Validation of Raised Cord Blood Interleukin-16 in Perinatal Asphyxia and Neonatal Hypoxic-Ischaemic Encephalopathy in the BiHiVE2 Cohort.

The role of inflammation is an important factor in the progression of hypoxic-ischaemic encephalopathy (HIE). We have previously shown that interleukin-16 (IL-16) is increased in infants with moderate and severe HIE and relates to poor neurodevelopmental outcomes. We aimed to validate IL-16 as a cord blood-based biomarker for HIE and to examine its relationship to long-term outcomes. The study sample consisted of 105 full-term infants who experienced perinatal asphyxia (PA) (with and without an encephalopathy) along with healthy, gestational age-matched newborn controls. Umbilical cord blood serum was processed and biobanked at delivery. Infants were assigned a modified Sarnat score at 24 h. Analysis of IL-16 cytokine cord blood levels was performed using the sandwich-based enzyme-linked immunosorbent assay (ELISA) technique. Cord blood-based IL-16 was increased in infants with PA and HIE relative to controls (p = 0.025). IL-16 was also increased in the HIE group relative to controls (p = 0.042). There was no significant difference in IL-16 across grades of HIE or in those with abnormal outcomes at 2 years of age. This study validates findings that cord blood-based IL-16 levels are increased in infants with PA, including those who go on to develop HIE.

DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits.

BACKGROUND: Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. RESULTS: SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following adjustment for multiple-testing, significant association (q ≤ 0.05) remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield) only. Notably, the single SNP in the bovine NESP55 gene (rs41694656) was associated (P ≤ 0.01) with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2) values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues. CONCLUSIONS: Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues from 8-10 week old bovine foetuses.

Single nucleotide polymorphisms within the bovine DLK1-DIO3 imprinted domain are associated with economically important production traits in cattle.

Previous studies show that DNA sequence variation within the mammalian DLK1-DIO3 imprinted domain influences production traits in domestic livestock, most notably the ovine callipyge phenotype. We assessed genotype-phenotype associations between 7 single nucleotide polymorphisms (SNPs) within the orthologous bovine DLK1-DIO3 domain and performance traits in 848 progeny-tested Holstein-Friesian dairy sires. One SNP (MEG3_01) located proximal to the maternally expressed 3 (MEG3/Gtl2) gene was associated with milk yield, subcutaneous fat levels, and progeny carcass conformation (P ≤ 0.01) and also tended to be associated with milk fat and protein yield (P ≤ 0.10). A single SNP (CLPG_01) within the putative CLPG1 locus was associated with progeny carcass fat (P ≤ 0.05), whereas a single SNP (PEG11_01) located proximal to the paternally expressed 11 (PEG11/Rtl) gene was associated with progeny carcass weight (P ≤ 0.05). The MEG3_01 SNP together with an additional 2 SNPs (MEG8_01 and MEG8_02) located proximal to the putative maternally expressed 8 (MEG8/Rian) ortholog were associated (P ≤ 0.05) with perinatal mortality. Finally, one SNP (MEG3_03) was associated (P ≤ 0.05) with gestation length, whereas both the CLPG_01 and MEG8_01 SNPs also tended to be associated with calving interval (P ≤ 0.10). Linkage disequilibrium analysis suggests that some phenotypic associations observed at these loci are independent. To our knowledge, this is one of the first studies demonstrating associations between the bovine DLK1-DIO3 domain and milk, carcass, fertility and, health traits in cattle. This imprinted domain may serve as a potential target for future genetic selection strategies.

Single nucleotide polymorphisms at the imprinted bovine insulin-like growth factor 2 (IGF2) locus are associated with dairy performance in Irish Holstein-Friesian cattle.

The imprinted insulin-like growth factor 2 gene (IGF2) encodes a fetal mitogenic hormone protein (IGF-II) and has previously been shown to be associated with performance in dairy cattle. In this study we assessed genotype-phenotype associations between four single nucleotide polymorphisms (SNPs) located within the bovine IGF2 locus on chromosome 29 and a range of performance traits related to milk production, animal growth and body size, fertility and progeny survival in 848 progeny-tested Irish Holstein-Friesian sires. Two of the four SNPs (rs42196909 and IGF2.g-3815A>G), which were in strong linkage disequilibrium (r2 = 0·995), were associated with milk yield (P ≤ 0·01) and milk protein yield (P ≤ 0·05); the rs42196901 SNP was also associated (P ≤ 0·05) with milk fat yield. Associations (P ≤ 0·05) with milk fat percentage and milk protein percentage were observed at the rs42196901 and IGF2.g-3815A>G SNPs, respectively. The rs42196909 and IGF2.g-3815A>G SNPs were also associated with progeny carcass conformation (P ≤ 0·05), while an association (P ≤ 0·01) with progeny carcass weight was observed at the rs42194733 SNP locus. None of the four SNPs were associated with body size, fertility and progeny survival. These findings support previous work which suggests that the IGF2 locus is an important biological regulator of milk production in dairy cattle and add to an accumulating body of research showing that imprinted genes influence many complex performance traits in cattle.

DNA sequence polymorphisms in a panel of eight candidate bovine imprinted genes and their association with performance traits in Irish Holstein-Friesian cattle.

BACKGROUND: Studies in mice and humans have shown that imprinted genes, whereby expression from one of the two parentally inherited alleles is attenuated or completely silenced, have a major effect on mammalian growth, metabolism and physiology. More recently, investigations in livestock species indicate that genes subject to this type of epigenetic regulation contribute to, or are associated with, several performance traits, most notably muscle mass and fat deposition. In the present study, a candidate gene approach was adopted to assess 17 validated single nucleotide polymorphisms (SNPs) and their association with a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. These SNPs are located proximal to, or within, the bovine orthologs of eight genes (CALCR, GRB10, PEG3, PHLDA2, RASGRF1, TSPAN32, ZIM2 and ZNF215) that have been shown to be imprinted in cattle or in at least one other mammalian species (i.e. human/mouse/pig/sheep). RESULTS: Heterozygosities for all SNPs analysed ranged from 0.09 to 0.46 and significant deviations from Hardy-Weinberg proportions (P ≤ 0.01) were observed at four loci. Phenotypic associations (P ≤ 0.05) were observed between nine SNPs proximal to, or within, six of the eight analysed genes and a number of performance traits evaluated, including milk protein percentage, somatic cell count, culled cow and progeny carcass weight, angularity, body conditioning score, progeny carcass conformation, body depth, rump angle, rump width, animal stature, calving difficulty, gestation length and calf perinatal mortality. Notably, SNPs within the imprinted paternally expressed gene 3 (PEG3) gene cluster were associated (P ≤ 0.05) with calving, calf performance and fertility traits, while a single SNP in the zinc finger protein 215 gene (ZNF215) was associated with milk protein percentage (P ≤ 0.05), progeny carcass weight (P ≤ 0.05), culled cow carcass weight (P ≤ 0.01), angularity (P ≤ 0.01), body depth (P ≤ 0.01), rump width (P ≤ 0.01) and animal stature (P ≤ 0.01). CONCLUSIONS: Of the eight candidate bovine imprinted genes assessed, DNA sequence polymorphisms in six of these genes (CALCR, GRB10, PEG3, RASGRF1, ZIM2 and ZNF215) displayed associations with several of the phenotypes included for analyses. The genotype-phenotype associations detected here are further supported by the biological function of these six genes, each of which plays important roles in mammalian growth, development and physiology. The associations between SNPs within the imprinted PEG3 gene cluster and traits related to calving, calf performance and gestation length suggest that this domain on chromosome 18 may play a role regulating pre-natal growth and development and fertility. SNPs within the bovine ZNF215 gene were associated with bovine growth and body conformation traits and studies in humans have revealed that the human ZNF215 ortholog belongs to the imprinted gene cluster associated with Beckwith-Wiedemann syndrome--a genetic disorder characterised by growth abnormalities. Similarly, the data presented here suggest that the ZNF215 gene may have an important role in regulating bovine growth. Collectively, our results support previous work showing that (candidate) imprinted genes/loci contribute to heritable variation in bovine performance traits and suggest that DNA sequence polymorphisms within these genes/loci represents an important reservoir of genomic markers for future genetic improvement of dairy and beef cattle populations.