Extended one-generation reproductive toxicity study of food-grade titanium dioxide E171 with emphasis on reproductive and endocrine endpoints.

Food-grade titanium dioxide E171 was administered in feed to Sprague Dawley rats in an extended one-generation reproductive toxicity (EOGRT) study (OECD Test 443). The dosed diet (0, 100, 300, or 1000mg/kg body weight/day) started 10 weeks before mating and continued throughout the study. After weaning, pups were allocated to Cohorts 1A/1B (to assess reproductive toxicity), 2A/2B (to assess developmental neurotoxicity), and 3 (to assess developmental immunotoxicity); in addition, Cohort 1B was mated to produce an F2 generation and satellite F0 animals were evaluated for colonic aberrant crypt foci (ACF). In F0 animals, there were no systemic toxicity or reproductive effects, no treatment-related histopathological changes, and no ACF in the colon. Serum estradiol or testosterone concentrations were not changed in F0 or F1 animals. No pre-/postnatal developmental changes related to treatment were noted in F1 animals, and the reproductive performance of F1 Cohort 1B animals was unaffected. F2 pups showed no abnormalities in pre- or postnatal development (postnatal days 4-8). No treatment-related developmental neurotoxicity was observed in Cohorts 2A/2B. Although no treatment-related immunotoxicity was observed in Cohort 3, the positive control did not induce the expected response; this segment of the study will be repeated. Analyses of blood and urine showed negligible systemic absorption of E171 from the gastrointestinal tract upon dietary ingestion. The no observed adverse effect level (NOAEL) for parental systemic toxicity, reproductive toxicity, offspring toxicity, and developmental neurotoxicity was considered 1000mg/kg body weight/day. For developmental immunotoxicity, a NOAEL was not determined owing to insufficient T-cell-dependent antibody response in the positive control. Our study provides robust data on the reproductive toxicity and preneoplastic potential of E171.

Phasing millions of samples achieves near perfect accuracy, enabling parent-of-origin classification of variants.

Haplotype phasing, the process of determining which genetic variants are physically located on the same chromosome, is crucial for various genetic analyses. In this study, we first benchmark SHAPEIT and Beagle, two state-of-the-art phasing methods, on two large datasets: > 8 million diverse, research-consented 23andMe, Inc. customers and the UK Biobank (UKB). We find that both perform exceptionally well. Beagle's median switch error rate (SER) (after excluding single SNP switches) in white British trios from UKB is 0.026% compared to 0.00% for European ancestry 23andMe research participants; 55.6% of European ancestry 23andMe research participants have zero non-single SNP switches, compared to 42.4% of white British trios. South Asian ancestry 23andMe research participants have the highest median SER amongst the 23andMe populations, but it is still remarkably low at 0.46%. We also investigate the relationship between identity-by-descent (IBD) and SER, finding that switch errors tend to occur in regions of little or no IBD segment coverage. SHAPEIT and Beagle excel at 'intra-chromosomal' phasing, but lack the ability to phase across chromosomes, motivating us to develop an inter-chromosomal phasing method, called HAPTIC ( HAP lotype TI ling and C lustering), that assigns paternal and maternal variants discretely genome-wide. Our approach uses identity-by-descent (IBD) segments to phase blocks of variants on different chromosomes. HAPTIC represents the segments a focal individual shares with their relatives as nodes in a signed graph and performs bipartite clustering on the signed graph using spectral clustering. We test HAPTIC on 1022 UKB trios, yielding a median phase error of 0.08% in regions covered by IBD segments (33.5% of sites). We also ran HAPTIC in the 23andMe database and found a median phase error rate (the rate of mismatching alleles between the inferred and true phase) of 0.92% in Europeans (93.8% of sites) and 0.09% in admixed Africans (92.7% of sites). HAPTIC's precision depends heavily on data from relatives, so will increase as datasets grow larger and more diverse. HAPTIC enables analyses that require the parent-of-origin of variants, such as association studies and ancestry inference of untyped parents.

Reconstructing parent genomes using siblings and other relatives.

Reconstructing the DNA of ancestors from their descendants has the potential to empower phenotypic analyses (including association and genetic nurture studies), improve pedigree reconstruction, and shed light on the ancestral population and phenotypes of ancestors. We developed HAPI-RECAP, a method that reconstructs the DNA of parents from full siblings and their relatives. This tool leverages HAPI2's output, a new phasing approach that applies to siblings (and optionally one or both parents) and reliably infers parent haplotypes but does not link the ungenotyped parents' DNA across chromosomes or between segments flanking ambiguities. By combining IBD between the reconstructed parents and the relatives, HAPI-RECAP resolves the source parent of these segments. Moreover, the method exploits crossovers the children inherited and sex-specific genetic maps to infer the reconstructed parents' sexes. We validated these methods on research participants from both 23andMe, Inc. and the San Antonio Mexican American Family Studies. Given data for one parent, HAPI2 reconstructs large fractions of the missing parent's DNA, between 77.6% and 99.97% among all families, and 90.3% on average in three- and four-child families. When reconstructing both parents, HAPI-RECAP inferred between 33.2% and 96.6% of the parents' genotypes, averaging 70.6% in four-child families. Reconstructed genotypes have average error rates < 10-3, or comparable to those from direct genotyping. HAPI-RECAP inferred the parent sexes 100% correctly given IBD-linked segments and can also reconstruct parents without any IBD. As datasets grow in size, more families will be implicitly collected; HAPI-RECAP holds promise to enable high quality parent genotype reconstruction.

Phase-free local ancestry inference mitigates the impact of switch errors on phase-based methods.

Local ancestry inference (LAI) is an indispensable component of a variety of analyses in medical and population genetics, from admixture mapping to characterizing demographic history. However, the accuracy of LAI depends on a number of factors such as phase quality (for phase-based LAI methods), time since admixture of the population under study, and other factors. Here we present an empirical analysis of four LAI methods using simulated individuals of mixed African and European ancestry, examining the impact of variable phase quality and a range of demographic scenarios. We found that regardless of phasing options, calls from LAI methods that operate on unphased genotypes (phase-free LAI) have 2.6-4.6% higher Pearson correlation with the ground truth than methods that operate on phased genotypes (phase-based LAI). Applying the TRACTOR phase-correction algorithm led to modest improvements in phase-based LAI, but despite this, the Pearson correlation of phase-free LAI remained 2.4-3.8% higher than phase-corrected phase-based approaches (considering the best performing methods in each category). Phase-free and phase-based LAI accuracy differences can dramatically impact downstream analyses: estimates of the time since admixture using phase-based LAI tracts are upwardly biased by ≈10 generations using our highest quality phased data but have virtually no bias using phase-free LAI calls. Our study underscores the strong dependence of phase-based LAI accuracy on phase quality and highlights the merits of LAI approaches that analyze unphased genetic data.