Ecological and biogeographic features shaped the complex evolutionary history of an iconic apex predator (Galeocerdo cuvier).

BACKGROUND: The tiger shark (Galeocerdo cuvier) is a large iconic marine predator inhabiting worldwide tropical and subtropical waters. So far, only mitochondrial markers and microsatellites studies have investigated its worldwide historical demography with inconclusive outcomes. Here, we assessed for the first time the genomic variability of tiger shark based on RAD-seq data for 50 individuals from five sampling sites in the Indo-Pacific (IP) and one in the Atlantic Ocean (AO) to decipher the extent of the species' global connectivity and its demographic history. RESULTS: Clustering algorithms (PCA and NMF), FST and an approximate Bayesian computation framework revealed the presence of two clusters corresponding to the two oceanic basins. By modelling the two-dimensional site frequency spectrum, we tested alternative isolation/migration scenarios between these two identified populations. We found the highest support for a divergence time between the two ocean basins of ~ 193,000 years before present (B.P) and an ongoing but limited asymmetric migration ~ 176 times larger from the IP to the AO (Nm ~ 3.9) than vice versa (Nm ~ 0.02). CONCLUSIONS: The two oceanic regions are isolated by a strong barrier to dispersal more permeable from the IP to the AO through the Agulhas leakage. We finally emphasized contrasting recent demographic histories for the two regions, with the IP characterized by a recent bottleneck around 2000 years B.P. and the AO by an expansion starting 6000 years B.P. The large differentiation between the two oceanic regions and the absence of population structure within each ocean basin highlight the need for two large management units and call for future conservation programs at the oceanic rather than local scale, particularly in the Indo-Pacific where the population is declining.

Ancestry-related distribution of Runs of homozygosity and functional variants in Qatari population.

BACKGROUND: Describing how genetic history shapes the pattern of medically relevant variants could improve the understanding of how specific loci interact with each other and affect diseases and traits prevalence. The Qatari population is characterized by a complex history of admixture and substructure, and the study of its population genomic features would provide valuable insights into the genetic landscape of functional variants. Here, we analyzed the genomic variation of 186 newly-genotyped healthy individuals from the Qatari peninsula. RESULTS: We discovered an intricate genetic structure using ancestry related analyses. In particular, the presence of three different clusters, Cluster 1, Cluster 2 and Cluster 3 (with Near Eastern, South Asian and African ancestry, respectively), was detected with an additional fourth one (Cluster 4) with East Asian ancestry. These subpopulations show differences in the distribution of runs of homozygosity (ROH) and admixture events in the past, ranging from 40 to 5 generations ago. This complex genetic history led to a peculiar pattern of functional markers under positive selection, differentiated in shared signals and private signals. Interestingly we found several signatures of shared selection on SNPs in the FADS2 gene, hinting at a possible common evolutionary link to dietary intake. Among the private signals, we found enrichment for markers associated with HDL and LDL for Cluster 1(Near Eastern ancestry) and Cluster 3 (South Asian ancestry) and height and blood traits for Cluster 2 (African ancestry). The differences in genetic history among these populations also resulted in the different frequency distribution of putative loss of function variants. For example, homozygous carriers for rs2884737, a variant linked to an anticoagulant drug (warfarin) response, are mainly represented by individuals with predominant Bedouin ancestry (risk allele frequency G at 0.48). CONCLUSIONS: We provided a detailed catalogue of the different ancestral pattern in the Qatari population highlighting differences and similarities in the distribution of selected variants and putative loss of functions. Finally, these results would provide useful guidance for assessing genetic risk factors linked to consanguinity and genetic ancestry.

A curated dataset of modern and ancient high-coverage shotgun human genomes.

Over the last few years, genome-wide data for a large number of ancient human samples have been collected. Whilst datasets of captured SNPs have been collated, high coverage shotgun genomes (which are relatively few but allow certain types of analyses not possible with ascertained captured SNPs) have to be reprocessed by individual groups from raw reads. This task is computationally intensive. Here, we release a dataset including 35 whole-genome sequenced samples, previously published and distributed worldwide, together with the genetic pipeline used to process them. The dataset contains 72,041,355 sites called across 19 ancient and 16 modern individuals and includes sequence data from four previously published ancient samples which we sequenced to higher coverage (10-18x). Such a resource will allow researchers to analyse their new samples with the same genetic pipeline and directly compare them to the reference dataset without re-processing published samples. Moreover, this dataset can be easily expanded to increase the sample distribution both across time and space.

Bayesian Skyline Plots disagree with range size changes based on Species Distribution Models for Holarctic birds.

During the Quaternary, large climate oscillations impacted the distribution and demography of species globally. Two approaches have played a major role in reconstructing changes through time: Bayesian Skyline Plots (BSPs), which reconstruct population fluctuations based on genetic data, and Species Distribution Models (SDMs), which allow us to back-cast the range occupied by a species based on its climatic preferences. In this paper, we contrast these two approaches by applying them to a large data set of 102 Holarctic bird species, for which both mitochondrial DNA sequences and distribution maps are available, to reconstruct their dynamics since the Last Glacial Maximum (LGM). Most species experienced an increase in effective population size (Ne , as estimated by BSPs) as well as an increase in geographical range (as reconstructed by SDMs) since the LGM; however, we found no correlation between the magnitude of changes in Ne and range size. The only clear signal we could detect was a later and greater increase in Ne for wetland birds compared to species that live in other habitats, a probable consequence of a delayed and more extensive increase in the extent of this habitat type after the LGM. The lack of correlation between SDM and BSP reconstructions could not be reconciled even when range shifts were considered. We suggest that this pattern might be linked to changes in population densities, which can be independent of range changes, and caution that interpreting either SDMs or BSPs independently is problematic and potentially misleading.

Climate shaped how Neolithic farmers and European hunter-gatherers interacted after a major slowdown from 6,100 BCE to 4,500 BCE.

The Neolithic transition in Europe was driven by the rapid dispersal of Near Eastern farmers who, over a period of 3,500 years, brought food production to the furthest corners of the continent. However, this wave of expansion was far from homogeneous, and climatic factors may have driven a marked slowdown observed at higher latitudes. Here, we test this hypothesis by assembling a large database of archaeological dates of first arrival of farming to quantify the expansion dynamics. We identify four axes of expansion and observe a slowdown along three axes when crossing the same climatic threshold. This threshold reflects the quality of the growing season, suggesting that Near Eastern crops might have struggled under more challenging climatic conditions. This same threshold also predicts the mixing of farmers and hunter-gatherers as estimated from ancient DNA, suggesting that unreliable yields in these regions might have favoured the contact between the two groups.

Multiple freshwater invasions of the tapertail anchovy (Clupeiformes: Engraulidae) of the Yangtze River.

Freshwater fish evolved from anadromous ancestors can be found in almost all continents. The roles of paleogeographic events and nature selection in speciation process often are under focus of research. We studied genetic diversity of anadromous and resident tapertail anchovies (Coilia nasus species complex) in the Yangtze River Basin using 4,434 nuclear loci, and tested the history of freshwater invasion of C. nasus. We found that both C. brachygnathus and C. nasus were valid species, but the resident C. nasus taihuensis and the anadromous C. nasus were not different genetically based on Bayes factor species delimitation (BFD*). Maximum likelihood tree, Network, PCA and STRUCTURE analyses all corroborated the results of BFD*. Two independent freshwater invasion events of C. nasus were supported, with the first event occurring around 4.07 Ma and the second happened around 3.2 Ka. The time of the two freshwater invasions is consistent with different paleogeographic events. Estimation showed that gene flow was higher within ecotypes than between different ecotypes. F-DIST analyses identified 120 disruptive outliers by comparing C. brachygnathus to anadromous C. nasus, and 21 disruptive outliers by comparing resident C. nasus to anadromous C. nasus. Nine outliers were found to be common between the two comparisons, indicating that independent freshwater invasion of C. nasus might involve similar molecular pathways. The results of this study suggest that adaptation to landlocked freshwater environment of migratory fish can evolve multiple times independently, and morphology of landlocked ecotypes may cause confusion in their taxonomy.

Ancient cattle genomics, origins, and rapid turnover in the Fertile Crescent.

Genome-wide analysis of 67 ancient Near Eastern cattle, Bos taurus, remains reveals regional variation that has since been obscured by admixture in modern populations. Comparisons of genomes of early domestic cattle to their aurochs progenitors identify diverse origins with separate introgressions of wild stock. A later region-wide Bronze Age shift indicates rapid and widespread introgression of zebu, Bos indicus, from the Indus Valley. This process was likely stimulated at the onset of the current geological age, ~4.2 thousand years ago, by a widespread multicentury drought. In contrast to genome-wide admixture, mitochondrial DNA stasis supports that this introgression was male-driven, suggesting that selection of arid-adapted zebu bulls enhanced herd survival. This human-mediated migration of zebu-derived genetics has continued through millennia, altering tropical herding on each continent.

Demographic inferences after a range expansion can be biased: the test case of the blacktip reef shark (Carcharhinus melanopterus).

The evolutionary history of species is a dynamic process as they modify, expand, and contract their spatial distributions over time. Range expansions (REs) occur through a series of founder events that are followed by migration among neighboring demes. The process usually results in structured metapopulations and leaves a distinct signature in the genetic variability of species. Explicitly modeling the consequences of complex demographic events such as REs is computationally very intensive. Here we propose an an alternative approach that requires less computational effort than a comprehensive RE model, but that can recover the demography of species undergoing a RE, by combining spatially explicit modelling with simplified but realistic metapopulation models. We examine the demographic and colonization history of Carcharhinus melanopterus, an abundant reef-associated shark, as a test case. We first used a population genomics approach to statistically confirm the occurrence of a RE in C. melanopterus, and identify its origin in the Indo-Australian Archipelago. Spatial genetic modelling identified two waves of stepping-stone colonization: an eastward wave moving through the Pacific and a westward one moving through the Indian Ocean. We show that metapopulation models best describe the demographic history of this species and that not accounting for this may lead to incorrectly interpreting the observed genetic variation as signals of widespread population bottlenecks. Our study highlights insights that can be gained about demography by coupling metapopulation models with spatial modeling and underscores the need for cautious interpretation of population genetic data when advancing conservation priorities.

Genetic Landscape of Slovenians: Past Admixture and Natural Selection Pattern.

The Slovenian territory played a crucial role in the past serving as gateway for several human migrations. Previous studies used Slovenians as a source population to interpret different demographic events happened in Europe but not much is known about the genetic background and the demographic history of this population. Here, we analyzed genome-wide data from 96 individuals to shed light on the genetic role and history of the Slovenian population. Y chromosome diversity splits into two major haplogroups R1b and R1a with the latter suggesting a genetic contribution from the steppe. Slovenian individuals are more closely related to Northern and Eastern European populations than Southern European populations even though they are geographically closer. This pattern is confirmed by an admixture and clustering analysis. We also identified a single stream of admixture events between the Slovenians with Sardinians and Russians around ∼2630 BCE (2149-3112). Using ancient samples, we found a significant admixture in Slovenians using Yamnaya and the early Neolithic Hungarians as sources, dated around ∼1762 BCE (1099-2426) suggesting a strong contribution from the steppe to the foundation of the observed modern genetic diversity. Finally, we looked for signals of selection in candidate variants and we found significant hits in HERC2 and FADS responsible for blue eye color and synthesis of long-chain unsaturated fatty acids, respectively, when Slovenians were compared to Southern Europeans. While the comparison was done with Eastern Europeans, we identified significant signals in PKD2L1 and IL6R which are genes associated with taste and coronary artery disease, respectively.

Ancient goat genomes reveal mosaic domestication in the Fertile Crescent.

Current genetic data are equivocal as to whether goat domestication occurred multiple times or was a singular process. We generated genomic data from 83 ancient goats (51 with genome-wide coverage) from Paleolithic to Medieval contexts throughout the Near East. Our findings demonstrate that multiple divergent ancient wild goat sources were domesticated in a dispersed process that resulted in genetically and geographically distinct Neolithic goat populations, echoing contemporaneous human divergence across the region. These early goat populations contributed differently to modern goats in Asia, Africa, and Europe. We also detect early selection for pigmentation, stature, reproduction, milking, and response to dietary change, providing 8000-year-old evidence for human agency in molding genome variation within a partner species.

Origins and genetic legacies of the Caribbean Taino.

The Caribbean was one of the last parts of the Americas to be settled by humans, but how and when the islands were first occupied remains a matter of debate. Ancient DNA can help answering these questions, but the work has been hampered by poor DNA preservation. We report the genome sequence of a 1,000-year-old Lucayan Taino individual recovered from the site of Preacher's Cave in the Bahamas. We sequenced her genome to 12.4-fold coverage and show that she is genetically most closely related to present-day Arawakan speakers from northern South America, suggesting that the ancestors of the Lucayans originated there. Further, we find no evidence for recent inbreeding or isolation in the ancient genome, suggesting that the Lucayans had a relatively large effective population size. Finally, we show that the native American components in some present-day Caribbean genomes are closely related to the ancient Taino, demonstrating an element of continuity between precontact populations and present-day Latino populations in the Caribbean.

Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

Historical introgression drives pervasive mitochondrial admixture between two species of pelagic sharks.

We use a genomic sampling of both nuclear and mitochondrial DNA markers to examine a pattern of genetic admixture between Carcharhinus galapagensis (Galapagos sharks) and Carcharhinus obscurus (dusky sharks), two well-known and closely related sharks that have been recognized as valid species for more than 100years. We describe widespread mitochondrial-nuclear discordance in which these species are readily distinguishable based on 2152 nuclear single nucleotide polymorphisms from 910 independent autosomal regions, but show pervasive mitochondrial admixture. The species are superficially morphologically cryptic as adults but show marked differences in internal anatomy, as well as niche separation. There was no indication of ongoing hybridization between the species. We conclude that the observed mitochondrial-nuclear discordance is likely due to historical mitochondrial introgression following a range expansion.

Signatures of human European Palaeolithic expansion shown by resequencing of non-recombining X-chromosome segments.

Human genetic diversity in Europe has been extensively studied using uniparentally inherited sequences (mitochondrial DNA (mtDNA) and the Y chromosome), which reveal very different patterns indicating sex-specific demographic histories. The X chromosome, haploid in males and inherited twice as often from mothers as from fathers, could provide insights into past female behaviours, but has not been extensively investigated. Here, we use HapMap single-nucleotide polymorphism data to identify genome-wide segments of the X chromosome in which recombination is historically absent and mutations are likely to be the only source of genetic variation, referring to these as phylogeographically informative haplotypes on autosomes and X chromosome (PHAXs). Three such sequences on the X chromosome spanning a total of ~49 kb were resequenced in 240 males from Europe, the Middle East and Africa at an average coverage of 181 ×. These PHAXs were confirmed to be essentially non-recombining across European samples. All three loci show highly homogeneous patterns across Europe and are highly differentiated from the African sample. Star-like structures of European-specific haplotypes in median-joining networks indicate past population expansions. Bayesian skyline plots and time-to-most-recent-common-ancestor estimates suggest expansions pre-dating the Neolithic transition, a finding that is more compatible with data on mtDNA than the Y chromosome, and with the female bias of X-chromosomal inheritance. This study demonstrates the potential of the use of X-chromosomal haplotype blocks, and the utility of the accurate ascertainment of rare variants for inferring human demographic history.

Population genomics of C. melanopterus using target gene capture data: demographic inferences and conservation perspectives.

Population genetics studies on non-model organisms typically involve sampling few markers from multiple individuals. Next-generation sequencing approaches open up the possibility of sampling many more markers from fewer individuals to address the same questions. Here, we applied a target gene capture method to deep sequence ~1000 independent autosomal regions of a non-model organism, the blacktip reef shark (Carcharhinus melanopterus). We devised a sampling scheme based on the predictions of theoretical studies of metapopulations to show that sampling few individuals, but many loci, can be extremely informative to reconstruct the evolutionary history of species. We collected data from a single deme (SID) from Northern Australia and from a scattered sampling representing various locations throughout the Indian Ocean (SCD). We explored the genealogical signature of population dynamics detected from both sampling schemes using an ABC algorithm. We then contrasted these results with those obtained by fitting the data to a non-equilibrium finite island model. Both approaches supported an Nm value ~40, consistent with philopatry in this species. Finally, we demonstrate through simulation that metapopulations exhibit greater resilience to recent changes in effective size compared to unstructured populations. We propose an empirical approach to detect recent bottlenecks based on our sampling scheme.

Great ape Y Chromosome and mitochondrial DNA phylogenies reflect subspecies structure and patterns of mating and dispersal.

The distribution of genetic diversity in great ape species is likely to have been affected by patterns of dispersal and mating. This has previously been investigated by sequencing autosomal and mitochondrial DNA (mtDNA), but large-scale sequence analysis of the male-specific region of the Y Chromosome (MSY) has not yet been undertaken. Here, we use the human MSY reference sequence as a basis for sequence capture and read mapping in 19 great ape males, combining the data with sequences extracted from the published whole genomes of 24 additional males to yield a total sample of 19 chimpanzees, four bonobos, 14 gorillas, and six orangutans, in which interpretable MSY sequence ranges from 2.61 to 3.80 Mb. This analysis reveals thousands of novel MSY variants and defines unbiased phylogenies. We compare these with mtDNA-based trees in the same individuals, estimating time-to-most-recent common ancestor (TMRCA) for key nodes in both cases. The two loci show high topological concordance and are consistent with accepted (sub)species definitions, but time depths differ enormously between loci and (sub)species, likely reflecting different dispersal and mating patterns. Gorillas and chimpanzees/bonobos present generally low and high MSY diversity, respectively, reflecting polygyny versus multimale-multifemale mating. However, particularly marked differences exist among chimpanzee subspecies: The western chimpanzee MSY phylogeny has a TMRCA of only 13.2 (10.8-15.8) thousand years, but that for central chimpanzees exceeds 1 million years. Cross-species comparison within a single MSY phylogeny emphasizes the low human diversity, and reveals species-specific branch length variation that may reflect differences in long-term generation times.

The Y-chromosome tree bursts into leaf: 13,000 high-confidence SNPs covering the majority of known clades.

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes.

Identification of the remains of King Richard III.

In 2012, a skeleton was excavated at the presumed site of the Grey Friars friary in Leicester, the last-known resting place of King Richard III. Archaeological, osteological and radiocarbon dating data were consistent with these being his remains. Here we report DNA analyses of both the skeletal remains and living relatives of Richard III. We find a perfect mitochondrial DNA match between the sequence obtained from the remains and one living relative, and a single-base substitution when compared with a second relative. Y-chromosome haplotypes from male-line relatives and the remains do not match, which could be attributed to a false-paternity event occurring in any of the intervening generations. DNA-predicted hair and eye colour are consistent with Richard's appearance in an early portrait. We calculate likelihood ratios for the non-genetic and genetic data separately, and combined, and conclude that the evidence for the remains being those of Richard III is overwhelming.

Human loci involved in drug biotransformation: worldwide genetic variation, population structure, and pharmacogenetic implications.

Understanding the role of inheritance in individual variation in drug response is the focus of pharmacogenetics (PGx). A key part of this understanding is quantifying the role of genetic ancestry in this phenotypic outcome. To provide insight into the relationship between ethnicity and drug response, this study first infers the global distribution of PGx variation and defines its structure. Second, the study evaluates if geographic population structure stems from all PGx loci in general, or if structure is caused by specific genes. Lastly, we identify the genetic variants contributing the greatest proportion of such structure. Our study describes the global genetic structure of PGx loci across the 52 populations of the Human Genome Diversity Cell-Line Panel, the most inclusive set of human populations freely available for studies on human genetic variation. By analysing genetic variation at 1,001 single nucleotide polymorphisms (SNPs) involved in biotransformation of exogenous substances, we describe the between-populations PGx variation, as well geographical groupings of diversity. In addition, with discriminant analysis of principal component (DAPC), we infer how many and which groups of populations are supported by PGx variation, and identify which SNPs actually contribute to the PGx structure between such groups. Our results show that intergenic, synonymous and non-synonymous SNPs show similar levels of genetic variation across the globe. Conversely, loci coding for Cytochrome P450s (mainly metabolizing exogenous substances) show significantly higher levels of genetic diversity between populations than the other gene categories. Overall, genetic variation at PGx loci correlates with geographic distances between populations, and the apportionment of genetic variation is similar to that observed for the rest of the genome. In other words, the pattern of PGx variation has been mainly shaped by the demographic history of our species, as in the case of most of our genes. The population structure defined by PGx loci supports the presence of six genetic clusters reflecting geographic location of samples. In particular, the results of the DAPC analyses show that 27 SNPs substantially contribute to the first three discriminant functions. Among these SNPs, some, such as the intronic rs1403527 of NR1I2 and the non-synonymous rs699 of AGT, are known to be associated with specific drug responses. Their substantial variation between different groups of populations may have important implications for PGx practical applications.

BCHE and CYP2D6 genetic variation in Alzheimer's disease patients treated with cholinesterase inhibitors.

PURPOSE: Cholinesterase inhibitors are commonly prescribed to patients with Alzheimer's disease (AD) to enhance cholinergic neurotransmission. Differential response to these treatments has been observed, and claims have been made that individual genetic variants may influence the pharmacokinetic and pharmacodynamic properties of these drugs. Here we assess the effects of genetic variation at two loci involved in the activity of cholinesterase inhibitors on longitudinal clinical change in AD patients being treated with donepezil, galantamine, and rivastigmine. METHODS: This was an open study in which 171 Italian AD patients treated with donepezil (n = 92), galantamine (n = 33), or rivastigmine (n = 46) were enrolled. Response to treatment was quantified by grading the patient's cognitive state (Mini-Mental State Examination) and the patient's ability to perform normal daily activities (Activities of Daily Living, Instrumental Activities of Daily Living) at baseline and after 6 and 12 months of treatment. Genetic variation was comprehensively characterized and analyzed at two loci: CYP2D6, which is involved in donepezil and galantamine metabolism, and BCHE, which codes for an enzyme (butyrylcholinesterase) which is both target and metabolizer of rivastigmine. APOE (coding for apolipoprotein E), which is associated with the risk of AD and inefficacy of specific AD treatments, was genotyped to control for patient stratification. The influence of the CYP2D6 and BCHE genotype on clinical changes after 12 months was evaluated by several tests of association. RESULTS: After 1 year of treatment, 29, 12, and 12 of the patients receiving donepezil, galantamine, and rivastigmine, respectively, showed a cognitive decrement, while eight patients interrupted the therapy before 12 months of treatment. No significant differences between the three treatments were observed in terms of response and tolerability. Non-responders show a higher proportion of BCHE and CYP2D6 mutated alleles, but genetic variation at the two loci was not a reliable predictor of clinical changes in AD patients treated with cholinesterase inhibitors. CONCLUSIONS: Individualized therapy based on CYP2D6 and BCHE genotypes is unlikely to be beneficial for treating Alzheimer's disease patients in routine clinical practice.