Unexpected Relationships and Inbreeding in HapMap Phase III Populations

Correct annotation of the genetic relationships between samples is essential for population genomic studies, which could be biased by errors or omissions. To this end, we used identity-by-state (IBS) and identity-by-descent (IBD) methods to assess genetic relatedness of individuals within HapMap phase III data. We analyzed data from 1,397 individuals across 11 ethnic populations. Our results support previous studies (Pemberton et al., 2010; Kyriazopoulou-Panagiotopoulou et al., 2011) assessing unknown relatedness present within this population. Additionally, we present evidence for 1,657 novel pairwise relationships across 9 populations. Surprisingly, significant Cotterman''s coefficients of relatedness K1 (IBD1) values were detected between pairs of known parents. Furthermore, significant K2 (IBD2) values were detected in 32 previously annotated parent-child relationships. Consistent with a hypothesis of inbreeding, regions of homozygosity (ROH) were identified in the offspring of related parents, of which a subset overlapped those reported in previous studies (Gibson et al. 2010; Johnson et al. 2011). In total, we inferred 28 inbred individuals with ROH that overlapped areas of relatedness between the parents and/or IBD2 sharing at a different genomic locus between a child and a parent. Finally, 8 previously annotated parent-child relationships had unexpected K0 (IBD0) values (resulting from a chromosomal abnormality or genotype error), and 10 previously annotated second-degree relationships along with 38 other novel pairwise relationships had unexpected IBD2 (indicating two separate paths of recent ancestry). These newly described types of relatedness may impact the outcome of previous studies and should inform the design of future studies relying on the HapMap Phase III resource.     

Short-lived corpora lutea syndrome in anoestrous ewes following 17β-oestradiol or MAP treatments applied before an allogenic sexual stimulation with rams and oestrous ewes

When induced to ovulate during anoestrus, ewes, does and cows frequently develop a short-lived corpora lutea (SLCL) syndrome associated to lack of previous progesterone. Exogenous progesterone precludes SLCL by blocking oxytocin endometrial receptors, thus inducing normal life-span CL (NLCL). Paradoxically, circa 50% of unprimed ewes do not develop SLCL. We report results from 3 trials assessing follicular, oestrous, ovulatory, and luteal end-points after 17β-oestradiol or MAP treatments. Oestradiol benzoate (50 μg) induced follicular turnover, provoked ovulation in 40% (24/60) of ewes treated (93% of which developed SLCL), but did not affect the incidence of SLCL (26/53) after an allogenic sexual stimulation (ASS) by rams and oestrous ewes. By the onset of the ASS, most NLCL ewes (26/27) had already experienced turnover of their largest follicle, had smaller largest and second largest follicles, and ovulated their largest follicle more frequently than SLCL ewes did. Most SLCL ewes (19/25) did not ovulate their largest follicles, ovulating instead smaller follicles of identical size to those of NLCL ewes. Priming (40 mg of MAP for 12 days) was partially effective at preventing SLCL even when terminated 14 days in advance of an ASS, but failed at completely preventing SLCL when terminated 6 or more days in advance. The coupling of a timed acquisition of full steroidogenic capability before ovulation with a system of endometrial oestradiol–progesterone–oxytocin receptors linked in an unstable equilibrium controlling the amplification of the luteolytic feed-forward loop of oxytocin and prostaglandin F₂α explains occurrence and relative incidences of both NLCL and SLCL, and links proximate and ultimate causes of the SLCL syndrome.     

Detection of endogenous biotin in various tissues: novel functions in the hippocampus and implications for its use in avidin-biotin technology

Significant amounts of endogenous biotin were detected by avidin-peroxidase in fixed rat kidney, liver, and brain. The staining was indistinguishable from the true signals of immunoreactivity and could not be consistently blocked by pretreatment with avidin. The finding that certain neurons in the hippocampus contain more biotin than neurons in other areas of the brain suggests that biotin might have novel functions in the brain other than its well-known role as cofactor of carboxylases. Critical examination of published immunohistochemical localization studies on rat kidney strongly suggests that many false-positive results have been considered as true signals. Interference of endogenous biotin in any study using avidin-biotin technology must be considered if biological tissues are involved. The published data obtained by this method should therefore be reevaluated. Furthermore, appropriate controls, blockers and caution in interpreting results must be exercised, not only in immunohistochemistry but also in any applications of avidin-biotin technology.     

SNPchip: R classes and methods for SNP array data

High-density single nucleotide polymorphism microarrays (SNP chips) provide information on a subject's genome, such as copy number and genotype (heterozygosity/homozygosity) at a SNP. While fluorescence in situ hybridization and karyotyping reveal many abnormalities, SNP chips provide a higher resolution map of the human genome that can be used to detect, e.g., aneuploidies, microdeletions, microduplications and loss of heterozygosity (LOH). As a variety of diseases are linked to such chromosomal abnormalities, SNP chips promise new insights for these diseases by aiding in the discovery of such regions, and may suggest targets for intervention. The R package SNPchip contains classes and methods useful for storing, visualizing and analyzing high density SNP data. Originally developed from the SNPscan web-tool, SNPchip utilizes S4 classes and extends other open source R tools available at Bioconductor. This has numerous advantages, including the ability to build statistical models for SNP-level data that operate on instances of the class, and to communicate with other R packages that add additional functionality. AVAILABILITY: The package is available from the Bioconductor web page at www.bioconductor.org. SUPPLEMENTARY INFORMATION: The supplementary material as described in this article (case studies, installation guidelines and R code) is available from http://biostat.jhsph.edu/~iruczins/publications/sm/     

Synaptotagmin I is a molecular target for lead

Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with specific molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.     

5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes.

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.