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.

DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing.

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the question of human identification. Previous recommendations published in 2000 addressed the analysis and interpretation of mitochondrial DNA (mtDNA) in forensic casework. While the foundations set forth in the earlier recommendations still apply, new approaches to the quality control, alignment and nomenclature of mitochondrial sequences, as well as the establishment of mtDNA reference population databases, have been developed. Here, we describe these developments and discuss their application to both mtDNA casework and mtDNA reference population databasing applications. While the generation of mtDNA for forensic casework has always been guided by specific standards, it is now well-established that data of the same quality are required for the mtDNA reference population data used to assess the statistical weight of the evidence. As a result, we introduce guidelines regarding sequence generation, as well as quality control measures based on the known worldwide mtDNA phylogeny, that can be applied to ensure the highest quality population data possible. For both casework and reference population databasing applications, the alignment and nomenclature of haplotypes is revised here and the phylogenetic alignment proffered as acceptable standard. In addition, the interpretation of heteroplasmy in the forensic context is updated, and the utility of alignment-free database searches for unbiased probability estimates is highlighted. Finally, we discuss statistical issues and define minimal standards for mtDNA database searches.

Estimated activity patterns in British 45 year olds: cross-sectional findings from the 1958 British birth cohort.

OBJECTIVE: To investigate patterns of, and associations between, physical activity at work and in leisure time, television viewing and computer use. SUBJECTS: 4531 men and 4594 women with complete plausible data, age 44-45 years, participating in the 1958 British birth cohort study. METHODS: Physical activity, television viewing and computer use (hours/week) were estimated using a self-complete questionnaire and intensity (MET hours/week) derived for physical activity. Relationships were investigated using linear regression and chi(2) tests. RESULTS: From a target sample of 11,971, 9223 provided information on physical activity, of whom 75 and 47% provided complete and plausible activity data on work and leisure time activity respectively. Men and women spent a median of 40.2 and 34.2 h/week, respectively in work activity, and 8.3 and 5.8 h/week in leisure activity. Half of all participants watched television for > or =2 h/day, and half used a computer for <1 h/day. Longer work hours were not associated with a shorter duration of leisure activity, but were associated with a shorter duration of computer use (men only). In men, higher work MET hours were associated with higher leisure-time MET hours, and shorter durations of television viewing and computer use. Watching more television was related to fewer hours or MET hours of leisure activity, as was longer computer use in men. Longer computer use was related to more hours (or MET hours) in leisure activities in women. CONCLUSIONS: Physical activity levels at work and in leisure time in mid-adulthood are low. Television viewing (and computer use in men) may compete with leisure activity for time, whereas longer duration of work hours is less influential. To change active and sedentary behaviours, better understanding of barriers and motivators is needed.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex--results of a collaborative EDNAP exercise.

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

Diagnosing post-mortem treatments which inhibit DNA amplification from US MIAs buried at the Punchbowl.

The US military is committed to recovering and identifying the remains of unknown military service members. Casualties of the Korean War were exhumed from the National Memorial Cemetery of the Pacific, or Punchbowl, and submitted to the Armed Forces DNA Identification Laboratory (AFDIL) for mtDNA sequencing. Contrary to AFDIL's experience on other samples from this era, most failed to yield amplifiable DNA. Suspicion fell on mortuary practices that may have been applied to the remains, evidenced by a white powder found with the bones, and general records suggesting the use of formaldehyde-based stablizing agents. To improve the chances of successful identification of the unknown individuals, we looked for the causes underlying this failure. We did this by examining the state of the collagen, the most abundant biomolecule in bone, by using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The DSC analyses showed collagens with a range of different thermal stabilities. When these results were compared with the DNA amplification results, a clear correlation between elevated thermal stability and amplification failure was evident. TEM analysis revealed that fibril integrity was maintained after thermal and acid treatments in the samples which failed amplification. Together these two approaches implicate a stabilization agent as the cause of problems with DNA analysis, presumably due to excessive cross-linking. Following the initial study, the ability of DSC to rapidly identify problem samples was tested in a blind study of 14 samples, the method successfully identifying all the problematic samples from Punchbowl. Within this unusual context, DSC analysis is a useful method to assess the likelihood of successful DNA extraction and amplification.

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.

DNA Commission of the International Society for Forensic Genetics (ISFG): recommendations regarding the role of forensic genetics for disaster victim identification (DVI).

The ISFG membership consists of scientists and medical professionals specialized in using genetic testing for kinship analysis and the individualization of biological material. This expertise makes the forensic geneticist a resource of advice to international and national organizations dealing with human identifications and causes many DNA laboratories to get involved in DVI tasks. The present recommendations are meant to educate more forensic geneticists about their potential involvement in mass fatality preparedness and possible DVI efforts, as well as to provide practical guidance for each of the laboratories' individual tasks. The idea to work on DNA-specific recommendations was born after a round table discussion dealing with the 2004 Tsunami disaster in south east Asia during the 21st congress of the International Society for Forensic Genetics on the Azores, Portugal, in September 2005. The ensuing discussion between scientists and pathologists that had been involved in the International Center in Khao Lak, Thailand, revealed the need for the scientific community to be better prepared to answer the local authorities' questions by formulating generally acceptable scientific standards for the most efficient use of DNA-based victim identification methods. These recommendations, as well as the many cited references, are intended to provide guidance on establishing preparedness for the forensic genetics laboratory, on collecting and storing ante-mortem and post-mortem samples suitable for DNA analysis, on DNA extraction and genetic typing strategies, on data management, and on issues related to the biostatistical interpretation and reporting of results.

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.

Physical activity, television viewing and body mass index: a cross-sectional analysis from childhood to adulthood in the 1958 British cohort.

OBJECTIVE: To investigate relationships between frequency of physical activity or television viewing and body mass index (BMI) cross-sectionally at six ages from childhood to adulthood, to better understand longitudinal relationships. To investigate how the relationships vary with age and gender and whether any relationships are due to confounding factors. METHODS: The 1958 British birth cohort includes all births (approximately 17 000) in one week in March 1958. BMI and physical activity frequency were recorded at 11, 16, 23, 33 and 42 y and television viewing frequency at 11, 16 and 23 y. A total of 11 109 subjects provided BMI and activity data at 42 y. Relationships between BMI and (in)activity were investigated using linear regression. RESULTS: At ages 11, 33 and 42 y in both sexes and at 23 y in female subjects, those who were more active had lower BMIs, and the relationships strengthened with age. At 42 y, the most active had a lower mean BMI than the least active, by 0.83 kg/m2 in men, and 1.03 kg/m2 in women. BMI and activity were unrelated at 16 y in female subjects, and 23 y in male subjects. At 16 y in males, the most active males had a mean BMI 0.25 kg/m2 higher than the least active. At 11 y in female subjects and 23 y in both sexes, those who watched television most frequently had higher BMIs. BMI and television viewing were unrelated at 11 y in males and at 16 y in both sexes. Relationships between BMI and activity or television viewing were largely unexplained by potential confounding factors. CONCLUSIONS: The relationship between BMI and physical activity changes with age. In early adolescence and in adulthood, a higher activity level, or lower frequency of television viewing was associated with a lower BMI. In later adolescence (16 y), television viewing and activity were unrelated to BMI, except for an unexpected BMI-activity relationship in males. We suspect this relationship in males is primarily due to selection effects, whereby physically bigger boys, with a larger BMI, are more likely to take part in exercise activity, and possibly also to BMI being a less accurate predictor of fatness in adolescent boys.

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.

Naming the Dead - Confronting the Realities of Rapid Identification of Degraded Skeletal Remains.

The Armed Forces DNA Identification Laboratory (AFDIL) is one of the leading laboratories in the world for the processing of degraded skeletal remains. Extended efforts have been made to develop protocols and standards that will hold up to the intense scrutiny of both the scientific world and the U.S. legal system. Presented in this paper are the specifics of the in-house systems and procedures that have allowed AFDIL to streamline the processing of degraded skeletal remains and family references for mitochondrial DNA (mtDNA) analysis. These include the development of our in-house bioinformatics systems by which every package and sample that passes through the laboratory is tracked; protocols designed specifically for both questioned and reference samples; and the difficulties inherent in this type of organization. Two case studies presented involve one of ancient remains and one on the recent event of September 11, 2001. Finally, future directions available to both AFDIL and the DNA analysis community as a whole are discussed.

Infant feeding and obesity through the lifecourse.

In the 1958 British birth cohort (n = 12,857 at age 7), breast feeding and BMI were unrelated in childhood. Breast feeding was protective against increased BMI at ages 16 and 33 years in females, and at 33 years in males, but this effect was markedly reduced and no longer significant after adjustment for confounding factors.

A new database of mitochondrial DNA hypervariable regions I and II sequences from 162 Japanese individuals.

A database of mitochondrial DNA (mtDNA) hypervariable region 1 (HV1) and region 2 (HV2) sequences of the mtDNA control region was established from 162 unrelated Japanese individuals. The random match probability and the genetic diversity for this database were 0.96% and 0.997, respectively. Length heteroplasmy in the C-stretch regions located around position 16189 in HVI and 310 in HV2 was observed in 37% and 38% of the samples, respectively. A strategy using internal sequencing primers was devised to obtain confirmed sequences in these length heteroplasmic individuals. This database, combined with other mtDNA sequence databases from the Japanese population, will permit the significance of mtDNA match results to be properly reported in mtDNA typing casework in Japan.

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.

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.

Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome.

The principal limitation in forensic mitochondrial DNA (mtDNA) testing is the low power of discrimination that is obtained when common "mtDNA types" are involved in a case. Currently, an "mtDNA type" refers to the sequence within hypervariable regions I and II (HV1/HV2) of the control region, approximately 610 bp. In Caucasians, the most common HV1/HV2 type is found in approximately 7% of the population and there are 12 additional types found at greater than approximately 0.5% (ignoring HV2 C-stretch polymorphism). We are performing large scale sequencing of the entire mtDNA genome (mtGenome), approximately 16,569 bp, of individuals who have common HV1/HV2 types. Of 31 individuals with the most common HV1/HV2 type, only 3 still match after mtGenome sequencing. Similar high discrimination is seen in other common HV1/HV2 types. The sites that discriminate the various common HV1/HV2 types are generally not those that are known to vary widely in more diverse population samples. This indicates that complete mtGenome sequencing of selected HV1/HV2 types may stand as the best way for identifying maximally useful single nucleotide polymorphism sites outside of the control region. Our strategy for identifying SNP sites is useful in resolving U.S. Caucasian, Hispanic, and African American mtDNAs is discussed. We also discuss the development of homogeneous fluorogenic polymerase chain reaction assays that target phenotypically neutral sites for practical use in casework.

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.

Point mutations of the mtDNA control region in normal and neurodegenerative human brains.

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.

Mitochondrial enteropathy: the primary pathology may not be within the gastrointestinal tract.

BACKGROUND: Mitochondrial DNA (mtDNA) defects are an important cause of disease. Although gastrointestinal symptoms are common in these patients, their pathogenesis remains uncertain. AIM: To investigate the role of the mtDNA defect in the production of gastrointestinal dysfunction. PATIENT: A 20 year old woman who presented at 15 years of age with recurrent vomiting and pseudo-obstruction, who did not respond to conservative management and ultimately had subtotal gastrectomy and Roux-en-y reconstruction. She subsequently presented with status epilepticus and was found to have a mitochondrial respiratory chain disorder due to a pathogenic mtDNA point mutation (A3243G). METHODS: Resected bowel was studied using light and electron microscopy and mtDNA analysed from both mucosal and muscular layers using polymerase chain reaction generated RFLP analysis. RESULTS: Histological and electron microscopic studies revealed no morphological abnormalities in the resected stomach, and molecular genetic analysis failed to identify the genetic defect in either the mucosal or muscle layers. CONCLUSION: This study suggests that in some individuals with gastrointestinal symptoms associated with established mitochondrial DNA disease, the primary pathology of the mitochondrial enteropathy lies outside the gastrointestinal tract.