In-Solution Hybridization for the Targeted Enrichment of the Whole Mitochondrial Genome.

A detailed protocol is presented for the targeted enrichment of whole mitochondrial genomes based on an in-solution hybridization strategy. Bait is produced in-house by sonication of two long-range PCR amplicons and ligation of biotinylated double-stranded adapters. Indexed target DNA is hybridized with the bait in a multiplex enrichment reaction and pulled down using magnetic streptavidin beads followed by subsequent post-enrichment PCR and sequencing on an Illumina MiSeq. This strategy removes the need for expensive commercial bait probes while allowing enrichment of multiple samples in a single hybridization reaction. The method is particularly suitable for degraded DNA as it is able to enrich short DNA fragments and is not susceptible to polymerase artifacts introduced during PCR-based assays.

High Y-chromosomal diversity and low relatedness between paternal lineages on a communal scale in the Western European Low Countries during the surname establishment.

There is limited knowledge on the biological relatedness between citizens and on the demographical dynamics within villages, towns and cities in pre-17th century Western Europe. By combining Y-chromosomal genotypes, in-depth genealogies and surname data in a strict genetic genealogical approach, it is possible to provide insights into the genetic diversity and the relatedness between indigenous paternal lineages within a particular community at the time of the surname adoption. To obtain these insights, six Flemish communities were selected in this study based on the differences in geography and historical development. After rigorous selection of appropriate DNA donors, low relatedness between Y chromosomes of different surnames was found within each community, although there is co-occurrence of these surnames in each community since the start of the surname adoption between the 14th and 15th century. Next, the high communal diversity in Y-chromosomal lineages was comparable with the regional diversity across Flanders at that time. Moreover, clinal distributions of particular Y-chromosomal lineages between the communities were observed according to the clinal distributions earlier observed across the Flemish regions and Western Europe. No significant indication for genetic differences between communities with distinct historical development was found in the analysis. These genetic results provide relevant information for studies in historical sciences, archaeology, forensic genetics and genealogy.

Increasing phylogenetic resolution still informative for Y chromosomal studies on West-European populations.

Many Y-chromosomal lineages which are defined in the latest phylogenetic tree of the human Y chromosome by the Y Chromosome Consortium (YCC) in 2008 are distributed in (Western) Europe due to the fact that a large number of phylogeographic studies focus on this area. Therefore, the question arises whether newly discovered polymorphisms on the Y chromosome will still be interesting to study Western Europeans on a population genetic level. To address this question, the West-European region of Flanders (Belgium) was selected as study area since more than 1000 Y chromosomes from this area have previously been genotyped at the highest resolution of the 2008 YCC-tree and coupled to in-depth genealogical data. Based on these data the temporal changes of the population genetic pattern over the last centuries within Flanders were studied and the effects of several past gene flow events were identified. In the present study a set of recently reported novel Y-SNPs were genotyped to further characterize all those Flemish Y chromosomes that belong to haplogroups G, R-M269 and T. Based on this extended Y-SNP set the discrimination power increased drastically as previous large (sub-)haplogroups are now subdivided in several non-marginal groups. Next, the previously observed population structure within Flanders appeared to be the result of different gradients of independent sub-haplogroups. Moreover, for the first time within Flanders a significant East-West gradient was observed in the frequency of two R-M269 lineages, and this gradient is still present when considering the current residence of the DNA donors. Our results thus suggest that an update of the Y-chromosomal tree based on new polymorphisms is still useful to increase the discrimination power based on Y-SNPs and to study population genetic patterns in more detail, even in an already well-studied region such as Western Europe.

Low historical rates of cuckoldry in a Western European human population traced by Y-chromosome and genealogical data.

Recent evidence suggests that seeking out extra-pair paternity (EPP) can be a viable alternative reproductive strategy for both males and females in many pair-bonded species, including humans. Accurate data on EPP rates in humans, however, are scant and mostly restricted to extant populations. Here, we provide the first large-scale, unbiased genetic study of historical EPP rates in a Western European human population based on combining Y-chromosomal data to infer genetic patrilineages with genealogical and surname data, which reflect known historical presumed paternity. Using two independent methods, we estimate that over the last few centuries, EPP rates in Flanders (Belgium) were only around 1­2% per generation. This figure is substantially lower than the 8­30% per generation reported in some behavioural studies on historical EPP rates, but comparable with the rates reported by other genetic studies of contemporary Western European populations. These results suggest that human EPP rates have not changed substantially during the last 400 years in Flanders and imply that legal genealogies rarely differ from the biological ones. This result has significant implications for a diverse set of fields, including human population genetics, historical demography, forensic science and human sociobiology.

Updating the Y-chromosomal phylogenetic tree for forensic applications based on whole genome SNPs.

The Y-chromosomal phylogenetic tree has a wide variety of important forensic applications and therefore it needs to be state-of-the-art. Nevertheless, since the last 'official' published tree many publications reported additional Y-chromosomal lineages and other phylogenetic topologies. Therefore, it is difficult for forensic scientists to interpret those reports and use an up-to-date tree and corresponding nomenclature in their daily work. Whole genome sequencing (WGS) data is useful to verify and optimise the current phylogenetic tree for haploid markers. The AMY-tree software is the first open access program which analyses WGS data for Y-chromosomal phylogenetic applications. Here, all published information is collected in a phylogenetic tree and the correctness of this tree is checked based on the first large analysis of 747 WGS samples with AMY-tree. The obtained result is one phylogenetic tree with all peer-reviewed reported Y-SNPs without the observed recurrent and ambiguous mutations. Nevertheless, the results showed that currently only the genomes of a limited set of Y-chromosomal (sub-)haplogroups is available and that many newly reported Y-SNPs based on WGS projects are false positives, even with high sequencing coverage methods. This study demonstrates the usefulness of AMY-tree in the process of checking the quality of the present Y-chromosomal tree and it accentuates the difficulties to enlarge this tree based on only WGS methods.

Genetic genealogy comes of age: perspectives on the use of deep-rooted pedigrees in human population genetics.

In this article, we promote the implementation of extensive genealogical data in population genetic studies. Genealogical records can provide valuable information on the origin of DNA donors in a population genetic study, going beyond the commonly collected data such as residence, birthplace, language, and self-reported ethnicity. Recent studies demonstrated that extended genealogical data added to surname analysis can be crucial to detect signals of (past) population stratification and to interpret the population structure in a more objective manner. Moreover, when in-depth pedigree data are combined with haploid markers, it is even possible to disentangle signals of temporal differentiation within a population genetic structure during the last centuries. Obtaining genealogical data for all DNA donors in a population genetic study is a labor-intensive task but the vastly growing (genetic) genealogical databases, due to the broad interest of the public, are making this job more time-efficient if there is a guarantee for sufficient data quality. At the end, we discuss the advantages and pitfalls of using genealogy within sampling campaigns and we provide guidelines for future population genetic studies.

In the name of the migrant father--analysis of surname origins identifies genetic admixture events undetectable from genealogical records.

Patrilineal heritable surnames are widely used to select autochthonous participants for studies on small-scale population genetic patterns owing to the unique link between the surname and a genetic marker, the Y-chromosome (Y-chr). Today, the question arises as to whether the surname origin will be informative on top of in-depth genealogical pedigrees. Admixture events that happened in the period after giving heritable surnames but before the start of genealogical records may be informative about the additional value of the surname origin. In this context, an interesting historical event is the demic migration from French-speaking regions in Northern France to the depopulated and Dutch-speaking region Flanders at the end of the sixteenth century. Y-chr subhaplogroups of individuals with a French/Roman surname that could be associated with this migration event were compared with those of a group with autochthonous Flemish surnames. Although these groups could not be differentiated based on in-depth genealogical data, they were significantly genetically different from each other. Moreover, the observed genetic divergence was related to the differences in the distributions of main Y-subhaplogroups between contemporary populations from Northern France and Flanders. Therefore, these results indicate that the surname origin can be an important feature on top of in-depth genealogical results to select autochthonous participants for a regional population genetic study based on Y-chromosomes.

Signature of selection on the rhodopsin gene in the marine radiation of American seven-spined gobies (Gobiidae, Gobiosomatini).

In comparison with terrestrial and freshwater ecosystems, information about speciation modes and the role of selection in marine environments is scarce. Recent studies have indicated that spectral adaptation could play an important role in the diversification of marine species flocks. Natural selection influences specific amino acids (AAs) that are involved in the spectral tuning mechanism of visual pigment genes. To study the wider occurrence and the characteristics of spectral adaptation in marine radiations, a reinterpretation of the rhodopsin (RH1) data of American seven-spined gobies (genus Elacatinus; Gobiidae; Teleostei) was carried out. Reanalysis revealed that some AAs, which are well known in the literature as spectral tuning sites, are variable in Elacatinus. Those crucial AA substitutions originated polyphyletically, indicating convergent evolution within the genus Elacatinus. Moreover, statistical tests based on the d(N)/d(S) ratio detected selection in several phylogenetic lineages and at specific AAs. Many of these AAs were previously shown to be under selection in other marine radiations. Therefore, the current phylogenetic approach provided an extended list of AAs that are probably involved in spectral tuning, and which should be validated by mutagenic experiments.

Mitochondrial DNA analysis of the putative heart of Louis XVII, son of Louis XVI and Marie-Antoinette.

According to official historiography, the 10-year-old Louis XVII died in the Temple of Paris on June 8, 1795. However, public rumour spread the theory that Louis XVII escaped and that his descendants would be alive today. One such putative 'Louis XVII' was Carl Wilhelm Naundorff, who died in 1845 in Delft (the Netherlands). Comparative mitochondrial DNA (mtDNA) analysis gave evidence that his remains could not be identified as those of Louis XVII. In the present study, mtDNA analysis was performed on the heart of the young boy who died in the prison of Paris in 1795. In order to obtain the strongest evidence possible, two laboratories independently analysed the heart. The results showed that the consensus mtDNA sequence of the heart was identical to that of the maternal relatives of Louis XVII.

Y-chromosomal STR haplotypes in three major population groups in Bulgaria.

A total of 280 unrelated males from the three largest population groups in Bulgaria: Bulgarians, Bulgarian Turks and Gypsies, were analyzed for seven Y-chromosome STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393). Comparison of the allele frequency distributions revealed significant differences between the three ethnic groups which were confirmed with haplotype analysis. This permits us to suggest that population differentiation should be taken into account in forensic case analysis and paternity testing in Bulgaria.

Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII.

Amplification of mtDNA D-loop fragments with a length of 200 bp or more from ancient and even from fairly recent biological samples, can lead to erroneous results. This was clearly illustrated in our investigation of the putative heart of Louis XVII. By selecting different sets of primers which amplified shorter fragments of mtDNA (length 109 bp-201 bp), authentic polymorphisms could be visualised which remained undetected with the more classical primers for fragment sizes > 210 bp. Here we have extended those findings to other biological materials. A competitive PCR assay for quantitation of the amount of mtDNA for different fragment lengths, using a 10 bp deletion construct, was applied to ancient material and on a set of hairs of various ages of sampling (1966 up to the present). The results showed that DNA degradation started a few years after sampling. In the DNA extracts of the older hair shafts (1983-1995), the proportion of the number of short fragments to the number of long fragments is on average 4 in contrast to the most recent hair shafts. The numbers of amplifiable mtDNA copies for the hairs from 1975 and older were too small to show a clear difference. Use of long PCR fragments in such cases can yield misleading results. Use of short PCR fragments for the analysis of mtDNA from shed hair, in combination with a competitive PCR assay to determine the state of degradation, should improve the reliability of forensic mtDNA analysis considerably.

Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language.

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant clines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. Principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low, nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift.

DNA analysis of a transplanted cryopreserved meniscal allograft.

SUMMARY: Meniscal transplantation is frequently performed in young patients with a single meniscal-deficient compartment as a result of previous total meniscectomy. Indications, operative techniques, and preservation of meniscal allografts have been studied extensively. In this study we compared the DNA profile of a meniscal allograft with that of the human recipient 1 year after transplantation. Applying techniques routinely used in forensic analysis, we were able to show that the DNA profile of the meniscal allograft was 95% identical to that of the human recipient. These findings indicate that 1 year after transplantation the meniscal allograft is nearly completely repopulated by host cells.

Genetic data obtained for two Chinese Han populations with a quadruplex fluorescent STR typing system (HUMVWA, HUMTH01, D21S11 and HPRT).

Abstract DNA typing of four tetrameric repeat loci (HUMVWA, HUMTH0I, D21SII and HPRT) was carried out in a Chinese Han population from Shanghai (East China) and one from Guangzhou (South-East China) using a quadruplex PCR amplification and detection of the fluorescent-labeled alleles on the ALF DNA sequencer. All loci were in accordance with Hardy-Weinberg equilibrium except for D21S11 in the Guangzhou population. A test for population differentiation showed no statistical difference in the allele frequency distribution between the two populations. Comparison of the allele frequency data with other Chinese Han populations from North and South-West China for the STR loci HUMVWA and HUMTH01 revealed heterogeneity between Northern Chinese Han and Southern Chinese Han, which is in accordance with previous studies on the basis of protein markers.

Mitochondrial DNA analysis on remains of a putative son of Louis XVI, King of France and Marie-Antoinette.

Carl Wilhelm Naundorff was buried in 1845 in Delft as Louis Charles, Duc de Normandie, 'Louis XVII'. However, the son of Louis XVI and Marie-Antoinette-Louis XVII--officially died in the Temple of Paris in 1795. In order to resolve the identity of Naundorff, mitochondrial DNA (mtDNA) D-loop sequences of his remains were compared with the sequences obtained from the hairs of two sisters of Marie-Antoinette, Marie-Antoinette herself, and with the sequences obtained from DNA samples of two living maternal relatives. The mtDNA sequence of a bone sample from Naundorff showed two nucleotide differences from the sequences of the three sisters and four differences from the sequences of living maternal relatives. Based on this evidence it becomes very unlikely that Naundroff is the son of Marie-Antoinette.

Quadruplex fluorescent STR typing system (HUMVWA, HUMTH01, D21S11 and HPRT) with sequence-defined allelic ladders identification of a new allele at D21S11.

A protocol for simultaneous amplification of the tetrameric STR loci HUMVWA, HUMTH01, D21S11 and HPRT has been developed in this study. Fluorescent amplified alleles were detected by laser scanning on the ALF DNA sequencer, and identified with locus specific allelic ladders. A sequencing survey of these STR loci was performed in 40 selected individuals from three major ethnic groups and confirmed the data reported previously. At the D21S11 locus, a new allele of 207 bp, was found in a Belgian Caucasian individual and designated as allele 25.2. Sequence analysis of this allele revealed that it contained a 14 bp deletion of (TCTA)3 TA at the beginning of the constant region. A nonconsensus allele of 245 bp, described and designated as allele 33.2.3(2x) by Brinkmann et al. (1996), was identified in an individual of central African origin. In addition, three new sequence variants of the allele 29, 30 and 32 were observed at D21S11.

Evaluation of a decontamination protocol for hair shafts before mtDNA sequencing.

Mitochondrial DNA (mtDNA) sequencing is a powerful and sensitive method to identify the donor of shed hairs found at a crime scene. Because of the low amounts of DNA in shed hair and the sensitivity of PCR, contaminating cells (e.g. saliva, blood), sometimes present on these hairs, will be co-amplified. This will result in ambiguous sequencing results and might even lead to erroneous exclusions of suspects. We have evaluated a strategy for effectively removing saliva and blood contamination from hair samples. Unambiguous mtDNA results were obtained by incubating the hair samples in a differential lysis buffer (which contains no DTT) prior to DNA extraction. Since the nuclear DNA of the hair root is affected, this procedure should be restricted to hair shaft proportions.

A single course of remission reinduction chemotherapy for acute myelogenous leukemia relapsing after allogeneic bone marrow transplantation is complicated by graft-versus-host disease and followed by sustained complete remission.

Graft-versus-host disease (GVHD) remains a major immunological complication after allogeneic bone marrow transplantation (allo-BMT), but also favors development of the beneficial graft-versus-leukemia (GVL) effect. A patient with AML-M4 (inv (16)) is described, who was given non-myeloablative remission reinduction therapy for leukemic relapse (inv (16), trisomy 8) diagnosed on day 184 after HLA-compatible sibling BMT. On day 236, ie about 6 weeks after completion of this course, a clinical syndrome suggestive of acute GVHD grade 3 had developed. Skin biopsy confirmed the clinical diagnosis of GVHD, with a compatible liver biopsy. Transfusion-associated GVHD was ruled out by analysis of short tandem repeat (STR) alleles in the skin biopsy, revealing alleles from donor and recipient but not from third party origin. Cyclosporin A (CsA) therapy, which had been tapered between days 150 and 175, was resumed, resulting in a favorable response and gradual transition to limited chronic GVHD. The patient has since remained in complete remission with an excellent performance status for more than 40 months, without further chemotherapy. Thus this biopsy proven case of GVHD was induced by marrow donor lymphocytes more than 200 days after transplantation and apparently triggered by remission reinduction chemotherapy. The case indicates that intensive non-myeloablative chemotherapy can cure AML relapsing after allo-BMT. The therapeutic effect in this case probably involved a direct pharmacological suppression of the leukemic clone followed by a GVL effect initiated by donor-derived alloreactive T lymphocytes.

Evaluation of the ALF DNA sequencer for high-speed sizing of short tandem repeat alleles.

DNA typing of short tandem repeat (STR) loci with automated real-time analysis of the fluorescent polymerase chain reaction (PCR) products has given forensic DNA analysis a new dimension. In the present work, the ALF DNA sequencer was evaluated for automated size determination of tetra-nucleotide STRs at high speed. Short gel plates, with a well-to-laser distance of 10 cm, allowed for the analysis of four STR loci (HUMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size determination was done with two external allelic ladders for each locus. Lane-to-lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR loci. The accuracy of sizing and allele detection within and between different gels was high (99.89%) for all four STR systems investigated and the gels could be reloaded without a decrease in accuracy of the allele size estimation. This way, the throughput of the system was increased, which is of interest for linkage studies, gene mapping, and population diversity studies.