide valuable insight in this regard.
3% for colour (Figure  S3 ). This is in spite of white spotting phenotypes being exclusively domesticated as well as exclusively categorised as pattern alterations. Thus, the disparity between colour and pattern phenotypes in terms of mutation type may be larger than this dataset suggests in non-domesticated evolution, particularly given the overall prevalence of domesticated studies in pigmentation research. Overall the Gephebase dataset supports a disproportionate role of cis-regulatory changes in the generation of pattern variation  (Orteu and Jiggins, 2020).
Alternatively, the categories representing downstream processes -melanosome formation, pigment biosynthesis and pigment trafficking/localisation -all exhibited a higher proportion of protein coding mutations. The overall trend appears to fit the hypothesis that for upstream processes such as pigment cell lineage specification, mutations are more likely to be cis-regulatory when compared with downstream cellular processes which do not affect cell viability, such as variable rates of pigment deposition. Table  3 : Summary of functional categories: assignment criteria for each functional category, and the total number of entries in the dataset for which that category was assigned. Each gene was assigned to only one functional category.
, 2014; Gunnarsson et al. , 2011). Sox10, and other sox family transcription factors, are highly pleiotropic and essential for neural crest specification, migration and differentiation  (Sarkar and Hochedlinger, 2013). No sox10 coding mutations were identified in this dataset. Conversely, multiple presumptive null coding mutations have been identified in mc1r, including one example of parallel evolution in cavefish. Thus, in contrast to sox10 and other transcription factors, highly specialised proteins such as mc1r may be less susceptible to pleiotropy arising from null mutations. As such, we expected that molecular functions that are less pleiotropic would exhibit higher cis-regulatory proportions, and vice versa. Using EBI's QuickGO mouse slimmer and manual combination of highly related molecular GO terms, we assigned each entry to one of 7 categories of GO molecular function (see Methods, Table  S1 , Figure  S4A  and S4B).
For most signaling receptor genes, the corresponding ligand was also present in the dataset in the signaling receptor binding GO category. This includes kit and its ligand, as well as multiple endothelin signaling receptors and their respective ligands. Our results suggest that the coding sequence of genes encoding signaling ligands are more constrained than that of their signaling receptors. This supports previous findings that cis-regulatory mutations in ligand genes drive morphological evolution  (Martin and Courtier-Orgogozo, 2017). The receptor/ligand pairing with the most entries in the dataset, mc1r/asip, supports this -mc1r has a cis-regulatory proportion of 1. 1%, in contrast with its ligand asip with 39. 4%. This trend is also observed when candidate gene studies are removed, with 5.
Divergent pigmentation systems have fewer shared evolutionary hotspots One of the major outcomes of the study of the genetic basis of evolutionary change is the discovery that the repeated co-option of the same genes underlies the evolution of convergent phenotypes. Genes that are repeatedly found as causal drivers of similar phenotypic changes in diverse taxa or populations have been referred to as genetic hotspots  (Martin and Orgogozo, 2013). Here, we hypothesised that similar pigmentation systems would be more likely to evolve through re-use of the same set of genes, whereas more divergent pigmentation systems would have fewer shared evolutionary hotspots. We therefore divided the dataset by clade and compared the genes shared between each pairing of clades. The overlap between clades was calculated by combining the entries corresponding to each shared gene, and normalising by the total number of entries in the respective clade (see Methods).
As the number of shared hotspot genes would be expected to reduce over evolutionary time, phylogenetic distance is likely to contribute to the observed overlap between clades  (Conte et al. , 2012). Although mc1r was highly represented in all clades, and thus contributed considerable hotspot overlap to all clade pairings, many other genes that were highly represented in the dataset were confined to mammals and birds only. The second, third, and fourth most represented genes in the overall datasetkit, asip, and tyrp1 -all had no teleost or squamate entries, and are all implicated in melanogenesis or melanogenic switching. However, certain melanogenic genes also exhibited overlap between teleost and squamate entries, such as oca2, a melanosomal transport protein (Table  S2 ). Oca2 comprised 15% (n = 3) of all teleost entries, and 12.
Discussion Gephebase is a powerful tool that can be used to compile data on phenotype-genotype associations to infer general patterns regarding the mechanisms underlying evolutionary change. Importantly, this dataset can be used to find knowledge gaps in the current literature, and identify novel directions and study systems for future research. Here, we analysed a large dataset of 363 Gephebase entries, each a pair of alleles linked to phenotypic variation in vertebrate pigmentation. Of the three forms of empirical evidence accepted by Gephebase, it was clear that the study methodology with higher ascertainment bias -candidate gene approach -identified more coding sequence mutations than the other methods. Irrespective of this bias, protein coding changes were still the majority of mutations identified with linkage and association mapping.
Strong selection for crypsis against variable soil colouration instead leads to high variance in agouti coding sequence, and rapid divergence driven by colonisation over the last ~4000 years  (Pfeifer et al. , 2018). The prevalence of intraspecific coding mutations in our dataset may therefore be related to the strength of selection typical for vertebrate pigmentation evolution. In the future it would be interesting to investigate the effect of relative selection strength on the role of each mutation type, and to perform the same analyses in multiple morphological traits to determine if these trends are pigmentation specific.