Monica Pirastru

Genomic signatures of adaptive introgression from European mouflon into domestic sheep

Mouflon (Ovis aries musimon) became extinct from mainland Europe after the Neolithic, but remnant populations from the Mediterranean islands of Corsica and Sardinia have been used for reintroductions across Europe since the 19th-century. Mouflon x sheep hybrids are larger-bodied than mouflon, potentially showing increased male reproductive success, but little is known about genomic levels of admixture, or about the adaptive significance of introgression between resident mouflon and local sheep breeds. Here we analysed Ovine medium-density SNP array genotypes of 92 mouflon from six geographic regions, along with data from 330 individuals of 16 domestic sheep breeds. We found lower levels of genetic diversity in mouflon than in domestic sheep, consistent with past bottlenecks in mouflon. Introgression signals were bidirectional and affected most mouflon and sheep populations, being strongest in one Sardinian mouflon population. Developing and using a novel approach to identify chromosomal regions with consistent introgression signals, we infer adaptive introgression from mouflon to domestic sheep related to immunity mechanisms, but not in the opposite direction. Further, we infer that Soay and Sarda sheep carry introgressed mouflon alleles involved in bitter taste perception and/or innate immunity. Our results illustrate the potential for adaptive introgression even among recently diverged populations.

The First Mitogenome of the Cyprus Mouflon (Ovis gmelini ophion): New Insights into the Phylogeny of the Genus Ovis.

Sheep are thought to have been one of the first livestock to be domesticated in the Near East, thus playing an important role in human history. The current whole mitochondrial genome phylogeny for the genus Ovis is based on: the five main domestic haplogroups occurring among sheep (O. aries), along with molecular data from two wild European mouflons, three urials, and one argali. With the aim to shed some further light on the phylogenetic relationship within this genus, the first complete mitochondrial genome sequence of a Cypriot mouflon (O. gmelini ophion) is here reported. Phylogenetic analyses were performed using a dataset of whole Ovis mitogenomes as well as D-loop sequences. The concatenated sequence of 28 mitochondrial genes of one Cypriot mouflon, and the D-loop sequence of three Cypriot mouflons were compared to sequences obtained from samples representatives of the five domestic sheep haplogroups along with samples of the extant wild and feral sheep. The sample included also individuals from the Mediterranean islands of Sardinia and Corsica hosting remnants of the first wave of domestication that likely went then back to feral life. The divergence time between branches in the phylogenetic tree has been calculated using seven different calibration points by means of Bayesian and Maximum Likelihood inferences. Results suggest that urial (O. vignei) and argali (O. ammon) diverged from domestic sheep about 0.89 and 1.11 million years ago (MYA), respectively; and dates the earliest radiation of domestic sheep common ancestor at around 0.3 MYA. Additionally, our data suggest that the rise of the modern sheep haplogroups happened in the span of time between six and 32 thousand years ago (KYA). A close phylogenetic relationship between the Cypriot and the Anatolian mouflon carrying the X haplotype was detected. The genetic distance between this group and the other ovine haplogroups supports the hypothesis that it may be a new haplogroup never described before. Furthermore, the updated phylogenetic tree presented in this study determines a finer classification of ovine species and may help to classify more accurately new mitogenomes within the established haplogroups so far identified.

A new unstable variant of the fetal hemoglobin HBG2 gene: Hb F-Turritana [Gγ64(E8)GlyAsp, HBG2:c.194G>A] found in cis to the Hb F-Sardinia gene [Aγ(E19)IleThr, HBG1:c.227T>C]

A new variant of the fetal hemoglobin (Hb) was observed in a newborn baby subjected to phototherapy due to jaundice, by means of electrophoretic and chromatographic techniques. The variant Hb resulted unstable by the isopropanol stability test. After HBG2 gene sequencing, the G to A transversion at codon 64, position eight of the E helix, was found, which corresponds to the Asp for Gly amino acid substitution. The new variant was called Hb F‐Turritana [Gγ64(E8)Gly→Asp, HBG2:c.194G>A]. Incoming aspartic acid residue, bulky and negatively charged, may be responsible for alteration of the heme pocket steric configuration and for instability. The new abnormal HBG2 gene was found to be associated in cis with the mutated HBG1 gene, which characterizes the Hb F‐Sardinia [Aγ (E19)Ile→Thr, HBG1:c.227T>C] variant.

Two Abnormal Fetal Hemoglobins Found in the Sardinian Population: The New Hb F-Osilo [Aγ119(GH2)Gly→Ser, GGC > AGC] and Hb F-Paulinia [Gγ80(EF4)Asp→Tyr, GAT > TAT] Already Described In The Brazilian Population

Two healthy newborns, heterozygous for two different γ-globin chain mutations, were observed during an electrophoretic screening for hemoglobinopathies in Sassari, North Sardinia (Italy). The variants were characterized by reversed phase high performance liquid chromatography (HPLC) and sequencing of amplified γ-globin genes. One of the two abnormalities was a novel Aγchain variant and the tetramer was named Hb F-Osilo [Aγ119(GH2)Gly→Ser]. The other was a Gγ chain variant, Hb F-Paulinia [Gγ80(EF4)Asp→Tyr], already described in a Brazilian baby of African ancestry. No functional studies could be performed.

Hb F‐Porto Torres [Aγ75(E19)Ile→Thr, 136(H14)Ala→Ser]: A Novel Variant of the Aγ Chain Having Two Substitutions, One Being that of Hb F‐Sardinia

The abnormal Hb F‐Porto Torres [Aγ75(E19)Ile→Thr, 136(H14)Ala→Ser] was observed during a cord blood survey for hemoglobinopathies in North Sardinia. This silent variant showed the same mobility as Hb F‐Sardinia in isoelectric focusing (IEF) of the tetramers, whereas the abnormal globin chain was clearly separated by acid‐urea‐Triton polyacrylamide gel electrophoresis (AUT‐PAGE) from the normal Gγ‐ and Aγ‐globin chains. Separation of the globin chains by reversed phase high performance liquid chromatography (HPLC) indicated the following percentages: Gγ 68.4, Aγ 14.0, Xγ 17.6, that strongly suggested the abnormal chain as being a variant of the Aγ‐globin. Sequencing of the γ‐globin genes indicated that the mutated gene was in fact an Aγ with two nucleotide replacements, one being the ATA→ACA (Ile→Thr) at codon 75 (the so‐called AγT of the rather common Hb F‐Sardinia) and the second the GCA→TCA (Ala→Ser) at codon 136. This new variant is the seventh having the sequence of the AγT chain with an additional mutation so far described and the third characterized by gene sequencing.

The C→G transition in the α2‐globin gene of a normal αα‐chromosome is responsible for the Hb G‐Philadelphia variant in Sardinians

Sequencing of α‐globin genes of 18 Sardinian heterozygotes for the Hb G‐Philadelphia [α68(E17)Asn→Lys] variant, with four active α genes and circulating level of the variant of about 27%, showed the AAC→AAG change at codon 68 of the α2‐globin gene (αGα/αα). Two heterozygotes with level of about 37% were the carriers of the same mutation on the same α2 gene, and of the α2α1 hybrid gene, because of the 3.7‐kb deletion, in trans (αGα/−α3.7). In Black people, the same C→G mutation occurs on the hybrid gene (−αG3.7), whereas in Caucasians the Lys for Asn change is because of the C→A transversion occurring on the α2 gene of a normal αα arrangement. The identification of the C→G mutation on the normal αα chromosome points to an undescribed genotype for this rather common variant, which is probably because of the high rate of recombination between the duplicated α‐globin genes.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δβ-Thalassemia Carrier.

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δβ-thalassemia condition. The probands hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α₂^Aγ₂ and α₂^Gγ₂ instead of nearly 100% α₂^Aγ₂. In order to explain the unusual γ-chain ratio, sequencing of the ^Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the ^Gγ promoter. Results indicated that the observed in vivo ^Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

The Hemoglobin Polymorphism in Sardinian Goats: Nucleotide Sequence and Frequency of ßA, ßD, ßD-Malta, and ßE Globin Genes

As in most vertebrates, the goat α-globin gene locus is duplicated though producing Iα and IIα globin chains that differ in three amino acid residues [[1]–[3]]. As the result of an unusual evolutionary history, however, a goat (β-globin gene cluster consists of 12 genes organized as a triplicated four-gene set located on chromosome 7: 5’-eI-eII-ψβX-βC-eIII-eIV-ψβY-βA-eV-eVI-ψβZ-βF-3’[[4]-[6]]. The βA-globin gene is expressed in adult animals, whereas βF and βC genes are expressed in the fetus and in juveniles up to 6 months of age, respectively [[7]]. During the first year of postnatal life, the synthesis of pre-adult βC-globin is supplanted by the synthesis of adult βA. The βC→βA switch is reversible and the reactivation of the βC synthesis, at the expense of βA, can be induced by anemia, hypoxia, or the administration of erythropoietin [[1],[8]]. Due to this unusual feature, which is also common in sheep hemoglobin (Hb) type A and in Sardinian mouflon [[5], [7], [9], [10]], the organization of globin genes, the nucleotide sequence, and the evolution of goat and sheep globin genes have been extensively studied [[1]–[2], [11]–[13]].

Molecular Characterization of β-Thalassemia Mutations in Central Vietnam

Abstract The molecular basis of β-thalassemia (β-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of β-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the β0 and two of the β+ type) in a total of 48 chromosomes. The most common was codon 26 (G>A) or Hb E (HBB: c.79 G>A) accounting for 29.2% of the total studied chromosomes, followed by codon 17 (A>T) (HBB: c.52 A>T) (25.0%), and codons 41/42 (–TTCT) (HBB: c.126_129delCTTT) (18.8%). Other mutations with appreciable frequencies (6.3–8.3%) were IVS-I-1 (G>T) (HBB: c.92+1 G>T), codon 26 (G>T) (HBB: c.79 G>T) and codons 71/72 (+A) (HBB: c.216_217insA). Relatively rarer (2.0%) were the promoter –28 (A>G) (HBB: c.78 A>G) mutation, the codon 95 (+A) (HBB: c.287_288insA), which is reported only in the Vietnamese, and the codons 14/15 (+G) (HBB: c.45_46insG) mutation, thus far observed only in Thailand. Results are relevant for implementing appropriate measures for β-thal prevention and control in the region as well as in the whole country.

Functional properties of the newly observed (G)γ-chain fetal hemoglobin variant Hb F-Monserrato-Sassari (HBG2:c.280T>C) or [(G)γ93 (F9) Cys→Arg].

BACKGROUND HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population. METHODS Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined. RESULTS Sequencing of the γ globin genes revealed the TGT→CGT transition at codon 93 in one of the two (G)γ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity. GENERAL SIGNIFICANCE Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T→R transition of the HbF tetramer. Substitutions at the F9 position of the (G)γ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.