A2 Variant Research Articles

Proteomics and Integrated Techniques to Characterize Organic Residues in Funerary Findings from Italic Populations of the First Millennium BC.

Multiple analytical techniques were combined to achieve a detailed characterization of organic residues in different typologies of funerary pottery, which were found at two separate archeological sites in the Campania Region (Italy) and both dated back to the first millennium BC. Gas chromatography-mass spectrometry (GC-MS) analysis of lipids provided inconclusive results. The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of encrustation on two glazed bowls of the 3rd to 4th century BC were comparable to those of fresh bone, revealing the presence of hydroxyapatite and proteins, which were identified as bovine collagen chains by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS)-based proteomics. This finding confirmed that Italic populations used to inhume the dead along with votive meat offerings. Proteomics was decisive for identifying bovine milk in an unusually shaped amphora unearthed from a grave that belonged to a woman at the necropolis of the Greek colony in Cuma (7th century BC). Peptidomic analysis demonstrated that the genetic variant A1 of β-casein was already present in the southern Mediterranean area at least 2500 years ago. Overall, these results depict an agropastoral system of Italic populations at the age of Magna Graecia based on a significant role of domesticated cattle.

Analysis of CSN2 variants in Friesian Holstein cows and their association with milk protein allergy and production traits

<p><strong>Objective: </strong>The objective of this study was to analyze <em>CSN2</em> variants in Indonesian Friesian Holstein (FH) cow and their association with milk protein allergy and production traits.</p><p><strong>Methods: </strong>Genomic DNA was extracted from bloods of twelve Indonesian FH cow. Exon 7 of the <em>CSN2</em> was amplified using novel primer pair to produce 683 bp amplicon. The primers were 5’-ACCCCAATTTCTTAACCAAACCA-3’ as a forward primer and 5’-CATCAGAAGTTAAACAGCACAGT-3’ as a reverse primer. The PCR products were analyzed to determine the nucleotide sequence of <em>CSN2</em> using Bioedit version 7.2.5. Moreover, Hardy-Weinberg equilibrium (HWE) was calculated and one-way analysis of variance (ANOVA) was conducted to associate between <em>CSN2</em> variants and milk production traits.</p><p><strong>Results: </strong>Two polymorphisms, c.350A>C and c.516G>C, were identified in the <em>CSN2 </em>exon 7. Base substitution from adenine (A) to cytosine (C) of c.350A>C changed amino acid codon from histidine (CAU) to proline (CCU), and base substitution guanine (G) to cytosine (C) of c.516G>C changed amino acid codon from arginine (AGG) to serine (AGC). The CC genotype frequency for c.350A>C SNP was 33% and they were able to produce A2 <em>CSN2</em> variant which is favorable for preventing lactose intolerance. In addition, there were no association between c.350 A>C and c.516 G>C SNP of the <em>CSN2</em> with milk production traits.</p><p><strong>Conclusions: </strong>In conclusion, A1 and A2 variants of <em>CSN2</em> were identified in Indonesian FH population and they did not associate with milk production in Indonesian FH.</p><p> </p>

The β-casein (CSN2) A2 allelic variant alters milk protein profile and slightly worsens coagulation properties in Holstein cows

Milk proteins genetic variants have long attracted interest as they are associated with important issues relating to milk composition and technological properties. An important debate has recently opened at an international level on the role of β-casein (β-CN) A1 and A2 polymorphisms, toward human health. For this reason, a lot of efforts has been put into the promotion of A2 milk by companies producing and selling A1-free milk, leading the farmers and breeders to switch toward A2 milk production without paying attention on the potential effect of the processability of milk into cheese. The aim of the present work was to evaluate the effects of β-CN, specifically the A1 and A2 allelic variants, on the detailed milk protein profile and cheese-making traits in individual milk samples of 1,133 Holstein Friesian cows. The protein fractions were measured with reversed-phase (RP)-HPLC (expressed in g/L and % N), and the cheese-making traits, namely milk coagulation properties, cheese yield, and curd nutrient recoveries assessed at the individual level, with a nano-scale cheese-making procedure. The β-CN (CSN2), κ-CN (CSN3), and β-lactoglobulin (LGB) genetic variants were first identified through RP-HPLC and then confirmed through genotyping. Estimates of the effects of protein genotypes were obtained using a mixed inheritance model that considered, besides the standard nuisance variables (i.e., days in milk, parity, and herd-date), the milk protein genes located on chromosome 6 (CSN2, CSN3) and on chromosome 11 (LGB), and the polygenic background of the animals. Milk protein genes (CSN2, CSN3, and LGB) explained an important part of the additive genetic variance in the traits evaluated. The β-CN A1A1 was associated with a significantly lower production of whey proteins, particularly of β-lactoglobulin (-8.2 and -6.8% for g/L and % N, respectively) and α-lactalbumin (-4.7 and -4.4% for g/L and % N, respectively), and a higher production of β-CN (6.8 and 6.1% for g/L and % N, respectively) with respect to the A2A2 genotype. Regarding milk cheese-making ability, the A2A2 genotype showed the worst performance compared with the other genotypes, particularly with respect to the BA1, with a higher rennet coagulation time (7.1 and 28.6% compared with A1A1 and BA1, respectively) and a lower curd firmness at 30 min. Changes in milk protein composition through an increase in the frequency of the A2 allele in the production process could lead to a worsening of the coagulation and curd firming traits.

Assessment of A1 and A2 variants in the CNS2 gene of some cattle breeds by using ACRS-PCR method

The aim of this study is to reveal β-casein polymorphism of some cattle breeds and also the potential to produce A2 milk from existing animals and to develop strategies in this area. Therefore, a total of 400 cattle, 100 animals from each breed of Holstein, Brown Swiss, Jersey and Simmental raised commonly in Turkey, were obtained, and C > A polymorphism in 67th amino acid in the 7th exons of β-casein gene was determined by TaqI enzyme with PCR-ACRS method. Blood samples were collected from dairy cattle farms raising Holstein, Brown Swiss and Jersey breeds from Konya province and Simmental breed from Kütahya province in Turkey. A1 and A2 allele frequencies in Holstein, Brown Swiss, Jersey and Simmental cattle breeds were determined as 0.475/0.525, 0.370/0.630, 0.215/0.785 and 0.440/0.560, respectively. The highest A2 allele frequency (0.785) was found in Jersey breed. A1A1 genotypes in Holstein, Brown Swiss, Jersey and Simmental breeds were 0.240, 0.150, 0.030 and 0.160, respectively; A1A2 genotypes were 0.470, 0.440, 0.370 and 0.560, respectively; A2A2 genotypes were determined as 0.290, 0.410, 0.600 and 0.280, respectively. In these breeds, the highest A2A2 genotype frequency was found in Jersey (0.600), the lowest A1A1 genotype frequency (0.030) was found in Jersey and the highest A1A2 genotype frequency (0.560) was found in Simmental. Holstein, Brown Swiss, Simmental and Jersey populations were at the level of Hardy-Weinberg in terms of β-casein gene (p > 0.05). The average Ho, He and PIC values were calculated as 0.460, 0.469 and 0.605, respectively. In conclusion, the frequency of commonly reared cattles in Turkey especially Brown Swiss, and Jersey breeds in A2A2 genotype are satisfactory, but it can be said that the use of animals with A2 allele in selection is extremely important for increasing A2 milk producing potential in the future.

Genotyping of HF Crossbred Breeding Bulls for A1/A2 Variant of β-Casein Gene

Background: The variants of β-casein (CSN 2) gene viz. A1 and A2 are supposed to be related with human health. Hence, to know the status of breeding bulls used for AI services were screened for A1 and A2 variants. Total 47 blood samples of crossbred breeding bulls from Frozen Semen Laboratory, Pune and Aurangabad were used for present work. Methods: The extraction of genomic DNA by phenol-chloroform method were carried out from the blood samples of 47 breeding bulls. Further, DNA based PCR-RFLP method was used to identify the A1, A2 variants. Pure DNA fragments of b-casein (CSN 2) gene were amplified by PCR using suitable pairs of primer to amplify 121 bp and 244 bp products. The primer used to amplify 244 bp was allele specific. The 121 bp fragment then subjected to restriction enzyme digestion. The restriction enzyme digestions resulted into two types of band pattern. The 244 bp fragment was used for validation of A1 and A2 allele of 121 bp. Result: Two types of genotypes i.e. A1A2 and A2A2 were revealed in the experimental population of HF crossbred breeding bulls. The genotypic frequency of A1A2 and A2A2 was estimated 65 and 35 per cent, respectively. The present frequency status of A1 and A2 allele in the breeding bulls may be suggested for planning future breeding strategy in dairy cattle.

Analysis of Tat protein characteristics in human immunodeficiency virus type 1 sub-subtype A6 (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus-1)

Tat protein is a major factor of HIV (human immunodeficiency virus) transcription regulation and has other activities. Tat is characterized by high variability, with some amino acid substitutions, including subtypespecific ones, being able to influence on its functionality. HIV type 1 (HIV-1) sub-subtype A6 is the most widespread in Russia. Previous studies of the polymorphisms in structural regions of the A6 variant have shown numerous characteristic features; however, Tat polymorphism in A6 has not been studied.Goals and tasks. The main goal of the work was to analyze the characteristics of Tat protein in HIV-1 A6 variant, that is, to identify substitutions characteristic for A6 and A1 variants, as well as to compare the frequency of mutations in functionally significant domains in sub-subtype A6 and subtype B. The nucleotide sequences of HIV-1 sub-subtypes A6, A1, A2, A3, A4, subtype B and the reference nucleotide sequence were obtained from the Los Alamos international database. Q54H and Q60H were identified as characteristic substitutions. Essential differences in natural polymorphisms between sub-subtypes A6 and A1 have been demonstrated. In the CPP-region, there were detected mutations (R53K, Q54H, Q54P, R57G) which were more common in sub-subtype A6 than in subtype B. Tat protein of sub-subtype A6 have some characteristics that make it possible to reliably distinguish it from other HIV-1 variants. Mutations identified in the CPP region could potentially alter the activity of Tat. The data obtained could form the basis for the drugs and vaccines development.

A1/A2 Milk Research in Indian Cattle

Bovine beta casein A1 and A2 are the most common variants in cattle breeds. Because of the bioactive peptide beta casomorphin-7 (BCM7) produced by raw or processed A1-milk, which has high affinity for opioid receptors and can exert regulatory activities, A1 variant is considered a risk factor for many human diseases, but not by A2-milk, during digestion. The frequency of A1/A2 allele of beta casein is breed dependent and can be very easily checked with simple PCR-RFLP technique. The aim of this article was to review the different studies and assess the status of A1/A2 in Indian native cattle breeds as well as crossbred/taurine populations and breeding bulls being used at different AI centres in India. Analysis of frequency pattern clearly indicated that all Indian native cattle breeds have high frequency of A2 allele and homozygous A2A2 genotype and hence are very good resource for A2 milk. Crossbred, taurine cattle populations and breeding bulls being used at different AI centres have a higher frequency of A2 allele and A1A2 genotype. The percentage of animals with homozygous A2A2 genotype is low. This suggests the need for screening of crossbred/exotic bulls being used in A1 and modify the existing breeding policy so as to drift the herds towards A2.

Hemoglobin A2 and Heterogeneous Diagnostic Relevance Observed in Eight New Variants of the Delta Globin Gene.

Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Molecular serological characteristics of ABO system A antigen

Background. One of the polymorphic antigens in the ABO system is antigen A, which includes many allelic variants with different expression. Immunological methods for determining the blood group of the ABO system have limitations in their use, including due to the presence of weekly expressed antigens in humans. For the correct determination of blood group according to the ABO system, genetic typing is becoming increasingly important. 89 alleles of the ABO*A gene are known. Knowledge of ABO*A gene polymorphisms and their prevalence contributes to the prevention of errors in determining the blood group of donors and recipients.Objective: to describe variants of ABO*A gene alleles in Russians and serological characteristics of the antigens encoded by them.Materials and methods. The blood of 14,000 people was examined. The blood group was determined using anti-A, anti- Aweak, anti-B, lectin (anti-A1) and gel cards. A molecular study of ABO*A gene polymorphisms was conducted in 151 people. Polymerase chain reaction with sequence-specific primers and direct Sanger sequencing were used.Results. 7 different ABO*A alleles were detected, including the ABO*A1.01 and ABO*A1.02 alleles. In 118 individuals with a weak A antigen, the ABO*A2.01 allele was the most frequent (87.29 %). Rare alleles ABO*A2.06 (5.93 %), ABO*AW.06 (4.23 %), ABO*A2.09 (0.85 %) and ABO*Ax (1.70 %) were found. Serological characteristics of A antigens variants depending on genotypes are described, variants A1, A2, A3 and very weak A were detected. Extraagglutinins α1 were absent in all persons with weakened A antigen.Conclusion. Small or mixed agglutination with Coliclones or red blood cell stratification in the gel suggest the presence of antigen A with weakened expression. Modern molecular methods make it possible to identify rare gene alleles and genotypes. Erythrocyte genomics helps to resolve the ambiguity of the serological results allows understanding the true mechanisms of particular phenotype formation and makes a contribution to ensuring the immunological safety of blood components transfusions.

Phage Display Functionally Defines Variants in the Von Willebrand Factor Platelet Binding Domain

Missense variants in the von Willebrand factor (VWF) platelet binding domain, A1, may pathologically hyperactivate or weaken interactions with its platelet receptor, GPIbα, and lead to von Willebrand disease (VWD) subtypes 2B or 2M, respectively. Variants identified in VWD patients and tested as recombinant VWF have supported genotype-phenotype associations and subtyping of VWD by genetic analyses. However, novel variants, most classified as variants of uncertain significance (VUS) are poorly defined. To functionally characterize a large subset of VWF A1 variants (P1254-L1460), we screened a phage display library for binding to a recombinant form of GPIbα used to clinically assess VWF platelet binding activity, GPIbM. Comprised of ~5x10 6 independent clones, the phage display library contained 1,427 unique, missense variants (~36% of all possible single amino acid substitutions) which could be scored for significant enrichment, depletion, or no change following selection for GPIbM binding. The enrichment of phage displayed VWD variants previously classified as VWD subtype 2B significantly segregated from reported 2M variants (mean fold change from preselected phage ~1.06 for 2B vs. ~0.68 for 2M, p < 0.005). To further validate these findings, five depleted, four unchanged, and seven enriched VWF A1 variants were introduced into the full length VWF sequence by site-directed mutagenesis and expressed by transient transfection of HEK293T cells. Conditioned media were collected and analyzed for VWF level (VWF:Ag) and activity (VWF:GPIbM). Of the sixteen variants examined, fourteen (87.5%) exhibited a VWF GPIbM:Ag ratio that was concordant with the phage display findings. Furthermore, the VWF GPIbM:Ag ratios were well correlated with the degree of enrichment by phage display (Pearson R = 0.69, p< 0.01). Taken together, these findings demonstrate phage display as a high content approach to measure and functionally define the platelet-binding activity of genetic variants within the VWF A1 domain. DisclosuresGinsburg: Takeda: Patents & Royalties.

Molecular screening of bovine β-casein (CSN2) A2 variant in Indonesian Holstein dairy cattle as attempt to produce digestive friendly milk

Cow’s milk-intolerance is a digestive problem on people who not able to digest milk. This problem may relate to the variant of (3-casein (CSN2), especially A1, suggested due to (3-casomorphins (BCM-7) formation during enzymatic digestion, for that selecting cattle free BCM-7 become a concern to produce digestive friendly milk. This study aimed to differentiate A1 and A2 allele variant of CSN2 gene in selected population of Indonesian Holstein cattle. In total 70 cows DNA were collected, and fragment of CSN2 exon 7 which contain Single Nucleotide Polymorphism (SNP) rs43703011 and rs43703013 were amplified. Variant analysis was done by mutation site analysis using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) with Mspl restriction enzyme and DNA sequence for confirmation. Result shows 2 allele variants of (3-casein that are B type, representing A1 family variant, and A2 in mutation site rs43703013C>G. We found A2 allele in the studied population is superior in frequency than A1 (0.916 vs. 0.084). Of that, 8.6% cattle were heterozygotes that is BA2 and 91.4% were homozygotes A2A2. Cattle which carry A1 allele variant should be excluded from dairy cattle breeding program for further milk production free of BCM-7.

Effect of Protein Genotypes on Physicochemical Properties and Protein Functionality of Bovine Milk: A Review.

Milk protein comprises caseins (CNs) and whey proteins, each of which has different genetic variants. Several studies have reported the frequencies of these genetic variants and the effects of variants on milk physicochemical properties and functionality. For example, the C variant and the BC haplotype of αS1-casein (αS1-CN), β-casein (β-CN) B and A1 variants, and κ-casein (κ-CN) B variant, are favourable for rennet coagulation, as well as the B variant of β-lactoglobulin (β-lg). κ-CN is reported to be the only protein influencing acid gel formation, with the AA variant contributing to a firmer acid curd. For heat stability, κ-CN B variant improves the heat resistance of milk at natural pH, and the order of heat stability between phenotypes is BB > AB > AA. The A2 variant of β-CN is more efficient in emulsion formation, but the emulsion stability is lower than the A1 and B variants. Foaming properties of milk with β-lg variant B are better than A, but the differences between β-CN A1 and A2 variants are controversial. Genetic variants of milk proteins also influence milk yield, composition, quality and processability; thus, study of such relationships offers guidance for the selection of targeted genetic variants.

β-Casein variants differently affect bulk milk mineral content, protein composition, and technological traits

Detailed milk protein composition has strong influence on milk quality. This study quantitatively investigated the effect of β-casein variants A1, A2 and B on composition, and technological traits of bulk milk. A total of 171 commercial herds located in northern and central Italy were visited, and 299 bulk milk samples were collected. Traditional milk quality traits, major minerals, milk coagulation properties were assessed. Detailed milk protein composition and β-casein genetic variants were determined by reversed-phase HPLC. Effects of β-casein variants were estimated by fitting a mixed model with variants concentration as a covariate. Results demonstrated that greater concentration of β-casein B is linked with greater protein, casein, and fat content. Likewise, greater concentration of variants A1 and B of β-casein had a significant positive effect on rennet coagulation time and curd firmness, and was linked to a greater amount of Ca and P content than was the A2 variant.

The Global Scenario of A1, A2 β-Casein Variant in Cattle and its Impact on Human Health

Beta casein (β-casein) has gained considerable interest among scientists and farmers as it provides promising factors to milk that enhance its quality and concentration. It is believed that the A1 β-casein variant shares an association with numerous human diseases. Since there are numerous hypotheses concerning the link between variants of β-casein and human diseases, many researchers across the globe have genotyped the cattle in terms of A1, A2 β-casein variants. This paper catalogs the A1 and A2 β-casein genotypes shared by different cattle breeds across the globe. Various literature encompassing the adverse effects of β-casein variants were assessed adequately. This paper suggests that the effects of undertaking the A1 β-casein variant on human health are ambiguous. Adequate research providing stronger evidence is needed for understanding the effects imparted by the consumption of both A1 and A2 β-casein variants on human health.

Indian cow and A2 beta-casein – A scientific perspective on health benefits

The cow has a special place in Hinduism since time immemorial due to the diverse health benefits of its products called Panchagavya. Ayurveda has well-described the medicinal benefits of cow milk, curd, ghee, dung and urine in indigestion, microbial infections, hormonal imbalance and neurological disorders. Indian cows are well-known for producing A2 beta-casein proteincontaining milk which is useful in hormonal disorders including diabetes, hypertension and dyslipidemia. On the other hand, milk containing A1 genotype of beta-casein has been considered to be harmful to health. Cow milk is proven to be a most nutritious food with the bio-protective effect which contains carotenes, vitamin A, vitamin B complex and vitamin C together with flavones, sterols and phenols. The present manuscript describes a general comparison between A1 and A2 variants of beta-casein based on scientific evidence. In view of the health benefits, the present work mainly emphasizes the conservation and propagation of indigenous Indian cow.

Ovine FABP4 Variation and Its Association With Flystrike Susceptibility.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and “dags” (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A1. The likelihood of flystrike occurrence decreased as copy number of A1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

“A2 milk” authentication using isoelectric focusing and different PCR techniques

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial “A2 milk” in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled “A2 milk”) to protect consumers from this unexpected fraud.

Genetic Variants in Cytosolic Phospholipase A2 Associated With Nonsteroidal Anti-Inflammatory Drug-Induced Acute Urticaria/Angioedema.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the main triggers of drug hypersensitivity reactions, probably due to their high consumption worldwide. The most frequent type of NSAID hypersensitivity is NSAID cross-hypersensitivity, in which patients react to NSAIDs from different chemical groups in the absence of a specific immunological response. The underlying mechanism of NSAID cross-hypersensitivity has been linked to cyclooxygenase (COX)-1 inhibition causing an imbalance in the arachidonic acid pathway. Despite NSAID-induced acute urticaria/angioedema (NIUA) being the most frequent clinical phenotype, most studies have focused on NSAID-exacerbated respiratory disease. As NSAID cross-hypersensitivity reactions are idiosyncratic, only appearing in some subjects, it is believed that individual susceptibility is under the influence of genetic factors. Although associations with polymorphisms in genes from the AA pathway have been described, no previous study has evaluated the potential role of cytosolic phospholipase A2 (cPLA2) variants. This enzyme catalyzes the initial hydrolysis of membrane phospholipids to release AA, which can be subsequently metabolized into eicosanoids. Here, we analyzed for the first time the overall genetic variation in the cPLA2 gene (PLA2G4A) in NIUA patients. For this purpose, a set of tagging single nucleotide polymorphisms (tagSNPs) in PLA2G4A were selected using data from Europeans subjects in the 1,000 Genomes Project, and genotyped with the iPlex Sequenom MassArray technology. Two independent populations, each comprising NIUA patients and NSAID-tolerant controls, were recruited in Spain, for the purposes of discovery and replication, comprising a total of 1,128 individuals. Fifty-eight tagSNPs were successfully genotyped in the discovery cohort, of which four were significantly associated with NIUA after Bonferroni correction (rs2049963, rs2064471, rs12088010, and rs12746200). These polymorphisms were then genotyped in the replication cohort: rs2049963 was associated with increased risk for NIUA after Bonferroni correction under the dominant and additive models, whereas rs12088010 and rs12746200 were protective under these two inheritance models. Our results suggest a role for PLA2G4A polymorphisms in NIUA. However, further studies are required to replicate our findings, elucidate the mechanistic role, and evaluate the participation of PLA2G4A variants in other phenotypes induced by NSAID cross-hypersensitivity.

HPV73 in cervical cancer and distribution of HPV73 variants in cervical dysplasia.

HPV73 is classified as possibly oncogenic and is not recognized by most commercial primary HPV screening platforms. The aim was to determine the prevalence of HPV73 among invasive cervical cancers, formalin-fixed paraffin embedded (FFPE) samples (N = 69), from southern Sweden during 2009 to 2010. Another aim was to determine proportions of HPV73 among Aptima HPV assay negative cervical cancers (N = 9, out of 206 cancers) and of high-grade cytological cervical diagnosis (N = 75, out of 5807 high grade lesions) in liquid-based cytology (LBC) samples collected between 2016 and 2019. We also investigated the distribution of HPV73 variants A1, A2 and B among HPV73-positive cases. HPV73 was detected by multiplex MGP-PCR and Luminex, and HPV73 variants were identified by sequencing PCR amplicons. HPV73 was detected in 2.9% (2/69, 95% CI: 0.18-9.9) of the FFPE cervical cancer series. Among the Aptima HPV-negative LBC samples, HPV73 was present in 55.5% (5/9) of the cancers and 29.3% (22/75) of the different grades of cervical diagnosis. The A1, A2 and B variants were present in 6.9% (2/29), 82.7% (24/29) and 10.3% (3/29) of the HPV73-positive women, respectively. Among the seven HPV73 cancer cases (two FFPE samples and five LBC samples), six A2 and one A1 isolate were detected. In summary, the A2 variant of HPV73 was most common in our region. In addition, the observed prevalence of HPV73 (2.9%) in cervical cancers and its relative high occurrence (55.5%) among Aptima HPV-negative cancers urge that detection of HPV73 should be included in future primary HPV screening programs.

Casein Intake Post Weaning Modulates Gut Microbial, Metabolic and Behavioral Profiles in Rats

The postnatal period is critical for brain and behavioral development and it is sensitive to environmental stimuli, such as nutrition. Prevention of weaning from maternal milk was previously shown to result in stress-induced immobility during the forced swim test (FST), indicative of depressive-like behavior, accompanied by neurochemical, gut microbial and metabolic alterations in rats. Separately, weaning was also found to act as a developmental trigger for a population of opioid receptor subtypes, an effect dependent on loss of dietary casein. In the present study, we explore the impact of exposure to the casein component of milk, and its two major genetic variants A1 and A2 β-casein, beyond weaning age. Rats exposed to casein-rich and casein-free milk, as well as to commercialized milk, containing a mixture of A1 and A2 β-casein, or exclusively A2 β-casein milk, from postnatal day (PND) 21 to PND25 were assessed for immobility behavior during the FST. Casein-rich and A1 β-casein containing milk but not casein-free and A2 β-casein milk increased stress-induced immobility compared to weaned control animals. This was concomitant with an increased abundance of bacteria from the Clostridium histolyticum group in the caecum and colon of the casein-rich group, region specific alterations of µ-opioid and oxytocin receptors in the brain, as well as modifications in urinary biochemical profiles. The A1 β-casein group additionally displayed a strong association between altered gut microbial metabolites in urine and brain metabolites. Our findings suggest that exposure to milk casein, in particular to A1 β-casein, beyond weaning age enhances stress-induced immobility during the FST, promoting a depressive-like phenotype, via a possible gut-brain axis dependent mechanism.