Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II.

In 1991, nine sets of skeletal remains were excavated from a mass grave near Yekaterinburg, Russia which were believed to include the Russian Tsar Nicholas II, the Tsarina Alexandra, and three of their daughters. Nuclear DNA testing of the remains verified such a family group, and mitochondrial DNA (mtDNA) sequences of the presumed Tsarina matched a known maternal relative, Prince Philip. mtDNA sequences from bone of the presumed Tsar matched two living maternal relatives except at a single position, where the bone sample had a mixture of matching (T) and mismatching (C) bases. Cloning experiments indicated that this mixture was due to heteroplasmy within the Tsar; nevertheless, the 'mismatch' fueled a lingering controversy concerning the authenticity of these remains. As a result, the official final report on the fate of the last Russian Royals has been postponed by Russian authorities pending additional, convincing DNA evidence. At the request of the Russian Federation government, we analysed the skeletal remains of the Tsar's brother Georgij Romanov in order to gain further insight into the occurrence and segregation of heteroplasmic mtDNA variants in the Tsar's maternal lineage. The mtDNA sequence of Georgij Romanov, matched that of the putative Tsar, and was heteroplasmic at the same position. This confirms heteroplasmy in the Tsar's lineage, and is powerful evidence supporting the identification of Tsar Nicholas II. The rapid intergenerational shift from heteroplasmy to homoplasmy, and the different heteroplasmic ratios in the brothers, is consistent with a 'bottleneck' mechanism of mtDNA segregation.

A high observed substitution rate in the human mitochondrial DNA control region.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

A compilation of tri-allelic SNPs from 1000 Genomes and use of the most polymorphic loci for a large-scale human identification panel.

In a directed search of 1000 Genomes Phase III variation data, 271,934 tri-allelic single nucleotide polymorphisms (SNPs) were identified amongst the genotypes of 2,504 individuals from 26 populations. The majority of tri-allelic SNPs have three nucleotide substitution-based alleles at the same position, while a much smaller proportion, which we did not compile, have a nucleotide insertion/deletion plus substitution alleles. SNPs with three alleles have higher discrimination power than binary loci but keep the same characteristic of optimum amplification of the fragmented DNA found in highly degraded forensic samples. Although most of the tri-allelic SNPs identified had one or two alleles at low frequencies, often single observations, we present a full compilation of the genome positions, rs-numbers and genotypes of all tri-allelic SNPs detected by the 1000 Genomes project from the more detailed analyses it applied to Phase III sequence data. A total of 8,705 tri-allelic SNPs had overall heterozygosities (averaged across all 1000 Genomes populations) higher than the binary SNP maximum value of 0.5. Of these, 1,637 displayed the highest average heterozygosity values of 0.6-0.666. The most informative tri-allelic SNPs we identified were used to construct a large-scale human identification panel for massively parallel sequencing, designed for the identification of missing persons. The large-scale MPS identification panel comprised: 1,241 autosomal tri-allelic SNPs and 29 X tri-allelic SNPs (plus 46 microhaplotypes adapted for genotyping from reduced length sequences). Allele frequency estimates are detailed for African, European, South Asian and East Asian population groups plus the Peruvian population sampled by 1000 Genomes for the 1,270 tri-allelic SNPs of the final MPS panel. We describe the selection criteria, kinship simulation experiments and genomic analyses used to select the tri-allelic SNP components of the panel. Approximately 5 % of the tri-allelic SNPs selected for the large-scale MPS identification panel gave three-genotype patterns in single individual samples or discordant genotypes for genomic control DNAs. A likely explanation for some of these unreliably genotyped loci is that they map to multiple sites in the genome - highlighting the need for caution and detailed scrutiny of multiple-allele variant data when designing future forensic SNP panels, as such patterns can arise from common structural variation in the genome, such as segmental duplications.

Unidirectional spread of secondary sexual plumage traits across an avian hybrid zone.

Theory predicts that traits under positive selection can rapidly cross a hybrid zone in spite of a substantial barrier to neutral gene flow between hybridizing taxa. An avian hybrid zone between Manacus candei (white-collared manakin) and M. vitellinus (golden-collared manakin) is reported here that displays an unusual pattern of noncoincident clines. Male secondary sexual traits of M. vitellinus have spread into populations that are genetically and morphometrically like M. candei. These birds have a lek breeding system in which male mating success is highly skewed, suggesting that sexual selection is driving male sexual traits across the zone.

Television viewing and obesity: a prospective study in the 1958 British birth cohort.

OBJECTIVE: To assess whether frequency of television viewing in adolescence (11 and 16 years) or early adulthood (23 years) affected subsequent changes in body mass index (BMI) through to mid-adulthood life, and waist-hip ratio in mid-adulthood. SUBJECTS: The 1958 British birth cohort includes all births in 1 week in March 1958 in England, Scotland and Wales. The main analyses included at least 11 301 participants. Outcome measures included BMI at 16, 23, 33 and 45 years and waist-hip ratio at 45 years. RESULTS: Watching television 'often' at 16 years (but not 11 years) was associated with a faster gain in BMI between 16 and 45 years in males (0.011 kg m(-2) per year, 95% confidence interval (CI) 0.003, 0.019) and females (0.013 kg m(-2) per year, 95%CI 0.003, 0.023). More frequent television viewing at 11, 16 and 23 years was associated with a faster gain in BMI between 23 and 45 years in females, but not in males. Television viewing at 23 years was associated with waist-hip ratio at 45 years: participants watching > or = 5 times per week had a waist-hip ratio 0.01 higher than those watching less often. At 45 years, those watching television for > or = 4 h day(-1) had a waist-hip ratio 0.03-0.04 higher than those watching for <1 h day(-1). CONCLUSIONS: More frequent television viewing in adolescence and early adulthood is associated with greater BMI gains through to mid-adulthood and with central adiposity in mid-life. Television viewing may be a useful behaviour to target in strategies to prevent obesity.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Changes in diet and physical activity in the 1990s in a large British sample (1958 birth cohort).

OBJECTIVES: To investigate whether adults studied in 1991 and 1999 (at ages 33 and 42 y) improved their diet and their physical activity level, in the direction of recommendations issued during the same period. DESIGN: Longitudinal 1958 British birth cohort study. SETTING: England, Scotland and Wales. PARTICIPANTS: All births, 3rd-9th March, 1958. A minimum of 11 341 participants provided data at 33 y, 11 361 at 42 y. MAIN OUTCOME MEASURES: Frequency of leisure time activity and consumption of (i) fried food, (ii) chips, (iii) wholemeal bread and (iv) fruit and salad/raw vegetables, at 33 and 42 y. RESULTS: Most people changed their physical activity and dietary habits over the 8-y period. About a third of men and women increased, and a third decreased their activity frequency. Findings for fried food consumption were similar. A significantly greater proportion of cohort members decreased their chips consumption (32%), rather than increased it (17%) and increased their fruit and salad consumption (30%), rather than decreased it (25%). In all, 26% of men and 33% of women consistently ate, or switched to eating mostly wholemeal bread, while 56% of men and 48% of women consistently ate less or switched to eating less. Social gradients were seen for activity and diet in 1991, but associations between social factors or body mass index and change in activity or diet were inconsistent. CONCLUSIONS: Lifestyle habits such as dietary intake and physical activity are slow to change. Current health promotion strategies may need to be supplemented with additional methods to affect the desired change in these habits.

Bone mineral mass consolidation in young British adults.

Forty males and 40 females, ages 18-21 years, were recruited into a prospective study to investigate the consolidation of bone mineral after cessation of linear growth and the influences of calcium intake, lifestyle factors, physical activity, and body composition on bone. Three sets of bone measurements were made annually at the spine, hip, and whole body (dual-energy X-ray absorptiometry) and at the wrist and midshaft radius (single photon absorptiometry). At baseline, the bone mineral content (BMC) was significantly greater in males than females at all sites (p < 0.004). After adjustment for scan area (BA), height, weight, and age, male BMC was significantly greater than female BMC at the midshaft radius (+7.4%; p < 0.01) but lower at the spine (-10.9%; p < 0.001) with no difference at the wrist, hip, or whole body. Positive effects of leanness on bone were observed in females but not in males. Dietary calcium, nutrient intakes, and physical activity levels were not size-independent determinants of BMC at any site. Significant increases in whole-body BMC were observed within individuals over the study period of 670 (SD 51) days, +1.3% in males and +2.1% in females (p < 0.01), mirrored by increases in BMC and BA at most skeletal sites. After allowing for changes in BA, significant increases (p < 0.01) were observed in adjusted BMC for the whole body (males, +1.1%; females, +0.6%), lumbar spine (males, +1.5%; females, +1.1%), and midshaft radius (males, +1.9%; females, +2.0%). No lifestyle or anthropometric factors were identified that influenced these longitudinal increases in bone mineral.

Reduced bone mass in Dutch adolescents fed a macrobiotic diet in early life.

This study investigated the effect of a macrobiotic (vegan-type) diet, low in calcium and vitamin D, consumed in early life, on bone mineral during adolescence. Bone mineral content (BMC) and bone area were measured in 195 adolescents (103 girls, 92 boys) aged 9-15 years, using dual-energy X-ray absorptiometry. Ninety-three adolescents (43 girls, 50 boys) had followed a macrobiotic diet in childhood, and 102 (60 girls, 42 boys) were control subjects. After adjustment for bone area, weight, height, percent body lean, age, and puberty, BMC was significantly lower in macrobiotic subjects, in boys and girls, respectively, at the whole body, -3.4% and -2.5%, spine, -8.5% and -5.0%, femoral neck, -8.0% and -8.2%, midshaft radius, -6.8% and -5.6%, and also in girls, at the trochanter, -5.8% (p < 0.05). No group differences were observed at the wrist. Group differences were not explained by current calcium adjusted bone mass at age 9-15 years, observations which may hold important implications for fracture risk in later life.

Uncritical use of bone mineral density in absorptiometry may lead to size-related artifacts in the identification of bone mineral determinants.

Epidemiological studies of bone mineral determinants rely heavily on measurements made with absorptiometric techniques such as single-photon absorptiometry and dual-energy x-ray absorptiometry. In general, absorptiometric data are expressed as areal densities (bone mineral density, BMD), obtained by dividing bone mineral content (BMC) by bone area or width (BA, BW). This size correction assumes that BMC and BA (BW) are directly proportional to one another, such that a 1% change in BA (BW) is matched by a 1% change in BMC. This is rarely the case, and the exact relationship depends on the population group, skeletal site, body size, instrumentation, and scanning conditions. Size adjustment determined by using predefined indexes, such as BMD and body mass index (BMI, wt/ht2), may fail to correct BMC fully for bone and body size, and may lead to spurious associations with other size-related variables such as calcium intake, energy expenditure, and grip strength. A general approach to size adjustment is described, in which BA (BW), weight, and height are incorporated in all regression models of BMC. Although BMD plays a valuable role in fracture-risk assessment and clinical management, we advocate that its use in epidemiological research be discontinued.

Association study of dopamine D3 receptor gene and schizophrenia.

Several groups have reported an association between schizophrenia and the MscI polymorphism in the first exon of the dopamine D3 receptor gene (DRD3). We studied this polymorphism using a North American sample (117 patients plus 188 controls) and an Italian sample (97 patients plus 64 controls). In the first part of the study, we compared allele frequencies of schizophrenia patients and unmatched controls and observed a significant difference in the total sample (P = 0.01). The second part of the study involved a case control approach in which each schizophrenia patient was matched to a control of the same sex, and of similar age and ethnic background. The DRD3 allele frequencies of patients and controls revealed no significant difference between the two groups in the Italian (N = 53) or the North American (N = 54) matched populations; however, when these two matched samples were combined, a significant difference was observed (P = 0.026). Our results suggest that the MscI polymorphism may be associated with schizophrenia in the populations studied.

Interaction of platelets and purified collagens in a laminar flow model.

Damage to the endothelial surface of the vessel wall can result in exposure of circulating blood components to collagen and other subendothelial structures. Collagen types I, III, IV, and V have been demonstrated in the vessel wall by chemical and immunohistological methods; type V is thrombin-sensitive, and is present on the endothelial cell surface. In an earlier study using a rocking model, both unstimulated and ADP-induced platelet adherence was reduced on wells coated with type V collagen in comparison to uncoated wells; and increased on plastic surfaces coated with types III and IV collagen in comparison to those coated with type V collagen. The present study was designed to determine the effect of erythrocytes and shear rate on platelet adherence to these purified collagen types in a laminar flow system. With platelet-rich plasma, adherence of labeled platelets was much lower in the laminar flow system compared with the rocking model. Erythrocytes significantly enhanced platelet adherence to surfaces that were untreated or absorbed with collagen types I, III, and IV. However, this enhancement was not seen in the presence of type V collagen. These studies provide additional evidence for the selectively nonthrombogenic nature of type V collagen.

Are levels of bone turnover related to lower bone mass of adolescents previously fed a macrobiotic diet?

Dutch adolescents who consumed a macrobiotic (vegan-type) diet in early life, demonstrate a lower relative bone mass than their omnivorous counterparts. We investigated whether subjects from the macrobiotic group showed signs of catching up with controls in terms of relative bone mass, reflected by higher levels of serum osteocalcin and alkaline phosphatase and lower levels of urinary cross-links. Group differences in calciotropic hormones and mineral excretion were also investigated. Bone measurements, blood, and urine samples were obtained from 69 macrobiotic (34 girls, 35 boys) and 99 control (57 girls, 42 boys) subjects, aged 9-15. Bone turnover markers and 1,25(OH)2D reached maximal levels at pubertal stages 3-4, and decreased thereafter. After adjusting for puberty, age, and lean body mass, no group differences were found in markers of bone turnover, 1,25(OH)2D, PTH, or calcium excretion, but phosphate excretion was 23% lower in macrobiotic girls. After adjustment for puberty, 1,25(OH)2D was positively related to osteocalcin. In summary, we found no evidence for group differences in bone turnover, or catch up in relative bone mass, which might be due to the fact that 60% of subjects were still in early stages of puberty.

Effects of preservation methods, parasites, and gut contents of black flies (Diptera: Simuliidae) on polymerase chain reaction products.

Molecular analysis of biological specimens usually requires extraction of high-molecular weight DNA free of foreign DNA contaminants. DNA was extracted from black flies at different life stages that had been preserved by 4 methods: larvae and adults in ethanol, larvae in Carnoy's solution, adults on card-points, and adults hand-swatted and sun-dried. Using specific primers for the mitochondrial ND4 gene, a 257-bp amplicon was obtained from specimens preserved by ethanol, card-point mounting, and sun-drying. Successful amplification often required DNA dilutions > or = 1:20 (< 1-10 ng). DNA from specimens preserved in Carnoy's solution (ethanol: acetic acid, 3:1) yielded degraded DNA, resulting in fewer successful amplications. Parasitic nematodes and, to a lesser extent, gut contents resulted in extra products when amplified with randomly amplified polymorphic DNA (RAPD) primers. Sufficient DNA was extracted from the head of a larva for a successful polymerase chain reaction (PCR), eliminating the need to remove the contaminating gut and parasites.

Diagnosis by random amplified polymorphic DNA polymerase chain reaction of four cryptic species related to Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) from Paraguay, Argentina, and Brazil.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was applied to samples from widespread populations of the poorly characterized Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga species complex, and 4 genetically differentiated species were distinguished. A screen of 65 random decamer oligonucleotide primers identified 12 primers, which produced 19 reproducible species-specific genetic markers and 4 markers common to 2 or more species. These markers were correlated in nearly all individuals of each species throughout the ranges sampled, including populations as far apart as 2,500 km. Each individual analyzed was from a different isofemale progeny brood, with associated morphological specimens. These specimens will facilitate studies to relate these species to previously reported chromosomal and enzymatic variation as well as to their feeding behavior and potential as malaria vectors. We hypothesize that 3 of the species have recognized valid names: An. (Nys.) albitarsis Lynch-Arribálzaga, An. (Nys.) marajoara Galvão and Damasceno, and An. (Nys.) deaneorum Rosa-Freitas, whereas the 4th is undescribed.

Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy.

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is often used for forensic identification due largely to the high genome copy number per cell. Literature from the "ancient DNA" field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size (1-4). Hand et al. have demonstrated the targeting and preferential amplification of authentic human DNA sequences with small amplicon products of 150 bp or less (1,2). Given this understanding of ancient DNA preservation and amplification, we report an improved approach to forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in highly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 11 extracts representing a range of sample quality were prepared from nonprobative forensic specimens. We demonstrate a significant increase in MPS amplification success when compared to testing methods using approximately 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unreported" due to mixed sequences provided potentially reportable sequence data from a single, authentic template with MPS testing.

Fetal and early life growth and body mass index from birth to early adulthood in 1958 British cohort: longitudinal study.

OBJECTIVES: To determine the influence of birth weight on body mass index at different stages of later life; whether this relation persists after accounting for potential confounding factors; and the role of indicators of fetal growth (birth weight relative to parental size) and childhood growth. DESIGN: Longitudinal study of the 1958 British birth cohort. SETTING: England, Scotland, and Wales. PARTICIPANTS: All singletons born 3-9 March 1958 (10 683 participants with data available at age 33). MAIN OUTCOME MEASURES: Body mass index at ages 7, 11, 16, 23, and 33 years. RESULTS: The relation between birth weight and body mass index was positive and weak, becoming more J shaped with increasing age. When adjustments were made for maternal weight, there was no relation between birth weight and body mass index at age 33. Indicators of poor fetal growth based on the mother's body size were not predictive, but the risk of adult obesity was higher among participants who had grown to a greater proportion of their eventual adult height by age 7. In men only, the effect of childhood growth was strongest in those with lower birth weights and, to a lesser extent, those born to lighter mothers. CONCLUSIONS: Maternal weight (or body mass index) largely explains the association between birth weight and adult body mass index, and it may be a more important risk factor for obesity in the child than birth weight. Birth weight and maternal weight seem to modify the effect of childhood linear growth on adult obesity in men. Intergenerational associations between the mother's and her offspring's body mass index seem to underlie the well documented association between birth weight and body mass index. Other measures of fetal growth are needed for a fuller understanding of the role of the intrauterine environment in the development of obesity.