Showing papers in "Investigative Genetics in 2014"

DNA fingerprinting in botany: past, present, future.

Abstract: Almost three decades ago Alec Jeffreys published his seminal Nature papers on the use of minisatellite probes for DNA fingerprinting of humans (Jeffreys and colleagues Nature 1985, 314:67–73 and Nature 1985, 316:76–79). The new technology was soon adopted for many other organisms including plants, and when Hilde Nybom, Kurt Weising and Alec Jeffreys first met at the very First International Conference on DNA Fingerprinting in Berne, Switzerland, in 1990, everybody was enthusiastic about the novel method that allowed us for the first time to discriminate between humans, animals, plants and fungi on the individual level using DNA markers. A newsletter coined “Fingerprint News” was launched, T-shirts were sold, and the proceedings of the Berne conference filled a first book on “DNA fingerprinting: approaches and applications”. Four more conferences were about to follow, one on each continent, and Alec Jeffreys of course was invited to all of them. Since these early days, methodologies have undergone a rapid evolution and diversification. A multitude of techniques have been developed, optimized, and eventually abandoned when novel and more efficient and/or more reliable methods appeared. Despite some overlap between the lifetimes of the different technologies, three phases can be defined that coincide with major technological advances. Whereas the first phase of DNA fingerprinting (“the past”) was dominated by restriction fragment analysis in conjunction with Southern blot hybridization, the advent of the PCR in the late 1980s gave way to the development of PCR-based single- or multi-locus profiling techniques in the second phase. Given that many routine applications of plant DNA fingerprinting still rely on PCR-based markers, we here refer to these methods as “DNA fingerprinting in the present”, and include numerous examples in the present review. The beginning of the third phase actually dates back to 2005, when several novel, highly parallel DNA sequencing strategies were developed that increased the throughput over current Sanger sequencing technology 1000-fold and more. High-speed DNA sequencing was soon also exploited for DNA fingerprinting in plants, either in terms of facilitated marker development, or directly in the sense of “genotyping-by-sequencing”. Whereas these novel approaches are applied at an ever increasing rate also in non-model species, they are still far from routine, and we therefore treat them here as “DNA fingerprinting in the future”.

Human paternal and maternal demographic histories: insights from high-resolution Y chromosome and mtDNA sequences

Abstract: Background: Comparisons of maternally-inherited mitochondrial DNA (mtDNA) and paternally-inherited non-recombining Y chromosome (NRY) variation have provided important insights into the impact of sex-biased processes (such as migration, residence pattern, and so on) on human genetic variation. However, such comparisons have been limited by the different molecular methods typically used to assay mtDNA and NRY variation (for example, sequencing hypervariable segments of the control region for mtDNA vs. genotyping SNPs and/or STR loci for the NRY). Here, we report a simple capture array method to enrich Illumina sequencing libraries for approximately 500 kb of NRY sequence, which we use to generate NRY sequences from 623 males from 51 populations in the CEPH Human Genome Diversity Panel (HGDP). We also obtained complete mtDNA genome sequences from the same individuals, allowing us to compare maternal and paternal histories free of any ascertainment bias. Results: We identified 2,228 SNPs in the NRY sequences and 2,163 SNPs in the mtDNA sequences. Our results confirm the controversial assertion that genetic differences between human populations on a global scale are bigger for the NRY than for mtDNA, although the differences are not as large as previously suggested. More importantly, we find substantial regional variation in patterns of mtDNA versus NRY variation. Model-based simulations indicate very small ancestral effective population sizes (<100) for the out-of-Africa migration as well as for many human populations. We also find that the ratio of female effective population size to male effective population size (Nf/Nm) has been greater than one throughout the history of modern humans, and has recently increased due to faster growth in Nf than Nm. Conclusions: The NRY and mtDNA sequences provide new insights into the paternal and maternal histories of human populations, and the methods we introduce here should be widely applicable for further such studies.

Validation of high throughput sequencing and microbial forensics applications

Abstract: High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

Metagenomic analyses of bacteria on human hairs: a qualitative assessment for applications in forensic science

Abstract: Mammalian hairs are one of the most ubiquitous types of trace evidence collected in the course of forensic investigations. However, hairs that are naturally shed or that lack roots are problematic substrates for DNA profiling; these hair types often contain insufficient nuclear DNA to yield short tandem repeat (STR) profiles. Whilst there have been a number of initial investigations evaluating the value of metagenomics analyses for forensic applications (e.g. examination of computer keyboards), there have been no metagenomic evaluations of human hairs—a substrate commonly encountered during forensic practice. This present study attempts to address this forensic capability gap, by conducting a qualitative assessment into the applicability of metagenomic analyses of human scalp and pubic hair. Forty-two DNA extracts obtained from human scalp and pubic hairs generated a total of 79,766 reads, yielding 39,814 reads post control and abundance filtering. The results revealed the presence of unique combinations of microbial taxa that can enable discrimination between individuals and signature taxa indigenous to female pubic hairs. Microbial data from a single co-habiting couple added an extra dimension to the study by suggesting that metagenomic analyses might be of evidentiary value in sexual assault cases when other associative evidence is not present. Of all the data generated in this study, the next-generation sequencing (NGS) data generated from pubic hair held the most potential for forensic applications. Metagenomic analyses of human hairs may provide independent data to augment other forensic results and possibly provide association between victims of sexual assault and offender when other associative evidence is absent. Based on results garnered in the present study, we believe that with further development, bacterial profiling of hair will become a valuable addition to the forensic toolkit.

DNA fingerprinting in zoology: past, present, future

Abstract: In 1962, Thomas Kuhn famously argued that the progress of scientific knowledge results from periodic ‘paradigm shifts’ during a period of crisis in which new ideas dramatically change the status quo Although this is generally true, Alec Jeffreys’ identification of hypervariable repeat motifs in the human beta-globin gene, and the subsequent development of a technology known now as ‘DNA fingerprinting’, also resulted in a dramatic shift in the life sciences, particularly in ecology, evolutionary biology, and forensics The variation Jeffreys recognized has been used to identify individuals from tissue samples of not just humans, but also of many animal species In addition, the technology has been used to determine the sex of individuals, as well as paternity/maternity and close kinship We review a broad range of such studies involving a wide diversity of animal species For individual researchers, Jeffreys’ invention resulted in many ecologists and evolutionary biologists being given the opportunity to develop skills in molecular biology to augment their whole organism focus Few developments in science, even among the subsequent genome discoveries of the 21st century, have the same wide-reaching significance Even the later development of PCR-based genotyping of individuals using microsatellite repeats sequences, and their use in determining multiple paternity, is conceptually rooted in Alec Jeffreys’ pioneering work

Pet fur or fake fur? A forensic approach

Abstract: In forensic science there are many types of crime that involve animals. Therefore, the identification of the species has become an essential investigative tool. The exhibits obtained from such offences are very often a challenge for forensic experts. Indeed, most biological materials are traces, hair or tanned fur. With hair samples, a common forensic approach should proceed from morphological and structural microscopic examination to DNA analysis. However, the microscopy of hair requires a lot of experience and a suitable comparative database to be able to recognize with a high degree of accuracy that a sample comes from a particular species and then to determine whether it is a protected one. DNA analysis offers the best opportunity to answer the question, ‘What species is this?’ In our work, we analyzed different samples of fur coming from China used to make hats and collars. Initially, the samples were examined under a microscope, then the mitochondrial DNA was tested for species identification. For this purpose, the genetic markers used were the 12S and 16S ribosomal RNA, while the hypervariable segment I of the control region was analyzed afterwards, to determine whether samples belonged to the same individual. Microscopic examination showed that the fibres were of animal origin, although it was difficult to determine with a high degree of confidence which species they belonged to and if they came from a protected species. Therefore, DNA analysis was essential to try to clarify the species of these fur samples. Macroscopic and microscopic analysis confirmed the hypothesis regarding the analyzed hair belonging to real animals, although it failed to prove with any kind of certainty which actual family it came from, therefore, the species remains unknown. Sequence data analysis and comparisons with the samples available in GenBank showed that the hair, in most cases, belonged to the Canidae family, and in one case only to Felidae.

Different waves and directions of Neolithic migrations in the Armenian Highland

Abstract: Background: The peopling of Europe and the nature of the Neolithic agricultural migration as a primary issue in the modern human colonization of the globe is still widely debated. At present, much uncertainty is associated with the reconstruction of the routes of migration for the first farmers from the Near East. In this context, hospitable climatic conditions and the key geographic position of the Armenian Highland suggest that it may have served as a conduit for several waves of expansion of the first agriculturalists from the Near East to Europe and the North Caucasus. Results: Here, we assess Y-chromosomal distribution in six geographically distinct populations of Armenians that roughly represent the extent of historical Armenia. Using the general haplogroup structure and the specific lineages representing putative genetic markers of the Neolithic Revolution, haplogroups R1b1a2, J2, and G, we identify distinct patterns of genetic affinity between the populations of the Armenian Highland and the neighboring ones north and west from this area. Conclusions: Based on the results obtained, we suggest a new insight on the different routes and waves of Neolithic expansion of the first farmers through the Armenian Highland. We detected at least two principle migratory directions: (1) westward alongside the coastline of the Mediterranean Sea and (2) northward to the North Caucasus.

Evaluating the Y chromosomal timescale in human demographic and lineage dating

Abstract: Y chromosome is a superb tool for inferring human evolution and recent demographic history from a paternal perspective. However, Y chromosomal substitution rates obtained using different modes of calibration vary considerably, and have produced disparate reconstructions of human history. Here, we discuss how substitution rate and date estimates are affected by the choice of different calibration points. We argue that most Y chromosomal substitution rates calculated to date have shortcomings, including a reliance on the ambiguous human-chimpanzee divergence time, insufficient sampling of deep-rooting pedigrees, and using inappropriate founding migrations, although the rates obtained from a single pedigree or calibrated with the peopling of the Americas seem plausible. We highlight the need for using more deep-rooting pedigrees and ancient genomes with reliable dates to improve the rate estimation.

The music of the genes.

Abstract: The most famous of musical dynasties is that of the Bachs. In Leipzig for a conference, I visited the Bach Museum opposite the Thomaskirche where the greatest Bach of all worked as Kapellmeister for 27 years, and is now buried. There, the family’s many names are laid out on a wall, from Johann Sebastian’s great-great-grandfather Veit, a miller and player of the cittern, born around 1550, via J.S. himself, and on to his grandson Wilhelm Friedrich Ernst. One by one, the names light up, and as each Bach is illuminated his music fills the air. The occurrence of so many great musicians in seven generations of one family might suggest musical ability in the genes. Francis Galton, in his Hereditary Genius[1], studied musicians alongside judges, statesmen, scientists, commanders, authors, poets and artists, as one of his classes of ‘eminent men’ (note, no women). As well as these brainy and artistic types, he cast wrestlers and rowers into the mix, too. Galton investigated the pattern of inheritance of exceptional ability within families, showing that the eminence of relatives of an eminent man declined with the degree of relatedness, and taking this as evidence of heritability. There are two difficulties with Galton’s approach to musical ability - the phenotype (general eminence) is complex and vaguely defined, and the influence of the environment is not accounted for. In the Bach family, for example, musical training at a young age was the norm, and clearly led to a good living, so alternative careers were not necessarily high on the agenda. Modern geneticists have also been interested in questions of musical inheritance, and have mostly focused on a simpler phenotype, absolute pitch (AP) - the ability to instantly recognize and correctly name the pitch of any of about seventy different notes in the middle of the auditory range. The phenotype is generally rare, and found in only approximately 1% to 2% of music students and musical professionals, so is far from being a proxy for musicianship. Clearly, AP requires some prior exposure to notes and their names, and an ability to fix and recall these associations. Oliver Sacks, in a chapter of his book Musicophilia[2] entitled ‘Papa Blows his Nose in G’, points out that the real wonder of AP is that for people who possess it, each tone has its own unique characteristic (for example, F-sharp-ness), which to them is often analogous to colour. Indeed, some composers, including Scriabin and Messiaen, explicitly linked notes and colours, possible examples of synesthesia, in which one kind of sensory stimulus evokes another. AP is a complex trait, involving both genetic and environmental factors: musical training during early development contributes to its acquisition, and it is more frequent in Asians than Europeans, a finding that some have attributed to early exposure to languages in which tone is particularly important. Genome-wide linkage analysis [3] in families showing both AP and synesthesia highlighted a shared region on chromosome 6, supporting the relationship between these phenotypes. Subsequent sequencing of candidate genes revealed otherwise rare amino-acid-changing variants in affected members of four families in EPHA7, a gene encoding a member of a family of cell-surface-bound receptor tyrosine kinases that may play an important role in neural differentiation and connectivity in the developing brain. One particular genetically defined group appears to exhibit a high natural ability for music. These are people with Williams-Beuren syndrome (WBS [4]). Carrying a 1.5- to 1.8-Mb deletion on the long arm of chromosome 7 that removes 26 to 28 genes, subjects suffer from a constellation of abnormalities, with a mean IQ of 55, and severe difficulties in spatial tasks such as solving jigsaw puzzles. However, there are compensating strengths in musical ability, with many showing skill in singing or playing instruments. There is disagreement about the incidence of absolute pitch in WBS: in a study by neuroscientist Howard Lenhoff [5], five young WBS subjects all had AP, but a more recent larger study has failed to find a convincing association [6]. Lenhoff’s own daughter, Gloria, has WBS and is a musical savant, singing almost 2,000 songs in many languages from memory, and performing with renowned orchestras. As well as phenotypes that enhance musical abilities, there are some that do the opposite. Congenital amusia (or tone deafness) is the failure to acquire the perception and recognition of music, despite having normal hearing, language, and intelligence. One patient seen by Sacks [2] could not recognize ‘Happy Birthday to You’, even though, as a school-teacher, she was obliged to play a recording of it at least 30 times a year. To her, the sound of music was ‘like pots and pans being thrown on the floor’. Family studies [7] show a genetic component, since 39% of first-degree relatives of subjects have amusia, compared to a population frequency of about 4%, but no gene hunts have yet been undertaken. Setting aside these unfortunate tone-deaf cases, music is a cultural universal, and findings of prehistoric bone flutes in southern Germany show that humans have been making sophisticated music for at least 42,000 years [8,9]. But what is it all for? Darwin believed that music evolved through sexual selection; by analogy with birds and their songs, the creation and appreciation of music was part of the complex process of attracting the opposite sex. ‘Music has a wonderful power … of recalling in a vague and indefinite manner, those strong emotions which were felt during long-past ages, when, as is probable, our early progenitors courted each other by the aid of vocal tones’ [10]. In Darwin’s opinion, language came after music. Herbert Spencer disagreed, claiming that music arose naturally from the cadences used in emotional speech. In modern times, Steven Pinker [11] has sided with Spencer, arguing that music is simply a useless byproduct of language (‘auditory cheesecake’). Steven Mithen [12], however, believes that music is too different from language to be a byproduct, and that its emotional power indicates a long and important evolutionary history. A consensus seems unlikely to emerge any time soon. The Leipzig conference in which I participated was concerned with language, and I learned that linguists disagree violently about some fundamental aspects of what modern languages can tell us about populations in the distant past. Funnily enough, music might help here. Analysing characters such as rhythm, pitch and dynamics in traditional vocal songs from nine indigenous populations of Thailand [13] allows a music-based distance measure to be calculated between them. Comparison of this to analogous distances based on languages and genetics (using maternally inherited mitochondrial DNA) shows that music is a better fit to genetics than language is. Likewise, a geographically broader study of Eurasian populations [14] shows that high musical similarity predicts high genetic similarity, and that the relationship is stronger for maternal than for paternal lineages. Perhaps, then, musicology could replace historical linguistics as a tool to find cultural connections that reflect deep shared ancestry - the language changes, but (like the legacy of Bach) the melody lingers on. Mothers sing to their babies, after all.

Editors' Pick: Contamination has always been the issue!

Abstract: In the middle of the 1980?s, I heard a highly profiled professor making a comment after a lecture about a brand new technique, the polymerase chain reaction (PCR). His comment was full of doubt about this novel technology, and the message was something like: ? it [PCR] can never become a widely used diagnostic tool due to the unavoidable contamination? . However, the PCR revolutionized life sciences from medicine to conservation genetics, the inventor was awarded with the Noble Prize, and many of us made careers using the very technique. Scientists, clinical diagnostics and forensic laboratories and others using PCR quickly learned to deal with contamination and build mechanisms to monitor for it. The contamination was there, but, it could be managed with the right laboratory environment, sample flow, and careful experimental design including proper sample handling and a set of controls.

Editors’ pick: Molecular genetic investigative leads to differentiate monozygotic twins

Abstract: Monozygotic twins arise from a single fertilized egg and, thus, for practical purposes are genetically identical. Therefore, in cases where one of the identical twins is associated with forensic biological evidence through DNA typing, the other twin cannot be excluded either. This conundrum of the ultimate “my brother did it” scenario has at times placed the legal community in a difficult position for solving the source of biological evidence in such cases. Indeed, identical twins are expected to have the same, for example, short tandem repeat loci, profile, no matter how highly individualizing the current forensic DNA diagnostic system is. Although the dogma is that monozygotic twins are genetically identical, it has been well-known for many years that there are genetic differences between such twins due to the accumulation of somatic mutations [1-8]. Undeniably, a few somatic DNA differences between twins are the norm, not the exception. The earlier in the embryonic development of an individual that a somatic mutation occurs, the more prevalent it will be among the tissues of that particular individual. Somatic mutations occur randomly, so it is extremely unlikely, if not impossible, that identical twins would share the same somatic mutations. These mutations, thus, potentially can serve as genetic markers to distinguish identical twins and thus resolve the dilemma of which one is likely to be the source of the biological evidence, if such a circumstance arises. Until recently, finding these few somatic mutations was not feasible routinely and very demanding technically. Over the last decade the advent of massively parallel sequencing (MPS) makes possible grand scale sequencing in which whole human genomes can be sequenced in a relatively rapid time frame and has ushered in a genetics revolution. Even forensic DNA analyses are being driven in this new direction with the promising capabilities provided by MPS. While much effort is being dedicated to the application of MPS in forensic DNA analysis, it is still considered by most in the development and validation testing phases and is not being used in casework analyses. However, recently Weber-Lehmann et al. published “Finding the needle in the haystack: Differentiating ’identical’ twins in paternity testing and forensics by ultra-deep next generation sequencing” [9] in which they exploit the power of MPS and show that the technology may assist even today in casework where pinpointing genetic differences between twins is desired. The authors sequenced reference DNA with the Illumina HiSeq 2000 platform using chemistry v3.0 and 2 × 100 bp paired-end read mode from sperm samples of two identical twins (monozygosity confirmed using PowerPlex 21 PCR Kit; Promega, Madison, WI) and from a blood sample of the child of one twin. With such high throughput of approximately 600 gigabases, whole genome sequence data were generated resulting in about 90X coverage on average per twin. The child’s genome was sequenced on average to 56X. After bioinformatics processing to remove noisy data, five single nucleotide polymorphisms (SNPs) were observed in the father and child pair that were not detected in the other twin (i.e., the child’s uncle). Once these SNPs were identified, directed PCR assays were readily developed and standard Sanger sequencing verified the SNPs. Thus, the validity of the SNPs was bolstered by orthogonal testing. The mother of the child was tested for these SNPs using the directed assay, and she was excluded as the source of the child’s SNPs. This study demonstrated that it is possible and feasible to identify somatic differences between twins. Four of the five SNPs that the twin father carried were in both his buccal and sperm DNA (ectodermal tissues). One of the SNPs was detected in both sperm and blood DNA. Lastly, one SNP was observed solely in sperm DNA. These findings support the hypothesis that the earlier in development of an embryo, rare SNPs may arise and be established not only in somatic tissues, but importantly in the germline. The implication of these findings for forensic investigations involving twins as the potential source is remarkable. Suppose there is a sexual assault case, semen is discovered, typed by standard DNA markers, and a “match” is observed between the evidence and an implicated twin. The primary question of differentiation of the twin who is the donor of the semen evidence can be resolved. Ideally, sequencing semen reference samples would be better for making comparisons by MPS; yet, obtaining such reference samples is unlikely. However, as long as the relevant somatic mutations that will be identified in a buccal or blood reference sample by MPS also reside in sperm DNA, the ability to differentiate the twins is highly probable. Using this investigative approach, the risk of false identification is infinitesimally low with the likely outcomes being differentiation or inconclusive. Indeed, only one or two SNPs would be needed to distinguish between the twins and there does not appear to be a need for additional statistical analyses of the outcome. Traditional STR typing already would have reduced the possible source of the biological evidence to be most likely that of one of the twins as opposed to other potential contributors. It is important to note that MPS is not being used to analyze the semen evidence with this approach. MPS is being used solely on reference samples to develop the investigative lead for identifying target SNPs. Once genetic variants are identified, typing of the evidence and the reference samples of the identical twins would be carried out using similar methodology which has been generally accepted for sequencing of mitochondrial DNA for almost two decades, i.e., targeted PCR and Sanger sequencing. Thus, the methodology that would be used for forensic analysis of the semen evidence and reference samples would be well-established and generally-accepted. Typing the evidence, and again the references samples, to detect those genetic variants using standard sequencing technology is an additional feature for supporting the reliability of the resultant SNPs. Lastly, using this approach, there is far less consumption of precious evidence than would be if MPS were used directly on the sperm DNA to identify SNPs. So, there will be, in at least some cases, sufficient DNA for retesting, if it were desired. Weber-Lehmann et al. [9] have shown that there currently are other practical uses of MPS for forensic investigations. The leads are those precious few markers that can differentiate monozygotic twins. While the authors use the catchy phrase “finding the needle in the haystack,” they have shown in actuality that it is not so difficult. But for now it likely will not be a cheap investigation as the genomes of each twin pair will have to be sequenced to identify the novel somatic SNPs that would differentiate them.

Evaluating the impacts of stressors of Pseudomonas syringae pathovar tomato on the effectiveness of multi-locus variable number tandem repeat analysis and multi-locus sequence typing in microbial forensic investigations

Abstract: Crops in the USA are vulnerable to natural and criminal threats because of their widespread cultivation and lack of surveillance, and because of implementation of growing practices such as monoculture. To prepare for investigation and attribution of such events, forensic assays, including determination of molecular profiles, are being adapted for use with plant pathogens. The use of multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST) in investigations involving plant pathogens may be problematic because the long lag periods between pathogen introduction and discovery of associated disease may provide enough time for evolution to occur in the regions of the genome employed in each assay. Thus, more information on the stability of the loci employed in these methods is needed. The MLVA fingerprints and MLST profiles were consistent throughout the experiment, indicating that, using a specific set of primers and conditions, MLVA and MLST typing systems reliably identify P.s. tomato DC3000. This information is essential to forensic investigators in interpreting comparisons between MLVA and MLST typing profiles observed in P.s. tomato isolates. Our results indicate that MLVA and MLST typing systems, utilizing the specified primers and conditions, could be employed successfully in forensics investigations involving P.s. tomato. Similar experiments should be conducted in the field and with other high-consequence plant pathogens to ensure that the assays are reliable for pathogens infecting plants in their natural environment and for organisms that may display faster rates of mutation.

Molecular clocks ticking in the court room

Abstract: Molecular phylogenies have been used in forensic molecular epidemiological investigations of transmission events of human immunodeficiency virus (HIV) in dental and medical practices and in rape cases [1-7]. Some of these investigations have resulted in criminal charges [4-7] and typically have involved only a few transmission events in a short period of time. Gonzales-Candelas et al. report in BMC Biology[8] a case of an anesthetist from Valencia, Spain, who was convicted of professional malpractice by infecting over 270 of his patients with hepatitis C virus (HCV). This case differs from most other viral infection cases in that the origin of transmissions date back approximately 25 years and it involved a large number of patients. Greater than 300 candidates, who had undergone a minor surgery in one of two hospitals, were tested, and for the first time a molecular clock was used to estimate the date of various transmission events. HCV is a single-stranded RNA virus and member of Flaviviridae family. It is transmitted by blood-to-blood contact, e.g. in intravenous drug use or unprotected sexual intercourse, but also during medical procedures under poor-quality conditions (e.g. non-screened blood transfusion and re-use of syringes or needles). Up to 20% of infected individuals develop severe medical complications, including liver cancer, cirrhosis with a consequent liver failure. An HCV infection is extremely devastating to an infected individual and an epidemic is costly for the society. As the first cases were detected in 1998, and an epidemiological investigation that there was a large outbreak with possibly hundreds of patients infected, public health officials suspected that an anesthetist, working in a public and private hospital, was the likely source of the HCV transmissions. The epidemiologists investigated data of over 66,000 individuals and common risk factors for infection in surgery, e.g., surgeon, surgery room, type of surgery, anesthesiologist, and type of anesthesia. The only significant factor (adjusted Odds Ratio 28.5, 95% CI 9.83 to 82.59) was this anesthetist. This observation was supported by the fact that of the initial 197 cases considered in the outbreak, 184 had been anesthetized by the suspected individual. Since the suspected epidemic involved an exceedingly large number of patients and the overall period or transmission events were unknown, the authorities, including the judge, tasked experts in evolutionary biology and viral epidemiology to address the following questions: 1) was the suspect the source responsible for the outbreak?; 2) Could they ascertain which patients had been infected from a common source and thus could be included in the outbreak?; 3) Alternatively, which patients could have been infected from other sources?; 4) Could they exclude these alternative sources or the existence of different but simultaneous outbreaks?; 5) Could they determine the duration of the outbreak?; 6) Could they date the time of infection for each patient in the outbreak?; and 7) Could they determine the date of infection of the anesthetist? Gonzales-Candelas et al. [8] tackled the questions by sequencing 229-nucleotides (nts) of a conservative non-structural NS5B gene by reverse transcription of RNA to DNA, followed by Sanger sequencing of the DNA in all patients, the anesthetist, and a number of control samples. The difficulty with genetic analysis of HCV is, like HIV, it is a very fast-evolving virus, and thus, even individuals harboring the virus with the same origin will have differences in their viral RNA sequences. However, those sequences from the same source are likely to be more similar than those of epidemiologically unassociated patients. Therefore, phylogenetic approaches could prove informative, as was practiced in some previous cases analyzing HIV sequences [1-6]. Gonzales-Candelas et al. [8] typed patients positive for the 320 HCV-1a and 290 HCV-1b strains and 44 HCV-1a positive patients serving as geographically local controls. The HCV from the anesthetist was genotypically consistent with HCV-1a, and a dual (HCV-1A and -1b) origin was excluded. The authors then analyzed only the HCV-1a positive patients, and compared them with the controls. The sequence differences along the targeted 229 nts of the NS5B gene among the patients vs among the controls compared to the anesthetist were clear: there were 0–19 differences among the patients of which half had exactly the same sequence as the anesthetist and three out of four had two or less differences. The challenge of determining the direction and timing of the infection still remained, since phylogenetic trees reconstructed from the relatively short and slowly evolving NS5B gene did not have sufficient resolving power to separate the branches of the patients and local controls. Analysis then was performed on a faster evolving 406-nt region in the structural envelope coding E1-E2 region. The samples from the anesthetist were taken on 12th Feb 1998, and no further sampling was possible under Spanish law. Altogether over 4000 cloned sequences were used for the phylogenetic analysis, and 134 of them represented the anesthetist with 28 different haplotypes, which clustered in two groups. Neighbor-joining (NJ) and maximum-likelihood (ML) based phylogenetic trees showed highly supported internal branching (100% and 96% bootstraps, respectively). These trees were used to define which patients were included in the outbreak. This analysis divided the group into 274 patients considered associated with the outbreak (victims), 47 patients initially thought to be in the outbreak that were excluded, and 47 controls. In order to assess the support for this grouping the authors calculated the likelihood ratio (LR) for alternating hypotheses that the sequences of the patients group with the outbreak vs they group with the controls. The LRs tended to favor the hypothesis that they fall within the outbreak group and ranged from 1.051 to 6.622 × 1095. Similarly, for those excluded from the outbreak the LRs ranged from 1.330 to 4.408 × 1084 supporting the alternate hypothesis. Both of the figures provided a very strong support for the conclusions. For estimating the timing of the transmission events Gonzales-Candelas et al.[8] used a Bayesian method implemented in the program BEAST (Bayesian Evolutionary Analysis by Sampling Trees). For each of the 274 patient they used the E1-E2 region sequences to estimate the time to the most recent common ancestor (MRCA). For the analysis they incorporated the dates of the sampling, and the dates of the infection of 24 patients, who visited the anesthetist only once. Sequences from these 24 patients were used to calibrate molecular clock estimates for the MRCA. The authors concluded that the estimated transmission events of the patients was between Jan 1987 and April 1998, compared to the one obtained for the anesthetist August 1984 to October 1991). In the court these data were compared with those obtained from the non-molecular investigation (documents and testimonies), and in two out of three cases the molecular data estimates were congruent with other data. The analysis in this case was systematic and rigorous. However, it is important to note that the data presented by Gonzales-Candelas et al.[8], like in many other DNA investigations presented in the court, were not used to prove if the anesthetist was guilty, but rather in concert with other evidence. Even though only a piece of the puzzle, analyses, such as carried out by Gonzales-Candelas et al., will likely be used increasingly to assist epidemiologic and criminal (also civil) investigations.

Trouble at the races.

Abstract: Recently, I signed a letter to the New York Times. This is not something I do very often, but I felt strongly about the subject. And it wasn’t just me - so did the other 140 or so signatories, all of them population geneticists or human evolutionary biologists. What aroused the ire of this dusty community of academics? A pop science book by Nicholas Wade, A Troublesome Inheritance: Genes, Race and Human History[1]. The objection to the book included its use of the population genetics literature to buttress claims for the existence of five major human races. The influential paper [2] that examined the population structure of microsatellite variation in the 1000 or so Human Genome Diversity Panel DNA samples looms large in this argument. When the program STRUCTURE [3] was asked to find five clusters in the data, these corresponded to Africa, Europe plus the Middle East, South/Central Asia, East Asia, the Americas and Oceania. The genetic differentiation of non-admixed human populations results from a serial founder effect from the out of Africa migrations, followed by the influence of isolation by distance. So it’s not surprising that continental groups are differentiable from each other. It doesn’t make them races, but according to A Troublesome Inheritance it does. A later study based on genome-wide SNPs [4] is also cited. From this ‘it might be reasonable to elevate the Indian and Middle Eastern groups to the level of major races, making seven in all. But then many more subpopulations could be declared races, so to keep things simple, the five-race, continent-based scheme seems the most practical’. Exactly. The book also makes much of the role of natural selection in humans, which is repeatedly described as ‘recent, copious and regional’. Examples are given of the EPAS1 gene and adaptation to altitude in Tibetans [5], lactase persistence, as well as more ancient adaptations involving pigmentation. If these, then why not behaviour? And why not complex behaviour, too, such as industriousness, tribalism or trust? Of course, it’s a shame we don’t have a clue about their genetic bases, but no doubt this will come in time, and if there are genes involved, ‘recent, copious and regional’ selection has no doubt been at work on them. This leads to the notion that there has been genetic adaptation within races involving behavioural genes that govern such traits: Africans are tribal because their genes are adapted to tribalism, and this explains why foreign aid is often ineffective. Let’s face it, there’s a simplistic view here about the genetic architecture of complex traits. The lessons we’ve learned from genome-wide association studies and their difficulties in explaining heritability are not explored. A lot of other population genetics literature is cited along the way, but with cherry-picking – the caveats and alternative explanations are airbrushed out, so Wade can tell the story he wants to tell. This unwillingness to capture disagreement amongst geneticists contrasts with a description of the failure of economic historians to concur about the causes of the Industrial Revolution. Because they can’t agree, we need a new explanation – hey presto, genetic adaptation! It’s striking that, after its paean to the power of twenty-first century genomics, the book ends up essentially siding with Linnaeus and his 18th century sub-species classifications of Homo sapiens europeus, asiaticus and afer: • Europeans: white, sanguine, muscular, inventive, governed by laws; • Asians: yellow, melancholy, severe, haughty, stingy, governed by opinions; • Africans: black, cunning, phlegmatic, ruled by impulse. Wade castigates those who claim that race is just a social construct, stating that these deluded folks are leftists and Marxists, and that their views are politically motivated, and not scientific. And he is partly right. Some of the influential writers on this question have indeed been leftists and Marxists. It is difficult, if not impossible, for anyone to entirely divorce their background and political views from their professional life. If they are lucky enough to work on transcriptional control in yeast, for example, this is probably not much of an issue, but human genetics is a different game. By contrast, the heterodox crew who work on the biological reality of race are characterised as ‘courageous’ seekers after truth. But I’d hazard a guess they all have their own social and political baggage, too. At the beginning of A Troublesome Inheritance, we are told that racism and eugenics are Bad Things, that Hitler was a Bad Man, and that ‘opposition to racism is well entrenched’. Many people would beg to differ with that last statement. Racism is alive and kicking (sometimes literally), and this book provides succour for racists by using speculation to support the idea that differences between population groups are in their genes. It also avers that linking the ‘success of the West’ to ‘Caucasian’ genetics is not racist, because ‘there is no assertion of superiority’. But from reading this book that is certainly not the message that comes across. Social psychologists have noted a significant increase in a belief in essential differences between whites and blacks as a result of genetic ancestry testing [6]. A Troublesome Inheritance, whatever its author’s motives, will do the same in spades. Its enthusiastic proponents already include some high profile white supremacists and a former Grand Wizard of the Ku Klux Klan.

The sperm’s tale

Abstract: Copenhagen is as flat as the proverbial pan-cake. A good way to appreciate this is to visit the Rundetaarn, a 35 m tower whose summit is reached by a spiral ramp designed in the 17th century to accommodate a horse and cart carrying books to its elevated library, and in more recent times inviting the competitive attention of puffing middle-aged blokes, and occasional maniacs on unicycles. Squeeze past the other tourists in the ascent of the final staircase, and you are treated to a view stretching out in all directions. On our recent visit we were pleased to see in the distance the televisually iconic Oresund Bridge connecting Sweden and Denmark, but there was not so much as a hint of a hill. This flatness perhaps goes some way to explain the enormous number of Copenhagen’s pedal-driven vehicles (mostly of the two-wheeled variety), which present a serious hazard to the unwary pedestrian. While you are unlikely to be terrorised by the aggressive lycra-clad warriors typical of London, you are under constant threat from a motley crowd of bicycles, including many with huge low-slung boxes on the front occupied by the cyclist’s family or friends, or some irritatingly stylish furniture items. However, we were disappointed not to encounter the city’s notorious Sperm Bike. This eccentric conveyance (Figure 1) has a blue frame, and instead of a cargo-box a liquid nitrogen tank in the shape of a sperm-head, full of (you guessed it) frozen semen samples. The monster sperm’s tail waves jauntily behind, arcing over the rear wheel like an axonemic mud-guard. This Sperm Bike criss-crosses Copenhagen carrying samples from donors to recipients, and is symptomatic of Denmark’s relaxed attitude to assisted reproductive therapy (ART). Rather as Switzerland has become the UK citizen’s destination of choice for assisted suicide, at the opposite end of life Denmark thrives in ‘fertility tourism’. This is partly because of a 2005 change in UK law, which meant that donors were no longer anonymous. This led to a fall in donations from the reserved Brits, and a sperm shortage. In a newspaper article [1] entitled ‘The Father’s a Viking’, a mother-to-be describes her experience of travelling to Copenhagen, and parting with £460; she says she will call the resulting daughter Freya, and comments that her own family are from the north and west of the British Isles, and therefore have Scandinavian blood already. Pity the future population geneticist who tries to understand the history of the Viking migrations. Figure 1 Copenhagen’s Sperm Bike in action. As a man gets older, his semen volume and sperm count decline [2], though elderly men still father healthy children, as the lively 96-year-old Ramjit Raghav demonstrated in 2012. But a more interesting effect of older fathers is the increased risk of passing mutations to the next generation. Births of children with short-limbed dwarfism (achondroplasia) were shown by Lionel Penrose in the 1950s [3] to become more likely as paternal age increased, and the same was later shown to be true of a suite of other dominantly-inherited genetic diseases. Today, next-generation sequencing can be used to detect de novo base substitutions directly by analysing the genomes of parents and their offspring. This shows that the paternal mutation rate is 3.9 times the maternal rate [4], and demonstrates a linear increase of about two new mutations per year due to ageing dads, but not mums. The widely accepted explanation comes down to the difference between the ways in which eggs and sperm are made - while a woman’s eggs have all been produced by the time she is born, a man goes on producing sperm throughout his life from spermatogonial stem-cells (SSCs), which divide about 23 times a year. Cell division means an opportunity for mistakes in DNA replication, so the errors keep accumulating with age. This explanation is probably right for mutation in general, though for some particular functional mutations, including those that cause achondroplasia, an exponential increase with age points to the selection of ‘selfish’ mutations in the testis that lead to clonal expansion of subsets of SSCs [5]. Because of the age effect, registered sperm donors in the UK must be 41 years old or younger. Furthermore, a rational course of action for young men nervous about the mutational load in their children might be to freeze their sperm for use in later life. Not very romantic, though. Sperm donors are also limited in the number of donations they can make, though this varies considerably (from 1 to 25) in different countries. The arguments for the appropriate number are based on keeping the likelihood of half-sibling matings among the donor’s progeny the same as that in the general population [6]. However, such matings would only happen if donors were anonymous, and as open-identity of donors becomes the norm, such calculations should become unnecessary. Unhappy incidents have occurred like the case of the Dutch donor who fathered 18 children before discovering he had a late-onset autosomal dominant cerebellar ataxia [7], and these rarities tend to lead to reductions in the per-donor limit, which is unfortunate given the general shortage of sperm samples available for ART. One genetically influenced trait that sperm donors are unlikely to pass on is male infertility. However, even this trait is now potentially heritable, thanks to intracytoplasmic sperm injection (ICSI). Sperm, either isolated directly from the testis, or from poor-quality semen, are directly injected into the oocyte, thus bypassing the barrier of natural selection. The method has been in use since the early 1990s, and thousands of ICSI babies born. On the whole, the method seems safe, but genetic causes of male infertility, such as Y-chromosomal deletions, are passed on to sons [8], and they in turn will require ICSI if they wish to have children. Given its current status as a premier ART destination, it is somewhat ironic that Copenhagen is also the original source of apocalyptic rumours about an inexorable fall in sperm-counts through time. An influential paper in 1992 [9] reported a drop of approximately 50% in global measures of sperm-count (and also in semen volume) between 1940 and 1990. There has been much subsequent debate over whether this phenomenon is real, or an artefact of a lack of standardised methods for counting [10]. There has also been a search for a reason for the decline, with theories ranging from environmental chemicals mimicking oestrogen, to the wearing of tight underwear. The latter appears to be true [11] - so bikers (Sperm-Bikers included) beware!

Editors’ Pick: milk sugar, migration and pastoralism in Africa

Abstract: Lactose persistence (LP), the ability to digest milk sugar (lactose), is one of the best examples of selection-based evolutionary change in humans from milk-drinking cultures. LP has been documented in populations with northern European ancestry and those originating in Central Asia, Middle East, the Arabian Peninsula, and Africa. Based on molecular genetic studies, it has been estimated that the selective change in the ability to digest lactose beyond childhood occurred within approximately the past 5,000-10,000 years [1]. The estimation is consistent with an advantage to LP in dairy farming-based subsistence, which came to prominence in certain geographical areas around the same time. The important enzyme in the breakdown of milk sugar, lactase (or more specifically lactase-phlorizin hydrolase (LPH)), is coded by the LCT gene. The molecular genetic data reported in recent years show several interesting aspects of LP. The first genetic variation described for European LP was not in the LCT gene, but in the in the intron 13 of the MCM6 gene, residing ~14 kb upstream of MCM6 [2]. However, it soon was realized that the MCM6 intron 13 T-13910 allele, which explained most LP in Europe, was absent in Africa [3,4]. Instead, three other SNPs in the same intron, C-14010, G-13907 and G-13915, were associated with lactase persistence in Africa. Also, these SNPs originated on different haplotype backgrounds from the European T-13910 allele and from each other providing a major model of convergent evolution in LP due to strong selective pressure [3,5]. To date, several SNPs associated with LP are known. In Europe, in addition to MCM6 intron 13 T-13910, intron 9 G-22018 is significantly associated with the LP trait. In Africa and the Arabian Peninsula, four SNPs (those above and G-14009), which are all located within 100 bp of T-13910 have been shown to be associated with the LP predominantly in pastoralist populations. Those variants do not, however, account for all the phenotypic variance of LP in Africa, implying that additional genetic variation may play a role. A new study by Ranciaro et al. in American Journal of Human Genetics[6] sought out new genetic variants associated with LP in Africa. They studied 819 individuals from 63 African populations, and 154 individuals from non-African populations from Europe, the Middle East and Asia. The authors sequenced MCM6 gene introns 9 and 13 and ~2 kb of the LCT gene promoter region in these samples. Additionally, in an effort to reconstruct the origin and spread of LP-associated alleles in Africa, four microsatellites were genotyped in a ~198 kb region in a subset of 252 individuals. The participants’ LP status was tested using a standardized blood test that tracks lactose digestion. With the new larger data set, and sampling from outside Africa, the authors confirmed the earlier observed association between the LP trait in Africa and three variants in intron 13, and they also found two additional LP-associated SNPs, one in intron 13 (G-12962) and one in the LCT promoter region (T-956). Further functional studies are naturally needed to prove whether these new SNPs are causative or just merely in linkage disequilibrium with the earlier identified variants. Ranciaro et al. [6] then used allele frequency and long-range linkage disequilibrium based neutrality tests to detect any signatures of selection. They found evidence for recent positive selection in eastern African populations and the Fulani from central Africa. While looking for the new variants for LP within Africa, the study by Ranciaro et al. [6] also considered that if LP patterns coincide with the rise in pastoralism, the LP-related alleles found in present-day populations should reveal past migrations of those populations. In order to do that, they analyzed a subset of 252 study participants using four microsatellites and combined these with the SNPs to create haplotypes. The haplotype analysis has some interesting insights about the migration of pastoralism groups in Africa. For example, the haplotype analysis supported an eastern African origin (Kenya and Tanzania) of the C-14010 LP-associated mutation now found in southern Africa in Bantu-speaking Xhosa and the hunter-gatherer San population. Also, the presence of Middle Eastern origin G-13915 allele in North African populations suggests migration between these regions. Interestingly, the data are supported by known historical interactions between these populations. Some of the data in Ranciaro et al. [6] is in agreement with earlier findings of distinct genetic LP variants that have arisen in various parts of Africa through convergent evolution. For example, the G-13907 was almost exclusively restricted to northern Kenya, Ethiopia and northern Sudan, as reported earlier. Last but not least, the authors also detected the traditionally European LP haplotype background in some West-and North-Central African populations. But, even with these new data from Ranciaro et al. [6], the genetic variants found and analyzed in various populations to date, are far from fully explaining LP in Africa.

Editors' Pick: What a pain…, or not!

Abstract: How nice, I thought as a child, would it be to not feel physical pain. Later I realized that sense of pain may exist for a reason; it was suggested that physical pain has evolved as an alarm system to protect the body from serious damage. Much later I read about human disorders involving severe insensitivity to physical pain. Still, the idea of not feeling pain remains appealing (especially anticipating my coming years when some painful age-related health problems are likely to kick in). Previously, it was demonstrated that a loss-of-function mutation in the sodium ion channel gene SCN9A leads to the inability to experience pain [1]. In a more recent study, Leipold et al.[2] identified another sodium ion channel gene, SCN11A, which, when harboring a particular missense mutation, causes loss of pain perception. SCN11A encodes the voltage-gated sodium ion channel NAv1.9, which is highly expressed in nociceptors (i.e., neurons that transmit sensory information from the body periphery to the spinal cord). The authors started out with whole-exome sequencing of healthy German parents and their child diagnosed with the congenital inability to experience pain. A heterozygote non-synonymous mutation in SCN11A was identified as the only de novo event detected in this affected child. Aiming to confirm this finding in independent samples, the authors performed Sanger sequencing of SCN11A exons in 58 additional individuals diagnosed with early-onset severe sensory loss, most of them representing sporadic cases. One Swedish individual showed the very same heterozygous SCN11A mutation, and the clinical history of both individuals was also similar. From these findings, the authors hypothesized that the identified mutation that leads to a replacement of leucine with proline (p.Leu811Pro) at the distal end of one of the transmembrane segments in domain II of NAv1.9 is disease causing and leads to loss of pain perception. Seeking molecular proof, the authors introduced the orthologous alteration (Leu799Pro) into the mouse Scn11a gene, but found no obvious morphological changes in sensory axons or in nerve fibers in the skin of the viable heterozygote knock-in mice. However, 11% of the knock-in mice had severe lesions that were not observed in the wild-type mice. Since the lesions also appeared in knock-in mice housed solitarily, it was assumed that they were self-inflicted. Also other experimental evidence pointed towards reduced pain sensitivity in these knock-in mice. Because NAv1.9 had been suggested to influence the excitability of dorsal root ganglia (DRG) neurons (i.e., their property to react to stimulation), the authors then investigated the electrical potential of DRG neurons isolated from wild-type and mutant mice. They found that a significant fraction of the mutant channels were active under resting conditions, and that the duration of action potentials was significantly reduced in the mutant neurons compared with wild-type neurons. In knock-out mice, however, only a minor effect on action potentials was observed. Additional experiments showed that the excessive activity at resting conditions caused sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. To study the effect of the mutation further, the authors heterologously expressed human NAv1.9 and NAv1.9 Leu811Pro mutant channels in ND7/23 cells (i.e., a mouse neuroblastoma x rat neuron hybrid cell line). They found that the phenotype of human NAv1.9 Leu811Pro mutant channels resembled that of mouse NAv1.9 Leu799Pro mutant channels, except in terms of steady-state inactivation. From the weak influence of the mutation on inactivation in the human channel, they concluded a stronger gain-of-function effect in humans than in knock-in mice. Besides impressively demonstrating the power of whole-exome sequencing to find rare candidate disease causing mutations, this study provides a new gain-of-function view on the molecular basis of pain loss, which was previously linked to loss-of-function mutations such as in the NAv1.7 channel encoded by SCN9A[1]. Furthermore, this finding may open up new avenues for the development of new painkilling drugs [3].