An alternative to the adenovirus inverted terminal repeat sequence increases the viral genome replication rate and provides a selective advantage in vitro.

During the development of human adenovirus 35-derived replication-incompetent (rAd35) vaccine vectors for prevention of infectious diseases, we detected mutations in the terminal 8 nt of the inverted terminal repeats (ITRs) of rAd35. The switch from the plasmid-encoded sequence 5'-CATCATCA-3' to the alternative sequence 5'-CTATCTAT-3' in the ITRs was found to be a general in vitro propagation phenomenon, as shown for several vectors carrying different transgenes or being derived from different adenovirus serotypes. In each tested case, the plasmid-encoded ITR sequence changed to exactly the same alternative ITR sequence, 5'-CTATCTAT-3'. The outgrowth of this alternative ITR version should result from a growth advantage conferred by the alternative ITR sequence. Indeed, replication kinetics studies of rAd35 harbouring either the original or alternative ITR sequence confirmed an increase in replication speed for rAd35 vectors with the alternative ITR sequence. These findings can be applied to generate recombinant adenoviral vectors harbouring the alternative ITR sequence, which will facilitate the generation of genetically homogeneous seed virus batches. Moreover, vector production may be accelerated by taking advantage of the observed improved replication kinetics associated with the alternative ITR sequence.

PHOX2B polyalanine repeat length is associated with sudden infant death syndrome and unclassified sudden infant death in the Dutch population.

Unclassified sudden infant death (USID) is the sudden and unexpected death of an infant that remains unexplained after thorough case investigation including performance of a complete autopsy and review of the circumstances of death and the clinical history. When the infant is below 1 year of age and with onset of the fatal episode apparently occurring during sleep, this is referred to as sudden infant death syndrome (SIDS). USID and SIDS remain poorly understood despite the identification of several environmental and some genetic risk factors. In this study, we investigated genetic risk factors involved in the autonomous nervous system in 195 Dutch USID/SIDS cases and 846 Dutch, age-matched healthy controls. Twenty-five DNA variants from 11 genes previously implicated in the serotonin household or in the congenital central hypoventilation syndrome, of which some have been associated with SIDS before, were tested. Of all DNA variants considered, only the length variation of the polyalanine repeat in exon 3 of the PHOX2B gene was found to be statistically significantly associated with USID/SIDS in the Dutch population after multiple test correction. Interestingly, our data suggest that contraction of the PHOX2B exon 3 polyalanine repeat that we found in six of 160 SIDS and USID cases and in six of 814 controls serves as a probable genetic risk factor for USID/SIDS at least in the Dutch population. Future studies are needed to confirm this finding and to understand the functional effect of the polyalanine repeat length variation, in particular contraction, in exon 3 of the PHOX2B gene.

Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.

Nonrecombining Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Although a key requirement for their application is reliable mutability knowledge, empirical data are only available for a small number of Y-STRs thus far. To rectify this, we analyzed a large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers. Following confirmation by DNA sequence analysis, the retrieved mutation data were modeled via a Bayesian approach, resulting in mutation rates from 3.78 × 10(-4) (95% credible interval [CI], 1.38 × 10(-5) - 2.02 × 10(-3)) to 7.44 × 10(-2) (95% CI, 6.51 × 10(-2) - 9.09 × 10(-2)) per marker per generation. With the 924 mutations at 120 Y-STR markers, a nonsignificant excess of repeat losses versus gains (1.16:1), as well as a strong and significant excess of single-repeat versus multirepeat changes (25.23:1), was observed. Although the total repeat number influenced Y-STR locus mutability most strongly, repeat complexity, the length in base pairs of the repeated motif, and the father's age also contributed to Y-STR mutability. To exemplify how to practically utilize this knowledge, we analyzed the 13 most mutable Y-STRs in an independent sample set and empirically proved their suitability for distinguishing close and distantly related males. This finding is expected to revolutionize Y-chromosomal applications in forensic biology, from previous male lineage differentiation toward future male individual identification.

A worldwide survey of human male demographic history based on Y-SNP and Y-STR data from the HGDP-CEPH populations.

We have investigated human male demographic history using 590 males from 51 populations in the Human Genome Diversity Project - Centre d'Etude du Polymorphisme Humain worldwide panel, typed with 37 Y-chromosomal Single Nucleotide Polymorphisms and 65 Y-chromosomal Short Tandem Repeats and analyzed with the program Bayesian Analysis of Trees With Internal Node Generation. The general patterns we observe show a gradient from the oldest population time to the most recent common ancestors (TMRCAs) and expansion times together with the largest effective population sizes in Africa, to the youngest times and smallest effective population sizes in the Americas. These parameters are significantly negatively correlated with distance from East Africa, and the patterns are consistent with most other studies of human variation and history. In contrast, growth rate showed a weaker correlation in the opposite direction. Y-lineage diversity and TMRCA also decrease with distance from East Africa, supporting a model of expansion with serial founder events starting from this source. A number of individual populations diverge from these general patterns, including previously documented examples such as recent expansions of the Yoruba in Africa, Basques in Europe, and Yakut in Northern Asia. However, some unexpected demographic histories were also found, including low growth rates in the Hazara and Kalash from Pakistan and recent expansion of the Mozabites in North Africa.

Three genome-wide association studies and a linkage analysis identify HERC2 as a human iris color gene.

Human iris color was one of the first traits for which Mendelian segregation was established. To date, the genetics of iris color is still not fully understood and is of interest, particularly in view of forensic applications. In three independent genome-wide association (GWA) studies of a total of 1406 persons and a genome-wide linkage study of 1292 relatives, all from the Netherlands, we found that the 15q13.1 region is the predominant region involved in human iris color. There were no other regions showing consistent genome-wide evidence for association and linkage to iris color. Single nucleotide polymorphisms (SNPs) in the HERC2 gene and, to a lesser extent, in the neighboring OCA2 gene were independently associated to iris color variation. OCA2 has been implicated in iris color previously. A replication study within two populations confirmed that the HERC2 gene is a new and significant determinant of human iris color variation, in addition to OCA2. Furthermore, HERC2 rs916977 showed a clinal allele distribution across 23 European populations, which was significantly correlated to iris color variation. We suggest that genetic variants regulating expression of the OCA2 gene exist in the HERC2 gene or, alternatively, within the 11.7 kb of sequence between OCA2 and HERC2, and that most iris color variation in Europeans is explained by those two genes. Testing markers in the HERC2-OCA2 region may be useful in forensic applications to predict eye color phenotypes of unknown persons of European genetic origin.

Inferring continental ancestry of argentineans from Autosomal, Y-chromosomal and mitochondrial DNA.

We investigated the bio-geographic ancestry of Argentineans, and quantified their genetic admixture, analyzing 246 unrelated male individuals from eight provinces of three Argentinean regions using ancestry-sensitive DNA markers (ASDM) from autosomal, Y and mitochondrial chromosomes. Our results demonstrate that European, Native American and African ancestry components were detectable in the contemporary Argentineans, the amounts depending on the genetic system applied, exhibiting large inter-individual heterogeneity. Argentineans carried a large fraction of European genetic heritage in their Y-chromosomal (94.1%) and autosomal (78.5%) DNA, but their mitochondrial gene pool is mostly of Native American ancestry (53.7%); instead, African heritage was small in all three genetic systems (<4%). Population substructure in Argentina considering the eight sampled provinces was very small based on autosomal (0.92% of total variation was between provincial groups, p = 0.005) and mtDNA (1.77%, p = 0.005) data (none with NRY data), and all three genetic systems revealed no substructure when clustering the provinces into the three geographic regions to which they belong. The complex genetic ancestry picture detected in Argentineans underscores the need to apply ASDM from all three genetic systems to infer geographic origins and genetic admixture. This applies to all worldwide areas where people with different continental ancestry live geographically close together.

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR Yfiler PCR amplification kit.

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR Yfiler polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730-1,764 DNA-confirmed father-son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son's birth) of fathers with mutations was with 34.40 (+/-11.63) years higher than that of fathers without ones at 30.32 (+/-10.22) years, a difference that is highly statistically significant (p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father's age on a statistically significant level (alpha = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father-son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.

Collaboration between key populations in a global partnership for health and human rights: Lessons learned from 'Bridging the Gaps'.

Because HIV and AIDS key populations share common social-cultural factors challenging their sexual health and rights (e.g. stigma, criminalisation), there is an assumed benefit of collaborative programmes where various key populations work in solidarity. In this paper, we reflect on how partners collaborated in a complex alliance of over 100 different NGOs, representing and supporting three key populations - lesbian, gay, bisexual and transgender (LGBT) people, sex workers and people who use drugs - working across 16 countries. We used a multiple-method approach of participant observation, qualitative interviews, and a survey to explore the benefits, facilitators and challenges of collaboration. Results show that motivators for collaboration included being part of the larger funding structure with applied impacts, a repressive human rights context, and intersectionality. Barriers for collaboration included identity politics, stigma, and constraints regarding the appropriate timing of new collaborations. Finally, facilitators include practical support for engagement, the framing of human rights in a medical agenda, and recognition of implicit differences. We conclude that for building the capacity for collaboration among socially marginalised groups it is important to develop trust and the ability to recognise strength in difference beyond the initial identification of shared norms and common goals.

Adenoviral Type 35 and 26 Vectors with a Bidirectional Expression Cassette in the E1 Region Show an Improved Genetic Stability Profile and Potent Transgene-Specific Immune Response.

Genetic vaccines based on replication-incompetent adenoviral (AdV) vectors are currently in clinical development. Monovalent AdV vectors express one antigen from an expression cassette placed in most cases in the E1 region. For many vaccines, inclusion of several antigens is necessary in order to raise protective immunity and/or target more than one pathogen or pathogen strain. On the basis of the current technology, a mix of several monovalent vectors can be employed. However, a mix of the standard monovalent AdV vectors may not be optimal with respect to manufacturing costs and the final dose per vector in humans. Alternatively, a variety of bivalent recombinant AdV vector approaches is described in the literature. It remains unclear whether all strategies are equally suitable for clinical development while preserving all the beneficial properties of the monovalent AdV (e.g., immunogenic potency). Therefore, a thorough assessment of different bivalent AdV strategies was performed in a head-to-head fashion compared with the monovalent benchmark. The vectors were tested for rescue efficiency, genetic stability, transgene expression, and potency to induce transgene-specific immune responses. We report that the vector expressing multiple antigens from a bidirectional expression cassette in E1 shows a better genetic stability profile and a potent transgene-specific immune response compared with the other tested bivalent vectors.

A new future of forensic Y-chromosome analysis: rapidly mutating Y-STRs for differentiating male relatives and paternal lineages.

The panels of 9-17 Y-chromosomal short tandem repeats (Y-STRs) currently used in forensic genetics have adequate resolution of different paternal lineages in many human populations, but have lower abilities to separate paternal lineages in populations expressing low Y-chromosome diversity. Moreover, current Y-STR sets usually fail to differentiate between related males who belong to the same paternal lineage and, as a consequence, conclusions cannot be drawn on the individual level as is desirable for forensic interpretations. Recently, we identified a new panel of rapidly mutating (RM) Y-STRs, composed of 13 markers with mutation rates above 1 × 10(-2), whereas most Y-STRs, including all currently used in forensics, have mutation rates in the order of 1 × 10(-3) or lower. In the present study, we demonstrate in 604 unrelated males sampled from 51 worldwide populations (HGDP-CEPH) that the RM Y-STRs provide substantially higher haplotype diversity and haplotype discrimination capacity (with only 3 haplotypes shared between 8 of the 604 worldwide males), than obtained with the largest set of 17 currently used Y-STRs (Yfiler) in the same samples (33 haplotypes shared between 85 males). Hence, RM Y-STRs yield high-resolution paternal lineage differentiation and provide a considerable improvement compared to Yfiler. We also find in this worldwide dataset substantially less genetic population substructure within and between geographic regions with RM Y-STRs than with Yfiler Y-STRs. Furthermore, with the present study we provide enhanced data evidence that the RM Y-STR panel is extremely successful in differentiating between closely and distantly related males. Among 305 male relatives, paternally connected by 1-20 meiotic transfers in 127 independent pedigrees, we show that 66% were separated by mutation events with the RM Y-STR panel whereas only 15% were with Yfiler; hence, RM Y-STRs provide a statistically significant 4.4-fold increase of average male relative differentiation relative to Yfiler. The RM Y-STR panel is powerful enough to separate closely related males; nearly 50% of the father and sons, and 60% of brothers could be distinguished with RM Y-STRs, whereas only 7.7% and 8%, respectively, with Yfiler. Thus, by introducing RM Y-STRs to the forensic genetic community we provide important solutions to several of the current limitations of Y chromosome analysis in forensic genetics.

Multiplex genotyping system for efficient inference of matrilineal genetic ancestry with continental resolution.

BACKGROUND: In recent years, phylogeographic studies have produced detailed knowledge on the worldwide distribution of mitochondrial DNA (mtDNA) variants, linking specific clades of the mtDNA phylogeny with certain geographic areas. However, a multiplex genotyping system for the detection of the mtDNA haplogroups of major continental distribution that would be desirable for efficient DNA-based bio-geographic ancestry testing in various applications is still missing. RESULTS: Three multiplex genotyping assays, based on single-base primer extension technology, were developed targeting a total of 36 coding-region mtDNA variants that together differentiate 43 matrilineal haplo-/paragroups. These include the major diagnostic haplogroups for Africa, Western Eurasia, Eastern Eurasia and Native America. The assays show high sensitivity with respect to the amount of template DNA: successful amplification could still be obtained when using as little as 4 pg of genomic DNA and the technology is suitable for medium-throughput analyses. CONCLUSIONS: We introduce an efficient and sensitive multiplex genotyping system for bio-geographic ancestry inference from mtDNA that provides resolution on the continental level. The method can be applied in forensics, to aid tracing unknown suspects, as well as in population studies, genealogy and personal ancestry testing. For more complete inferences of overall bio-geographic ancestry from DNA, the mtDNA system provided here can be combined with multiplex systems for suitable autosomal and, in the case of males, Y-chromosomal ancestry-sensitive DNA markers.

Contrasting signals of positive selection in genes involved in human skin-color variation from tests based on SNP scans and resequencing.

BACKGROUND: Numerous genome-wide scans conducted by genotyping previously ascertained single-nucleotide polymorphisms (SNPs) have provided candidate signatures for positive selection in various regions of the human genome, including in genes involved in pigmentation traits. However, it is unclear how well the signatures discovered by such haplotype-based test statistics can be reproduced in tests based on full resequencing data. Four genes (oculocutaneous albinism II (OCA2), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), and KIT ligand (KITLG)) implicated in human skin-color variation, have shown evidence for positive selection in Europeans and East Asians in previous SNP-scan data. In the current study, we resequenced 4.7 to 6.7 kb of DNA from each of these genes in Africans, Europeans, East Asians, and South Asians. RESULTS: Applying all commonly used neutrality-test statistics for allele frequency distribution to the newly generated sequence data provided conflicting results regarding evidence for positive selection. Previous haplotype-based findings could not be clearly confirmed. Although some tests were marginally significant for some populations and genes, none of them were significant after multiple-testing correction. Combined P values for each gene-population pair did not improve these results. Application of Approximate Bayesian Computation Markov chain Monte Carlo based to these sequence data using a simple forward simulator revealed broad posterior distributions of the selective parameters for all four genes, providing no support for positive selection. However, when we applied this approach to published sequence data on SLC45A2, another human pigmentation candidate gene, we could readily confirm evidence for positive selection, as previously detected with sequence-based and some haplotype-based tests. CONCLUSIONS: Overall, our data indicate that even genes that are strong biological candidates for positive selection and show reproducible signatures of positive selection in SNP scans do not always show the same replicability of selection signals in other tests, which should be considered in future studies on detecting positive selection in genetic data.

Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms.

We analyzed 67 short tandem repeat polymorphisms from the non-recombining part of the Y-chromosome (Y-STRs), including 49 rarely studied simple single-copy (ss)Y-STRs and 18 widely used Y-STRs, in 590 males from 51 populations belonging to 8 worldwide regions (HGDP-CEPH panel). Although autosomal DNA profiling provided no evidence for close relationship, we found 18 Y-STR haplotypes (defined by 67 Y-STRs) that were shared by two to five men in 13 worldwide populations, revealing high and widespread levels of cryptic male relatedness. Maximal (95.9%) haplotype resolution was achieved with the best 25 out of 67 Y-STRs in the global dataset, and with the best 3-16 markers in regional datasets (89.6-100% resolution). From the 49 rarely studied ssY-STRs, the 25 most informative markers were sufficient to reach the highest possible male lineage differentiation in the global (92.2% resolution), and 3-15 markers in the regional datasets (85.4-100%). Considerably lower haplotype resolutions were obtained with the three commonly used Y-STR sets (Minimal Haplotype, PowerPlex Y, and AmpFlSTR Yfiler. Six ssY-STRs (DYS481, DYS533, DYS549, DYS570, DYS576 and DYS643) were most informative to supplement the existing Y-STR kits for increasing haplotype resolution, or - together with additional ssY-STRs - as a new set for maximizing male lineage differentiation. Mutation rates of the 49 ssY-STRs were estimated from 403 meiotic transfers in deep-rooted pedigrees, and ranged from approximately 4.8 x 10(-4) for 31 ssY-STRs with no mutations observed to 1.3 x 10(-2) and 1.5 x 10(-2) for DYS570 and DYS576, respectively, the latter representing the highest mutation rates reported for human Y-STRs so far. Our findings thus demonstrate that ssY-STRs are useful for maximizing global and regional resolution of male lineages, either as a new set, or when added to commonly used Y-STR sets, and support their application to forensic, genealogical and anthropological studies.

Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development.

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Relating two deep-rooted pedigrees from Central Germany by high-resolution Y-STR haplotyping.

Y-STR haplotyping is a powerful forensic and anthropological tool for identifying male lineages. We used high-resolution Y-STR haplotyping to evaluate the possibility of a blood relationship between two deep-rooted paternal genealogies with the same surname and originating from the same geographical region in Central Germany. One pedigree comprised 13 generations covering >450 years, the other comprised nine generations covering >300 years. Of the 68 loci tested, 64 (94%) consistently had the same allele in all males in the two pedigrees (except for some unambiguously sporadic mutations within pedigrees). Only four Y-STRs had a consistent allelic difference of exactly one repeat between the two pedigrees. These findings suggested that the two pedigrees were paternally related, and a conservative assessment taking average mutation rates and the available local haplotype frequencies for nine loci into account yielded a likelihood ratio of 8.2:1 in favour of this hypothesis. Our study thus highlights the power of Y-STR haplotyping to identify male lineages. It also shows that families can be linked to common ancestors on the basis of Y-STR data, even if these individuals lived several hundred years ago. However, the potential of Y-STR haplotyping could still not be fully exploited in our case due to a lack of appropriate population frequency data for all analysed Y-STR loci. This shortcoming makes a strong case for more comprehensive haplotype databases, including more samples and larger numbers of loci.