Distinctive patterns of evolution of the δ-globin gene (HBD) in primates.

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and ß-globin family members. In placental mammals, the ß-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψß(HBBP1), and the late expressed genes, δ (HBD) and ß (HBB). While HBB encodes the major adult ß-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult ß-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the ß-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult ß-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.

DivStat: a user-friendly tool for single nucleotide polymorphism analysis of genomic diversity.

Recent developments have led to an enormous increase of publicly available large genomic data, including complete genomes. The 1000 Genomes Project was a major contributor, releasing the results of sequencing a large number of individual genomes, and allowing for a myriad of large scale studies on human genetic variation. However, the tools currently available are insufficient when the goal concerns some analyses of data sets encompassing more than hundreds of base pairs and when considering haplotype sequences of single nucleotide polymorphisms (SNPs). Here, we present a new and potent tool to deal with large data sets allowing the computation of a variety of summary statistics of population genetic data, increasing the speed of data analysis.

Evolutionary constraints in the ß-globin cluster: the signature of purifying selection at the δ-globin (HBD) locus and its role in developmental gene regulation.

Human hemoglobins, the oxygen carriers in the blood, are composed by two α-like and two ß-like globin monomers. The ß-globin gene cluster located at 11p15.5 comprises one pseudogene and five genes whose expression undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition. HBD encodes the δ-globin chain of the minor adult hemoglobin (HbA2), which is assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. In this study, we sought a detailed portrait of the genetic variation within the ß-globin cluster in a large human population panel from different geographic backgrounds. We resequenced the coding and noncoding regions of the two adult ß-globin genes (HBD and HBB) in European and African populations, and analyzed the data from the ß-globin cluster (HBE, HBG2, HBG1, HBBP1, HBD, and HBB) in 1,092 individuals representing 14 populations sequenced as part of the 1000 Genomes Project. Additionally, we assessed the diversity levels in nonhuman primates using chimpanzee sequence data provided by the PanMap Project. Comprehensive analyses, based on classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and nonredundant. Moreover, in the light of recent studies on the chromatin conformation of the ß-globin cluster, we present evidence sustaining that the strong functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but instead are likely due to a regulatory role in ontogenic switches of gene expression.

Relative frequency of known causes of multiple mtDNA deletions: two novel POLG mutations.

Diseases affecting mtDNA stability, termed nuclear-mitochondrial intergenomic communication disorders, are caused by a primary nuclear gene defect resulting in multiple mtDNA deletions. The aim of this study was to estimate the frequency of known etiologies and the spectrum of mutations in a cohort of 21 patients harboring multiple mtDNA deletions in skeletal muscle. We showed that 10 cases (48%) display mutations in POLG, including eight previously reported variants and two novel mutations (namely, p.Trp585X and p.Arg1081Gln). The novel mutations affect evolutionary conserved residues and were absent in a large set of control chromosomes. These findings expand the array of mutations associated with multiple rearranged mtDNA attributed to mutations in POLG. The relatively high diagnostic yield (about one in two cases) supports the notion that it is recommended to test POLG routinely in diagnostic laboratories whenever multiple mtDNA deletions are present, regardless of the age of onset of patients and their clinical phenotype.

Gains, losses and changes of function after gene duplication: study of the metallothionein family.

Metallothioneins (MT) are small proteins involved in heavy metal detoxification and protection against oxidative stress and cancer. The mammalian MT family originated through a series of duplication events which generated four major genes (MT1 to MT4). MT1 and MT2 encode for ubiquitous proteins, while MT3 and MT4 evolved to accomplish specific roles in brain and epithelium, respectively. Herein, phylogenetic, transcriptional and polymorphic analyses are carried out to expose gains, losses and diversification of functions that characterize the evolutionary history of the MT family. The phylogenetic analyses show that all four major genes originated through a single duplication event prior to the radiation of mammals. Further expansion of the MT1 gene has occurred in the primate lineage reaching in humans a total of 13 paralogs, five of which are pseudogenes. In humans, the reading frame of all five MT1 pseudogenes is reconstructed by sequence homology with a functional duplicate revealing that loss of invariant cysteines is the most frequent event accounting for pseudogeneisation. Expression analyses based on EST counts and RT-PCR experiments show that, as for MT1 and MT2, human MT3 is also ubiquitously expressed while MT4 transcripts are present in brain, testes, esophagus and mainly in thymus. Polymorphic variation reveals two deleterious mutations (Cys30Tyr and Arg31Trp) in MT4 with frequencies reaching about 30% in African and Asian populations suggesting the gene is inactive in some individuals and physiological compensation for its loss must arise from a functional equivalent. Altogether our findings provide novel data on the evolution and diversification of MT gene duplicates, a valuable resource for understanding the vast set of biological processes in which these proteins are involved.

Evolutionary history and functional diversification of phosphomannomutase genes.

Phosphomannomutases (PMMs) catalyze the interconversion of mannose-6-phosphate to mannose-1-phosphate. In humans, two PMM enzymes exist--PMM1 and PMM2; yet, they have different functional specificities. PMM2 presents PMM activity, and its deficiency causes a Congenital Disorder of Glycosylation (PMM2-CDG). On the other hand, PMM1 can also act as glucose-1,6-bisphosphatase in the brain after stimulation with inosine monophosphate and thus far has not been implicated in any human disease. This study aims to refine the evolutionary time frame at which gene duplication gave rise to PMM1 and PMM2, and to identify the most likely amino acid positions underlying the proteins' different functions. The phylogenetic analysis using available protein sequences, allowed us to establish that duplication occurred early in vertebrate evolution. In order to understand the molecular basis underlying the functional divergence, conserved and most likely functional divergence-related sites were identified, through the analysis of site-specific evolutionary rates. This analysis indicates that most of the sites known to be important in the homodimer formation and in the catalytic activity are conserved in both proteins. Among those potentially related to functional divergence, two positions (183 and 186 in human PMM1) emerge as the most interesting ones. The residues at these positions have different side-chain conformations in the protein structure in the unbound and bound states, and are highly but differently conserved in PMM1 and in PMM2 proteins. Altogether, these results provide new data into the evolutionary history of PMM1 and PMM2 duplicates and highlight the most probable sites that evolved to distinct functional specificities.

Epistatic interactions modulate the evolution of mammalian mitochondrial respiratory complex components.

BACKGROUND: The deleterious effect of a mutation can be reverted by a second-site interacting residue. This is an epistatic compensatory process explaining why mutations that are deleterious in some species are tolerated in phylogenetically related lineages, rendering evident that those mutations are, by all means, only deleterious in the species-specific context. Although an extensive and refined theoretical framework on compensatory evolution does exist, the supporting evidence remains limited, especially for protein models. In this current study, we focused on the molecular mechanism underlying the epistatic compensatory process in mammalian mitochondrial OXPHOS proteins using a combination of in-depth structural and sequence analyses. RESULTS: Modeled human structures were used in this study to predict the structural impairment and recovery of deleterious mutations alone and combined with an interacting compensatory partner, respectively. In two cases, COI and COIII, intramolecular interactions between spatially linked residues restore the folding pattern impaired by the deleterious mutation. In a third case, intermolecular contact between mitochondrial CYB and nuclear CYT1 encoded components of the cytochrome bc1 complex are likely to restore protein binding. Moreover, we observed different modes of compensatory evolution that have resulted in either a quasi-simultaneous occurrence of a mutation and corresponding compensatory partner, or in independent occurrences of mutations in distinct lineages that were always preceded by the compensatory site. CONCLUSION: Epistatic interactions between individual replacements involving deleterious mutations seems to follow a parsimonious model of evolution in which genomes hold pre-compensating states that subsequently tolerate deleterious mutations. This phenomenon is likely to have been constraining the variability at coevolving sites and shaping the interaction between the mitochondrial and the nuclear genome.