Notomelia and related neural tube defects in a baby born in Niger: case report and literature review.

INTRODUCTION: Notomelia associated with neural tube defects are rare diseases. CASE REPORT: A baby was born in Niger with multiple congenital embryonic malformations on the posterior midline. The most rostral malformation was an accessory limb (polymelia) at the level of the lumbar vertebrae composed of two long bones, a foot and three toes. Accessory male genitalia were present at the base of this malformed accessory limb which had no apparent motor or sensory innervation. The second malformation was a sacral vestigial appendage with an adjacent dermal sinus opening onto the posterior midline and extending internally to the dura through a defect of the vertebral arches. From the published literature and this particular case, we conclude that notomelia is a rare clinical sequela of a neural tube defect (NTD) and is correctly classified as a dysraphic appendage. CONCLUSION: The recent occurrence of three similar cases in the same ethnic group from Niger, three from consanguineous parents, suggests that genetic factors are likely to contribute significantly to the genesis of this syndrome, consistent with a recent report that mutation of the bovine NHLRC2 gene resulting in a V311A substitution at a highly conserved locus in the NHLRC2 protein is, when homozygous, causally associated with several forms of polymelia including notomelia, with heteropagus conjoined twinning and with other NTD-related embryonic malformations. Detailed genome-wide studies of children with dysraphic appendages are indicated.

Effects of timing and duration of test period and diet type on intake and feed efficiency of Charolais-sired cattle.

Objectives of this experiment were to: 1) determine appropriate test length, timing, and repeatability of DMI, ADG, and efficiency over different biological time points; 2) determine the efficacy of using decoupled performance and intake data to generate accurate feed efficiency measures; and 3) determine the relationship between forage-and grain-feed efficiency measures. Over 2 yr, Charolais crossbred heifers ( = 308) and steers ( = 320) were fed for two 70 d periods and DMI, ADG, and 12th rib fat thickness were recorded. Steers were fed grain-based diets during the growing and finishing periods to determine the effects of test period and timing on DMI and feed efficiency. Heifers were fed forage during the growing period and grain during the finishing period to test the effect of diet type on measures of DMI and feed efficiency. For each 70 d test period, individual DMI was recorded using the GrowSafe (Airdrie, AB) system. Residual feed intake (RFI) was calculated for each test period. Total feeding period ADG (FP_ADG) was calculated for steers by regressing all weights taken from feedlot arrival to final BW, which was calculated by dividing HCW by a standard dressing percentage (63%). Dry matter intake and RFI were correlated (r 0.56; < 0.01, and 0.63; < 0.01, respectively) for the growing and finishing periods of grain-fed steers. Average daily gain was not repeatable ( = 0.11; 0.06) across both test periods for steers. However, growing and finishing ADG were correlated ( = 0.58; < 0.01, and = 0.69; < 0.01, respectively) to FP_ADG. To assess the potential of shortening the intake test, DMI was analyzed in 7 d increments for grain-fed steers during the growing and finishing periods. Regardless of test length, from 7 to 70 d, DMI was strongly correlated ( ≥ 0.87; < 0.01) to total DMI during the growing period. Heifer forage DMI was correlated ( = 0.58; < 0.01) to grain DMI; subsequently, forage and grain RFI were moderately correlated ( = 0.40; < 0.01). This study suggests that DMI is repeatable across varying stages of maturity in cattle, and accurate feed efficiency measures can be obtained in either the growing or finishing period. The relationship of forage and grain DMI and efficiency in heifers suggests that measures of DMI and feed efficiency in heifers are relevant, regardless of diet fed. Intake evaluation periods can be shortened with minimal effects on the accuracy of predicting individual animal DMI.

Increased prenatal IGF2 expression due to the porcine intron3-G3072A mutation may be responsible for increased muscle mass.

A SNP (IGF2 G3072A) within intron 3 of disrupts a binding site for the repressor zinc finger BED-type containing 6 (ZBED6), leading to increased carcass lean yields in pigs. However, the relative contributions of prenatal as opposed to postnatal increased IGF2 expression are unclear. As muscle fiber number is set at birth, prenatal and neonate skeletal muscle development is critical in determining mature growth potential. Therefore, the objectives of this study were to determine the contributions of hyperplasia and hypertrophy to increased muscle mass and to delineate the effect of the mutation on the expression of myogenic genes during prenatal and postnatal growth. Sows (IGF2 A/A) were bred to a single heterozygous (IGF2 A/G) boar. For fetal samples, sows were euthanized at 60 and 90 d of gestation (d60 and d90) to obtain fetuses. Male and female offspring were also euthanized at birth (0d), weaning (21d), and market weight of approximately 130 kg (176d). At each sampling time, the LM, psoas major (PM), and semitendinosus (ST) muscles were weighed. Samples of the LM were used to quantify the expression of IGF family members, myogenic regulatory factors (MRF), myosin heavy chain isoforms, and growth factors, myostatin, and . Liver samples were used to quantify and expression. At 176d, weights of LM, PM, and ST muscles were all increased approximately 8% to 14% (P < 0.01) in pigs with paternal A (A(Pat)) alleles compared with those with paternal G (G(Pat)) alleles. Additionally, total muscle fiber number in the ST at 176d tended to be greater (P = 0.10), whereas muscle fiber cross-sectional area tended to be reduced ( P= 0.08) in A(Pat) pigs compared with G(Pat) pigs. In addition to the expected 2.7- to 4.5-fold increase (P ≤ 0.02) in expression in the LM in A(Pat) compared with G(Pat) pigs at postnatal sampling times (21d and 176d), IGF2 expression was also increased (P ≤ 0.06) 1.4- to 1.5-fold at d90 of gestation and at birth. At d90, expression of myogenic factor 5 (MYF5), a MRF expressed in proliferating myoblasts, in the LM was greater (P = 0.01) in A (Pat) pigs than in G(Pat) pigs. Interestingly, at 21d hepatic expression was greater (P = 0.01), whereas expression decreased (P = 0.01) in A(Pat) pigs compared with G(Pat) pigs; however, there were no differences (P ≥ 0.18) in hepatic expression between genotypes at 0d and 176d. These data suggest that prenatal hyperplasia of muscle fibers stimulated by increased IGF2 expression may contribute to increased muscle mass of A(Pat) pigs.

Effects of the porcine IGF2 intron 3-G3072A mutation on carcass cutability, meat quality, and bacon processing.

A SNP in a regulatory region of intron 3 within the porcine IGF2 gene (IGF2-G3072A) is associated with increased lean deposition and decreased fat deposition in pigs with paternal A alleles (APat) compared with pigs with paternal G alleles (GPat). However, data regarding fresh and processed meat quality characteristics of pigs with different alleles for this polymorphism are limited. A single heterozygote (AG) boar was bred to homozygous (AA) commercial Yorkshire-cross sows producing F1 barrows and gilts with either GPat or APat. Two farrowing groups of barrows and gilts were group housed, provided ad libitum access to a diet that met or exceeded NRC nutrient recommendations throughout production, and slaughtered at 176 d (±4 d) of age. Fresh LM quality and estimated percent fat-free lean measurements were taken on the left side of carcasses, while carcass cutouts were completed with right sides. Fresh belly and bacon processing traits were characterized for only block 1 pigs. Pig was treated as the experimental unit for all analyses. Ending live weight and HCW were not affected by IGF2 allele; however, 10th rib backfat thickness was 0.41 cm less (P=0.01), loin eye area was 4.0 cm2 greater (P=0.01), and predicted fat-free lean was over 2 percentage units greater (P<0.01) in APat pigs compared with GPat pigs. Furthermore, boneless lean cuts from the shoulder, loin, and ham were heavier (P<0.05) in APat pigs compared with GPat pigs. Minolta L* value was 2.36 units greater (P=0.03) but cooking loss was 1.82 percentage units greater (P<0.01) in APat pigs compared with GPat pigs. Additionally, despite reductions in subcutaneous fat, extractable intramuscular lipid from the LM was 0.64 percentage units greater (P=0.02) in APat pigs compared with GPat pigs. Bellies were 7.17 mm thinner (P=0.01), had 7.27 cm less flop distance (P=0.05), and tended to have 1.34 units greater iodine value (P=0.09) in APat pigs compared with GPat pigs. While not statistically different (P=0.30), the magnitude of difference in slicing yield as a percentage of green weight was 1.57 percentage units between bellies from APat pigs (85.83%) and bellies from GPat pigs (87.40%). Pigs with GPat had superior belly quality that may positively impact commercial bacon production. However, pigs with APat yielded a greater amount of lean product at the expense of producing lighter LM color and increased cooking loss.

A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

Investigating the genetic basis of pork tenderness: genomic analysis of porcine CAST.

A quantitative trait locus (QTL) affecting pork tenderness was recently detected within the Illinois Meat Quality Pedigree (IMQP) and fine-mapped to the region of porcine chromosome 2 (SSC2) harbouring the functional candidate gene calpastatin (CAST). To identify molecular variation that may underlie the observed differences in tenderness phenotypes, we characterized the porcine CAST gene and analysed allelic variation within the F(1) boars of the IMQP. The complete genomic sequence of porcine CAST has been determined, and was found to contain 35 exons spanning nearly 123 kb. Using the rapid amplification of cDNA ends (RACE) method, calpastatin transcript types I-III, as well as a putative novel transcript type, were detected within porcine skeletal muscle. Variability in transcription initiation and termination sites was observed, and alternative splicing of exons 1y and 3 was evident. Nearly 77.6% of the CAST gene, including all exons, was re-sequenced from each of six IMQP F(1) boars, and almost 900 polymorphisms were identified. The heterozygosity of nearly 400 polymorphisms appeared to be concordant with the previous QTL data, and the location of this variation within the CAST gene suggests that a causative mutation is likely to be regulatory. Functional characterization of CAST variation should enhance understanding of the molecular basis of pork tenderness, and thus allow for genetic improvement of pork products. The effectiveness of CAST polymorphisms for marker-assisted selection of pork tenderness can now be assessed.

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11.

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.

A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep.

Ovine hereditary chondrodysplasia, or spider lamb syndrome (SLS), is a genetic disorder that is characterized by severe skeletal abnormalities and has resulted in substantial economic losses for sheep producers. Here we demonstrate that a non-synonymous T>A transversion in the highly conserved tyrosine kinase II domain of a positional candidate gene, fibroblast growth factor receptor 3 (FGFR3), is responsible for SLS. We also demonstrate that the mutant FGFR3 allele has an additive effect on long-bone length, calling into question the long-standing belief that SLS is inherited as a strict monogenic, Mendelian recessive trait. Instead, we suggest that SLS manifestation is determined primarily by the presence of the mutant FGFR3 allele, but it is also influenced by an animal's genetic background. In contrast to FGFR3 mutations causing dwarfism in humans, this single-base change is the only known natural mutation of FGFR3 that results in a skeletal overgrowth phenotype in any species.

Evaluation of approaches to detect quantitative trait loci for growth, carcass, and meat quality on swine chromosomes 2, 6, 13, and 18. II. Multivariate and principal component analyses.

The merits of complementary multivariate techniques to identify QTL associated with multiple traits were evaluated. Records from 806 F2 pigs pertaining to a Berkshire x Duroc three-generation population were available. Six multitrait groups on SSC 2, 6, 13, and 18 with information on 30 markers were studied. Multivariate techniques studied included multivariate models and principal components analysis of each multitrait group. All models included, in addition to systematic effects, additive, dominance, and imprinting coefficients corresponding to a one-QTL model and a random family effect. Multivariate analysis identified QTL associated with genomewise significant variation in four of the multitrait groups. The majority of the multivariate analysis provided greater precision of parameter estimates and higher statistical significance in some cases than univariate approaches, because of the greater parameterization of the multivariate models and moderate information content of the data. Principal component analysis results were consistent with univariate and multivariate analyses and recovered the levels of statistical significance observed in univariate analyses on the original data. In addition, principal component analysis was able to provide a location associated with LM area not detected by other analyses. The relative advantage of multivariate over the univariate approaches varied with the level of genetic covariance between traits because of the modeled QTL effect and information contained in the data; however, multivariate approaches have the unique capability to identify pleiotropic effects or multiple linked QTL.

Evaluation of approaches to detect quantitative trait loci for growth, carcass, and meat quality on swine chromosomes 2, 6, 13, and 18. I. Univariate outbred F2 and sib-pair analyses.

Results from univariate outbred F2 interval mapping and sib-pair analyses of 12 growth and 28 carcass traits to identify QTL on SSC 2, 6, 13, and 18 were compared. Phenotypic and genetic data were recorded on a three-generation resource population including 832 F2 pigs from a cross between three Berkshire sires and 18 Duroc dams. Thirty markers with an average spacing of approximately 16 cM were genotyped across the four chromosomes. The outbred F2 mixed model included the effects of sex, birth month, and year, one-QTL additive, dominance and imprinting coefficients calculated every 1 cM using interval mapping, and a random family effect. The general sib-pair model used to describe the phenotypic differences between sib-pairs included the same systematic and random effects and a one-QTL additive coefficient calculated every 1 cM. The outbred F2 analysis found significant evidence of QTL on SSC 2 associated with 105-d weight, backfat thicknesses, LM area, fat percent, shear force, juiciness, marbling, and tenderness. In addition, QTL were identified on SSC 6 relating to 42-d weight and LM area, and on SSC 18 for fat and moisture percents. In most instances, the outbred F2 approach offered greater power to detect QTL; however, the sib-pair analysis offered greater power in several instances. The trait-specific superiority could be due to the relative advantage of each model within a trait data set. The two approaches provided complementary evidence for QTL segregating between the Berkshire and Duroc breeds used in the study that may be used to aid marker-assisted introgression and selection and candidate gene studies to improve swine growth and meat quality characteristics.

Identification of a mutation associated with factor XI deficiency in Holstein cattle.

An autosomal recessive deficiency of blood coagulation factor XI (FXI) has been described in Holstein cattle. Current testing methods are unsuitable for accurately identifying carriers (heterozygotes) of the disease. To identify the molecular basis of this deficiency, a polymerase chain reaction (PCR)-based strategy was implemented to clone and sequence the bovine FXI gene (F11) from animals of different genotypes. Approximately 14 kb of genomic DNA sequence and 1.8 kb of cDNA sequence, corresponding to exon 3 through the 3'-UTR, of the bovine gene were obtained. Comparison of sequences derived from homozygous normal and deficient individuals revealed that FXI deficiency in Holsteins is associated with the insertion of a 76 bp segment [AT(A)(28)TAAAG(A)(26)GGAAATAATAATTCA] within exon 12. This insertion introduces a stop codon that results in a mature FXI protein lacking the functional protease domain encoded by exons 13, 14 and 15. Based on these data, a DNA-based diagnostic test has been developed for accurate genotyping. Using this method, the frequency of the mutated allele has been determined to be 1.2% in a contemporary population of the USA Holstein sires.

Differential expression of the GTL2 gene within the callipyge region of ovine chromosome 18.

The inheritance pattern of the skeletal muscle hypertrophy phenotype caused by the callipyge gene has been characterized as polar overdominance. We hypothesized that this trait may be caused by a gain or loss of gene expression because of the reversible nature of the phenotype in paternal vs. maternal inheritance. Suppression subtraction cDNA probes were made from skeletal muscle mRNA of normal (NN) and callipyge (C(Pat)N(Mat)) animals and hybridized to Southern blots containing bacterial artificial chromosomes (BACs) that comprise a physical contig of the callipyge region. The CN-NN probes hybridized to two ovine and seven bovine BACs. Sequence analysis of fragments within those BACs indicated short regions of similarity to mouse gene trap locus (gtl2). Northern blots analysis of RNA from hypertrophy-responsive muscles show a population of GTL2 mRNA centred around 2.4 kb that were abundantly expressed in 14-day prenatal NN and C(Pat)N(Mat) lambs but were down-regulated in day 14 and day 56 postnatal NN lambs. The expression of GTL2 remained elevated in 14- and 56-day-old C(Pat)N(Mat) lambs as well as in 56-day-old N(Pat)C(Mat) and CC lambs. Expression of GTL2 in the supraspinatus, which does not undergo hypertrophy, was very low for all genotypes and ages. Isolation of cDNA sequences show extensive alternative splicing and a lack of codon bias suggesting that GTL2 does not encode a protein. The mutation of the callipyge allele has altered postnatal expression of GTL2 in muscles that undergo hypertrophy and will help identify mechanisms involved in growth, genomic imprinting and polar overdominance.

Development and mapping of microsatellites from a microdissected BTA 11-specific DNA library.

A chromosome-specific library was developed for Bos taurus autosome 11 by chromosome microdissection and microcloning using a bovine primary fibroblast culture, obtained from a t(X;23) heifer, that spontaneously developed a translocation chromosome involving bovine chromosome 11. The library was screened using (AC)12 oligos, positive clones selected, sequenced and primers developed to generate bovine chromosome 11-specific microsatellite markers. This study suggests that chromosome-specific libraries have great potential for development of microsatellite markers for the construction of marker-saturated linkage maps for each chromosome.

Fine-mapping and construction of a bovine contig spanning the ovine callipyge locus.

The callipyge (CLPG) gene was fine-mapped by linkage analysis to a 4.6-cM chromosome interval on distal ovine OAR18q, flanked by microsatellite markers IDVGA30 and OY3. The OAR18q linkage map and human HSA14q transcript map were aligned by genotyping two bovine-hamster whole-genome radiation hybrid panels with the microsatellite markers, as well as with sequences corresponding to HSA 14q genes. Using Type I loci mapping to the IDVGA30-OY3 interval as anchor points, we have constructed a 1.4-Mb bovine BAC contig containing the IDVGA30-OY3 interval. We demonstrate that the IDVGA30-OY3 interval spans approximately 770 kb and contains at least four genes: YY1, WARS, DLK1, and GTL2.

Phylogenomic analysis of the alpha proteasome gene family from early-diverging eukaryotes.

We employed a phylogenomic approach to study the evolution of alpha subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven alpha proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple alpha subunit sequences generated single alpha proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct alpha proteasome genes each. The presence of seven distinct alpha proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the alpha proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the alpha proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented.

Bovine chromosome 4 workshop: consensus and comprehensive linkage maps.

The report of the bovine chromosome 4 (BTA4) workshop is presented. Six laboratories contributed a total of 30,168 informative meioses from 62 loci. Twenty-two loci were typed by at least two independent laboratories and were used to construct a consensus linkage map of BTA4. The remaining 40 loci were subsequently incorporated into a comprehensive map. The sex-averaged consensus map covered 131.4 cM. The female map was 124.3 cM in length, while the male map was 134.3 cM. The comprehensive sex-averaged map spanned 141.6 cM. The length of the female and male comprehensive maps were 123.1 cM and 156.4 cM, respectively. Average genetic distance between loci was 6 and 2.3 cM for the consensus and comprehensive linkage maps, respectively.

Localization of the locus causing Spider Lamb Syndrome to the distal end of ovine Chromosome 6.

Spider Lamb Syndrome (SLS) is a semi-lethal congenital disorder, causing severe skeletal abnormalities in sheep. The syndrome has now been disseminated into several sheep breeds in the United States, Canada, and Australia. The mode of inheritance for SLS is autosomal recessive, making the identification and culling of carrier animals difficult due to their normal phenotype. Two large pedigrees segregating for the SLS mutation were established, and a genome scan with genetic markers from previously published genome maps of cattle and sheep was used to map the locus causing SLS. Genetic linkage between SLS and several microsatellite markers, OarJMP8, McM214, OarJMP12, and BL1038, was detected, thereby mapping the SLS locus to the telomeric end of ovine Chromosome (Chr) 6. Alignment of ovine Chr 6 with its evolutionary ortholog, human Chr 4, revealed a positional candidate gene, fibroblast growth factor receptor 3 (FGFR3).