A2 Variant Research Articles

Genetic variation in bovine milk proteins: Implications for functional and nutritional properties

The six major bovine milk proteins are encoded by highly polymorphic chromosomes. Genetic variation has become a topic of interest and controversy in recent years due to supposed adverse health effects, beginning with the A2 variant of β‐casein. The functional implications of genetic variants have been well characterised, specifically heat stability and coagulation (i.e. rennet and acid) properties of milk, which have found that the B variant of β‐casein, κ‐casein and β‐lactoglobulin are the most beneficial. Genetic variants of milk proteins provide considerable potential to create new dairy products to add value to milk.

Case Report: Hemoglobin Hasharon with Concurrent Alpha Thalassemia in a Patient with Maple Syrup Urine Disease

Abstract Hemoglobin Hasharon (Hb Hasharon) is an unstable hemoglobin variant stemming from a mutation in the alpha globin gene. This mutation involves the replacement of aspartic acid with histidine at position 47 of the alpha globin chain. Initially identified in 1967 in Israel, Hb Hasharon has since been sporadically reported in various populations, including in Italy, Greece, Germany, and among Latin Americans of Mediterranean descent. Only three English articles have reported several cases of the combined alpha thalassemia deletion and Hasharon mutation, all between 1978-1980. Notably, there has been no prior report of Hb Hasharon with concurrent alpha thalassemia in patients with maple syrup urine disease (MSUD). We hereby present the first case of Hb Hasharon with alpha thalassemia in a patient diagnosed with MSUD. This patient, a 16-month-old female, received a prenatal diagnosis of MSUD through amniocentesis, confirming homozygosity for a known pathogenic variant in DBT (c.75_76delAT, p.C26wfsX2). Prompt initiation of formula feeding has effectively controlled her MSUD. The patient was referred for further evaluation due to hemoglobin newborn screen showing hemoglobin A, Hb F and “variant hemoglobin”. Her parents denied any family history of hemoglobinopathy. Her complete blood count was predominantly normal except for mild microcytosis, with a mean corpuscular volume of 69.1 fl (normal range 70.0-86.0 fl). The patient didn’t exhibit neonatal jaundice or any other signs or symptoms of hemolysis. The reticulocyte count was also within normal limits. The hemoglobin electrophoresis results revealed a complex pattern, with 28.8% of an unknown hemoglobin between zone 4 and 5 on capillary zone electrophoresis (CZE) as well as a possible A2 variant in zone 1. High-performance liquid chromatography (HPLC) indicated a 29.1% peak at a retention time of 4.72 minutes, along with several smaller peaks, that were indicative of the degradation of the unstable unknown hemoglobin. Although the zones and retention time aligned with Hb Hasharon, the percentage differed from the expected range of 19-20% in CZE and 18-21% in HPLC for this variant, prompting the team to order alpha and beta globin sequencing tests. Beta globin gene complete sequencing yielded negative results, while the sequencing of alpha globin gene revealed that the patient carries one copy of the -alpha3.7 alpha-globin deletion and one copy of the c.142G>C (p.Asp48His) variant in the alpha2-globin (HBA2) gene, resulting in an -alpha/alpha2alpha1 genotype with Hb Hasharon. While Hb Hasharon is generally not clinically significant, its co-occurrence with alpha-thalassemia can lead to hemolytic anemia. In our patient, clinic and chemical parameters were predominantly within normal ranges, except for mild microcytosis. The potential interaction between MSUD and Hb Hasharon with concurrent alpha thalassemia remains unknown. Continued patient monitoring will enable us to evaluate whether these conditions may influence each other’s clinical and biochemical profiles.

Genotypic profiling of A1 and A2 β-casein alleles in Punganur cattle using PCR-RFLP

Milk is regarded as nature’s perfect food due to the presence of essential nutrients required for the growth and development of mammals. The consumption of milk with a specific β casein variant either A1 or A2 had a relationship with the risk of diseases has been identified. Considering the importance of β casein variants with the health significance, the present study was undertaken in Punganur cattle (N=175) by using PCR-RFLP with Dde I restriction enzyme that showed the distribution of A1A2 (121 bp, 86 bp, and 35 bp) in 5 animals, A2A2 (86 bp and 35 bp) in 170 animals with no evidence of A1A1 genotype. The genotypic frequency of the A1A2 heterozygous β-casein variant of exon7 of the CSN2 gene was 0.029, whereas, for the A2A2 homozygous β-casein variant was 0.971 and the A1A1 homozygous β-casein variant is absent in Punganur cattle. The A1 allelic frequency of exon7 of the CSN2 gene was 0.014, whereas, the A2 gene frequency is 0.986 in Punganur cattle. The study indicates that there is a minimum genetic variability in the tested population of the Punganur cattle breed suggesting less or lower level of crossbreeding activities with exotic animals.

Comparison of early and late aseptic baseplate failure in primary reverse shoulder arthroplasty with and without structural glenoid autograft

Early reverse total shoulder arthroplasty (RTSA) designs demonstrated high glenoid baseplate complication and revision rates. While contemporary designs have reduced the incidence of glenoid baseplate failures, there are reports of elevated failure risks in RTSA with glenoid bone grafting within the first 2 years. This study aims to evaluate the incidence and etiology of aseptic glenoid baseplate failure with a contemporary central screw baseplate. The null hypothesis is that majority of the baseplate failure occurs within the first 2 years and use of glenoid bone grafting does not lead to higher risk of baseplate failure. In 2014 - 2019, 753 consecutive patients who underwent primary RSA using the same inlay press-fit humeral stem and monoblock central screw baseplate were retrospectively reviewed. Fracture and septic arthropathy cases were excluded. All patients underwent preoperative radiographic and computed tomography evaluation. If there was significant glenoid erosion (Walch A2, B2, B3, C1, C2, E2, E3, and/or E4 variants), patient-specific structural glenoid bone grafting was performed. All patients underwent standardized radiographic follow-up and failure was strictly defined as any hardware breakage and/or shift in glenoid baseplate position. Failures were defined as "early" if occurring within 2 years and "late" if occurring greater than 2 years after surgery. Comparative analysis was performed to evaluate demographics, glenoid graft use, and graft union rates between the cohorts. There were 23 patients with baseplate failures (23/753, 3.0%) at mean of 23 months. Twenty-two failures (96%) occurred in patients who received structural glenoid bone grafting. Only 1 failure (0.2%) occurred when bone grafting was not indicated (p<0.001). The most common failure pattern was associated with B2 glenoid (16/23, 70%). There were 5 (22%) early failures and 18 (78%) late failures. There were no differences in any patient demographic characteristics between cohorts. All 5 early failures had graft nonunion and 4/5 occurred without trauma. In the 18 late failures , 9/18 (50%) occurred without trauma (p=0.135). Seventeen of these patients had glenoid grafting, of which 9/17 (53%) had graft nonunion. Contemporary RTSA glenoid baseplate designs have an acceptably low incidence of failure. However, the addition of structural bone graft to correct glenoid wear leads to higher aseptic baseplate failure rate. The majority of these patients suffer failure after the 2-year postoperative mark, highlighting the necessity of longer follow-up. Further analysis is necessary to quantify glenoid characteristics (severity of glenoid erosion, critical size of graft) associated with failure.

The Effect of A2 Milk on Gastrointestinal Symptoms in Comparison to A1/A2 Milk: A Single-center, Randomized, Double-blind, Cross-over Study.

β-Casein, a major protein in cow's milk, is divided into the A1 and A2 type variants. Digestion of A1 β-casein yields the peptide β-casomorphin-7 which could cause gastrointestinal (GI) discomfort but A2 milk containing only A2 β-casein might be more beneficial than A1/A2 (regular) milk. The aim of this study was to evaluate the differences in GI discomfort after ingestion of A2 milk and A1/A2 milk. A randomized, double-blind, cross-over human trial was performed with 40 subjects who experienced GI discomfort following milk consumption. For each intervention period, either A2 milk first (A2→A1/A2) or A1/A2 milk was first consumed for 2 weeks (A1/A2→A2) following a 2-week washout period. GI symptom rating scale (GSRS) scores, questionnaire for digestive symptoms, and laboratory tests including fecal calprotectin were evaluated. For symptom analysis, generalized estimating equations gamma model was used. A2 milk increased bloating (P = 0.041) and loose stools (P = 0.026) compared to A1/A2 milk in GSRS. However, A2 milk caused less abdominal pain (P = 0.050), fecal urgency (P < 0.001) and borborygmus (P = 0.007) compared to A1/A2 milk in questionnaire for digestive symptoms. In addition, fecal calprotectin also decreased or less increased after consumption of A2 milk compared to A1/A2 milk (P = 0.030), and this change was more pronounced in males (P = 0.005) than in females. There were no significant adverse reactions during the trial. A2 milk alleviated digestive discomfort in Koreans following A2 milk consumption (ClinicalTrials.gov NCT06252636 and CRIS KCT0009301).

Occurrence of beta-casein allele A1 in Bovidae species

In the last two decades bovine beta-casein variant A1 has attracted attention in the dairy industry as a source of beta-casomorphin-7 opioid-like peptide with potentially harmful effects on human health. Because the origin of this variant is unknown, the aim of the study was to investigate its occurrence in different species of the Bovidae family. Blood samples were taken from 137 animals belonging to 17 Bovidae species. All animals were genotyped in beta-casein locus by Illumina Bovine MDv2 Chip. The results shown that allele A1 was present in Bos taurus cattle, including primitive cattle (Watusi and Ukraine) and in a modern dairy breed (Holstein). In Dahomey cattle, two heterozygous A1A2 individuals were found. In all remaining species, no animal carrying allele A1 of beta-casein was identified. It may be concluded that allele A2 is ancestral to allele A1 and was spread especially within modern dairy cattle

Effects of A1 Milk, A2 Milk and the Opioid-like Peptide β-Casomorphin-7 on the Proliferation of Human Peripheral Blood Mononuclear Cells.

Special attention is given to cow's milk and its variants, with ongoing discussions about health-related impacts primarily focusing on the A1 variant in contrast to the A2 variant. The difference between these variants lies in a single amino acid alteration at position 67 of β-casein. This alteration is presumed to make the A1 variant more susceptible to enzymatic breakdown during milk digestion, leading to an increased release of the peptide β-casomorphin-7 (BCM-7). BCM-7 is hypothesized to interact with µ-opioid receptors on immune cells in humans. Although BCM-7 has demonstrated both immunosuppressive and inflammatory effects, its direct impact on the immune system remains unclear. Thus, we examined the influence of A1 and A2 milk on Concanavalin A (ConA)-stimulated human peripheral blood mononuclear cells (PBMCs), as well as the effect of experimentally digested A1 and A2 milk, containing different amounts of free BCM-7 from β-casein cleavage. Additionally, we evaluated the effects of pure BCM-7 on the proliferation of ConA-stimulated PBMCs and purified CD4+ T cells. Milk fundamentally inhibited PBMC proliferation, independent of the β-casein variant. In contrast, experimentally digested milk of both variants and pure BCM-7 showed no influence on the proliferation of PBMCs or isolated CD4+ T cells. Our results indicate that milk exerts an anti-inflammatory effect on PBMCs, regardless of the A1 or A2 β-casein variant, which is nullified after in vitro digestion. Consequently, we deem BCM-7 unsuitable as a biomarker for food-induced inflammation.

BCM-7: Opioid-like Peptide with Potential Role in Disease Mechanisms.

Bovine milk is an essential supplement due to its rich energy- and nutrient-rich qualities. Caseins constitute the vast majority of the proteins in milk. Among these, β-casein comprises around 37% of all caseins, and it is an important type of casein with several different variants. The A1 and A2 variants of β-casein are the most researched genotypes due to the changes in their composition. It is accepted that the A2 variant is ancestral, while a point mutation in the 67th amino acid created the A1 variant. The digestion derived of both A1 and A2 milk is BCM-7. Digestion of A2 milk in the human intestine also forms BCM-9 peptide molecule. The opioid-like characteristics of BCM-7 are highlighted for their potential triggering effect on several diseases. Most research has been focused on gastrointestinal-related diseases; however other metabolic and nervous system-based diseases are also potentially triggered. By manipulating the mechanisms of these diseases, BCM-7 can induce certain situations, such as conformational changes, reduction in protein activity, and the creation of undesired activity in the biological system. Furthermore, the genotype of casein can also play a role in bone health, such as altering fracture rates, and calcium contents can change the characteristics of dietary products. The context between opioid molecules and BCM-7 points to a potential triggering mechanism for the central nervous system and other metabolic diseases discussed.

The A1/A2 β-casein genotype of cows, but not their horn status, influences peptide generation during simulated digestion of milk

The effect of the horn status of cows on their milk composition and quality is a controversial research topic. In this study, 128 milk samples from 64 horned and 64 disbudded Brown Swiss and Original Braunvieh cows were collected from alpine farms where both horned and disbudded cows were grazing on mountain pastures. The samples were analyzed for their detailed composition and protein digestion in a simulated in vitro digestion (INFOGEST). To exclude probable influences on digestion, the β-CN genotype with its variants A1 and A2 was also included in the study. The effects of horn status and β-CN genotype were investigated in linear mixed models, which included additional influencing random factors such as Original Braunvieh blood proportion, stage of lactation, and farm. Horn status did not have any effect on milk composition or digestion. In contrast, milk from A1A1 cows showed a different protein digestion than milk of A1A2 and A2A2 cows in the gastric phase, including smaller amounts of β-casomorphin(BCM)21-associated peptides and larger amounts of BCM11-associated peptides. Abundances of BCM7 did not differ between β-CN genotypes. At the end of the intestinal phase, the digested milk of A1A1 and A2A2 β-CN genotypes did not differ.

Analysis of β-casein gene for A1 and A2 allelic variants in Holstein-Local crossbred cattle of Bangladesh using allele-specific PCR

Beta-casein (CSN2) is one of the important bovine milk proteins synthesized by β-casein gene. The aim of this study was to investigate the A1 and A2 allelic variants of CSN2 gene in Holstein-Friesian and Local (HF×L) crossbred cattle of Bangladesh. Allele specific polymerase chain reaction (AS-PCR) was carried out for genotyping of allelic variants. A total of 390 blood samples of HF×L crossbred cattle was included from govt., military and private dairy farms. Six different crossbred genotypes were identified in the studied population as 50%HF×50%L, 62.5%HF×37.5%L, 68.75%HF×31.25%L, 75%HF×25%L, 81.25%HF×18.75%L and 87.5%HF×12.5%L. The mean frequencies of the A1A1, A1A2, and A2A2 genotypes were 0.11, 0.48, and 0.41, respectively, while the corresponding frequencies of the A1 and A2 alleles were 0.34 and 0.66, respectively. The frequency of desirable A2A2 genotype reduced from 0.60 (50%HF×50%L) to 0.38 (87.5%HF×12.5%L) with the advancement of exotic HF inheritance from 50 to 87.5%. In contrast, the frequency of A1A1 and A1A2 genotypes increased proportionately with the progression of HF inheritance level. The undesirable A1A1 genotype frequency was found highest in the HF crossbreds of private dairy farms (0.13) compared to government (0.08) and military dairy farm (0.07) that signifies the availability of upper graded cows in the private dairy farms. Altogether, this study provides current scenario of A1 and A2 allelic variants of HF×L crossbreds of Bangladesh that could be utilized to design suitable breeding plans in crossbreeding programs for production of premium quality milk.

A discussion on A1-free milk: Nuances and comments beyond implications to the health.

This chapter provides an overarching view of the multifaceted aspects of milk β-casein, focusing on its genetic variants A1 and A2. The work examines the current landscape of A1-free milk versus regular milk, delving into health considerations, protein detection methods, technological impacts on dairy production, non-bovine protein, and potential avenues for future research. Firstly, it discussed ongoing debates surrounding categorizing milk based on A1 and A2 β-casein variants, highlighting challenges in establishing clear regulatory standards and quality control methods. The chapter also addressed the molecular distinction between A1 and A2 variants at position 67 of the amino acid chain. This trait affects protein conformation, casein micelle properties, and enzymatic susceptibility. Variations in β-casein across animal species are acknowledged, casting doubt on non-bovine claims of "A2-like" milk due to terminology and genetic differences. Lastly, this work explores the burgeoning field of biotechnology in milk production.

GENOTYPING OF CATTLE BY ALLELIC VARIANTS A1 AND A2 OF THE BETA-CASEIN GENE: EMPLOYING DIFFERENT METHODOLOGICAL APPROACHES (AS-PCR AND ACRS-PCR)

This article addresses the comparative analysis of the efficiency of cattle genotyping based on allelic variants A1 and A2 of the beta-casein gene, employing different methodological approaches. The primary methods employed include AS-PCR (AS-PCR 244 bp and AS-PCR 854 bp) and ACRS-PCR (ACRS-PCR DdeI and ACRS-PCR TaqI). Bioinformatics and laboratory diagnostics methods were used for a comparative analysis of genotyping efficiency. The study results unveiled the advantages and disadvantages of each methodological approach employed, it identified the specificity and accuracy of flanking the experimental fragment of the bovine beta-casein gene and underscored the necessity to optimize typing algorithms based on prevailing conditions when utilizing model objects. Based on the results of the research, an effective general typing algorithm was developed using the AS-PCR and ACRS-PCR methods. The allele-specific PCR method is proposed as the primary approach for routine genotyping of cattle, with ACRS-PCR suggested as a tool to verify results in cases of ambiguous findings and for blind typing of samples, among other applications.

Impact of A2 milk on human health and the dairy industry - a review

In recent years, a new type of cow's milk, called "A2 milk," has been introduced to the market. This type of milk was first marketed in New Zealand and has since gained a presence in the markets of several countries. It mainly contains two types of β-casein: the A1 and A2 variants. In recent years, researchers have studied the possible implications of the composition of the β casein protein fraction for the manifestation of a new intolerance: milk protein intolerance. Casein is the main component of milk proteins, of which approximately 30-35% is beta-casein. A2 beta-casein has proline at position 67 of the protein amino acid chain, while A1 beta-casein has histidine at that position; this is associated with a possibility of gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) released during gastrointestinal digestion. The purpose of this review is to provide an update on the impact of A2 milk on human health as well as on its many technological qualities for the production of dairy products with improved health benefits for consumers.

Abstract PO-026: Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer

Abstract Human papillomavirus (HPV) integration has been considered as one of the driver factors for cancer progression, but its pattern and role in carcinogenesis is still unclear. Whole genome sequencing data of 14 Japanese HPV16+ OPSCC and 13 HPV+ OPSCC from Pan-Cancer Analysis of Whole Genomes were used to identify integrations and somatic mutations. The phylogenetic analysis of HPV16 revealed that in tumors of 14 Japanese subjects, the A4 variant was the most common (8/14, 57.1%), followed by the A5 variant (2/14, 14.3%), which is considered unique to HPV16 in East and Southeast Asian, and the A2/A3/D2/D3 variants (1/14, 7.1%), showing a significant difference from the distribution of the HPV16 sublineages in Europe and the United States, where the A1 variant accounts for half. A total of 250 integration breakpoints (BPs) in 20 of the 27 tumors (74.1%) were detected. We estimated the cancer cell fraction of integration BPs and found that HPV integration also shows intra-tumor heterogeneity, indicating that nearly half of the integration events occurred after carcinogenesis. Clonal BPs were more likely to occur in the E1 (P = 0.036), confirming that the disruption of the E1 gene may result in abnormal negative regulation of the E6/E7 oncogenes and promote OPSCC carcinogenesis. Subclonal BPs were less likely to occur in the E6 (P = 0.045), suggesting that the E6 may also play an important role in the process of cancer evolution after carcinogenesis. Four states of the HPV genome were identified: (1) episomal-only (7/27, 25.9%), (2) integrated-only (4/27, 14.8%), (3) clonally-mixed (episomal + clonally integrated) (11/27, 40.7%), and (4) subclonally-mixed (episomal + subclonally integrated) (5/27, 18.5%). Since nearly half of these cancers have developed with just episomal copies of HPV, integration itself is not essential for carcinogenesis and can occur during and after carcinogenesis. Interestingly, the E6/E7 were conserved in all four cancers with integrated-only HPV, while the E2 and E1 were disrupted or deleted. This confirms that the constitutive expression of E6/E7 is essential for the carcinogenesis of HPV+ OPSCC. These results suggest that some integrations may be drivers of carcinogenesis with disruption of E1/E2, as in these four cancers, and others may arise randomly due to genomic instability, as in the cancers with the E6/E7 deleted integrants. The number of BPs of structural variations (SVs) occurred in the overlapping 100 kb regions flanking integration BPs (ITG regions) was positively correlated with the number of integration BPs (Spearman’s rank correlation ρ = 0.78; Permutation test P = 9.9 × 10−5). The genomic instability that causes clonal integrations during carcinogenesis is considered to cause clonal SVs, mainly large duplications, and subclonal integrations and SVs such as large deletions. Clonal SVs, mainly large duplications, were also observed in the regions where only subclonal integrations occurred, suggesting that large duplications occur before integrations. Citation Format: Noah Sasa, Hirotaka Eguchi, Hidenori Tanaka, Takahito Fukusumi, Motoyuki Suzuki, Yukinori Takenaka, Yukinori Okada, Hidenori Inohara. Intra-tumor heterogeneity of HPV integration and its association with focal genomic instability in oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-026.

Influence of β-casein genotype on physicochemical properties and functionality of bovine milk

Several studies have been focused on the effect of milk protein genetic variants on milk physicochemical properties and functionality in recent years. β-casein, an important protein related to milk processibility, has been reported to have 2 main genetic variants A1 and A2, for which cows may be homozygous or heterozygous. In this study, several physicochemical properties of milk with β-casein variants A1A1, A1A2, and A2A2 from 3 collection occasions were analyzed. Higher manganese content and lower pH were found to be associated with the A1A1 variant compared with the other 2 genotypes. Better rennet and acid coagulation were found in A1A1 milk compared with A1A2 and A2A2 milk (although P > 0.05), whereas A2A2 milk was more stable to creaming compared with the other 2 genotypes, which may be linked to its smaller fat globule size. Thus, milk from cows with A1A1 genotype could be preferable for cheese making, while that with A2A2 variant can be used in formulations requiring good stability against creaming, and for example, yogurt making, where the softer yogurt texture may be easier to digest.

Identification of β‐casein phenotypes (A1/A2) in the milk of the Indian Jersey crossbreed bovine using the high‐resolution accurate mass spectrometer

SummaryFoodomics is an emerging probing method of phenotype investigation of the different milk proteins and their subtypes. The polymorphic nature of the β‐casein (β‐Cn) protein has shown fourteen different protein variants to date in bovines. The analysis of the β‐Cn genetic polymorphism from the milk of the crossbred dairy animals is crucial for the quality assurance of the consumers from the various health concerns, especially those linked with the A1 phenotype which yields β‐casomorphin‐7 on in vivo digestion. Jersey‐crossed Indian cattle have been widely utilised in dairy because of their better milk production and survival performance trait. In this investigation, an SDS‐PAGE coupled with a high‐resolution accurate mass spectrometry‐based proteomics approach has been applied to identify the presence of specific phenotype of the β‐Cn protein in the milk of the 24 Indian crossbred (Jersey crossed) animals. Amino acid sequential analysis has been done using different search modules, as MS Amanda and Sequest HT showed 17 cows are producing A2 β‐Cn (Pro~67) while only seven animals yielded the A1 variant (His~67). The maximum number of Indian Jersey‐crossed animals are lactating milk having A2 β‐Cn. The A2 milk from the crossbred animals is free from the negative impact on health caused by β‐casomorphin‐7 (BCM‐7) released during digestion of the A1 phenotype. Among the molecular biology techniques, top‐down proteomics has been an intriguing technique for the identification of protein genetic polymorphic products.

Generation and characterization of avian IgY antibodies for detecting beta-casein A1 in bovine milk

Beta-casein is a primary milk protein that constitutes approximately 30% of the casein in bovine milk, with the two most common types in cattle being A1 and A2. The A2 protein differs from the A1 version due to a mutation in the codon at position 67, resulting in a histidine to proline substitution. However, the bioactive peptide, beta-casomorphine-7 (BCM7), which originates from partial proteolysis of the A1 variant, has been linked to several gastrointestinal disorders in humans. Production of A1 beta casein-free products is increasing demand in the milk market, worldwide. This study generated and characterized a polyclonal IgY antibody that specifically recognizes the A1 beta-casein protein present in cow's milk. A commercially available IgY anti-A1 antibody was used as a positive control, and the sensitivity and specificity of both the commercial and produced anti-A1 antibodies were evaluated. The results showed 100% sensitivity and specificity of 100% of the commercial IgY anti-A1. The in-house produced anti-A1 antibody demonstrated a sensitivity of 95.2% and a specificity of 100%, indicating its potential as a reliable and cost effective tool for detecting A1 beta-casein protein in milk samples.

SIGNIFICANCE OF GENETIC CASEIN POLYMORPHISM IN ANIMAL HUSBANDRY

: Milk proteins are a rich source of peptides which are bioactive. Beta-casomorphin-7 ( BCM-7) is produced by successive proteolytic ingestion of beta- casein variants A1 and B, while not observed in A2 variant. The large amount of proteins in bovine’s milk are caseins, which consist of four categories ( αs1, αs2, β, and κ) encrypted by corresponding genes ( CSN1S1, CSN1S2, CSN2, and CSN3, respectively). Ingestion of A1 beta-casein showed the way for the emergence of beta-casomorphin-7 (BCM-7), a peptide that has been proposed to be a major cause of various human hazards.The relative profuseness of proline, methionine, and α-lactose were the highest in A2 variant of β-casein, while glycine, citric acid, choline, and cAMP (cyclic Adenosine Monophosphate) exhibited the highest abundance in A1 variant. These results thus come up with novel insights into the effect of casein variants, further expediting research into the biogenesis of milk ingredients and the prospective physiological activity of milk correlated with variants of β-casein.

GENOTYPING OF THE BETA-CASEIN A1 AND A2 VARIANTS IN CHILEAN DAIRY CATTLE

&lt;b&gt;Bovine milk is a very important food for human health and nutrition. However, its consumption can also be associated with dysfunctions or pathologies in some consumers. The objective of this work was to evaluate the distribution of the -casein A1 and A2 variants in the main dairy cattle breeds used in Chile. Blood samples were obtained from 134 cattle from southern Chile, belonging to different breeds: Holstein-Friesian (HF), Jersey (JE), Montbliarde (MB), Overo Colorado (OC), and crossbreds (HYB). Genomic DNA was purified from the blood, and genotyping of the A1 and A2 variants was performed using allele-specific PCR. Genotypic and allelic frequencies were estimated by direct count, and the Hardy-Weinberg equilibrium was tested for using the chi-squared test, for a significance level of P &lt; 0.05. Heterozygosity was evaluated by means of Wright's fixation index (FIS) using GenePop software. Genotyping of the variants showed variability in their distribution, with prevalence of A2 in almost all breeds (including HF), with the exception of OC. The 2 value indicated that the animal populations were in Hardy-Weinberg equilibrium with the exception of MB and JE (p &lt; 0.05), with the latter showing a significant and positive FIS value suggesting inbreeding. In conclusion, our results, although preliminary, suggest that there is an opportunity to produce A2 milk in southern Chile and thus reduce the presentation of gastrointestinal dysfunctions in consumers who are affected by conventional milk.

Prospective screening for δ-hemoglobinopathies associated with decreased hemoglobin A2 levels or hemoglobin A2 variants: A single center experience

Backgroundδ-hemoglobinopathies may lead to misdiagnosis of several thalassemia syndromes especially β-thalassaemia carrier, it is important to evaluate the δ-globin gene defects in areas with high prevalence of globin gene disorders. We describe a prospective screening for δ-hemoglobinopathies in a routine setting in Thailand. MethodsStudy was done on a cohort of 8,471 subjects referred for thalassemia screening, 317 (3.7%) were suspected of having δ-globin gene defects due to reduced hemoglobin (Hb) A2 levels and/or appearance of Hb A2-variants on hemoglobin analysis. Hematologic and DNA analysis by PCR and related assays were carried out. ResultsDNA analysis of δ-globin gene identified seven different δ-globin mutations in 24 of 317 subjects (7.6%). Both known mutations; δ−77(T>C) (n = 3), δ−68(C>T) (n = 1), δ−44(G>A) (n = 8), Hb A2-Melbourne (n = 5), δIVSII−897(A>C) (n = 5), and Hb A2-Troodos (n = 1) and a novel mutation; the Hb A2-Roi-Et (n = 1) were identified. This Hb A2-Roi-Et, results from a double mutations in-cis, δCD82(AAG>AAT) and δCD133(GTG>ATG), was interestingly found in combination with an in trans, 12.6 kb deletional δβ0-thalassemia in an adult Thai woman who had no Hb A2 and elevated Hb F. A multiplex-allele-specific PCR was developed to detect these novel δ-globin gene defects. ConclusionsThe result confirms a diverse heterogeneity of δ-hemoglobinopathies in Thailand which should prove useful in a prevention and control program of thalassemia in the region.