Genetic reference materials and their application to haematology

Genetic investigations are becoming increasingly useful and widespread in many areas of human health. However, there is a worldwide lack of certified reference materials for use in genetic testing, meaning that tests are being run without well validated controls and new assays are more difficult to develop and validate. We have responded to this challenge by starting a programme of developing genetic reference materials (GRMs) for international accreditation and worldwide distribution. Our approach has been to make materials for disorders where testing is commonplace and genotyping errors have been demonstrated. To ensure a continuing supply of DNA, cell lines are established from consenting, phenotypically well-characterised patients and are then grown up in bulk for genomic DNA extraction to yield up to 100 milligrams of DNA. In most cases the DNA is then formulated, freeze-dried and sealed in glass ampoules to ensure greater stability over time and obviate the need for chilled transportation. In this paper we explore the options and routes available to the production of DNA reference materials and describe the establishment of the first internationally recognised reference materials for human genomic DNA, with particular reference to some genetic tests carried out frequently within haematological and cardiovascular laboratories.     

Are wild boars roaming Ireland once more?

Wild boars (Sus scrofa) have been increasingly sighted in the wild in Ireland during the last few years, likely due to illegal releases and/or escapees. The species has since been designated an invasive species in Ireland, which is seen as controversial by some because of uncertainties about the historic status of the species in Ireland. However, just as pertinent to the argument is the genetic purity of these individuals currently found in Ireland: are these pure wild boars? We carried out a genetic assessment of 15 wild boars shot in Ireland between 2009 and 2012 using 14 microsatellites and mitochondrial DNA (mtDNA). These were compared to European wild boar, domestic pig breeds and a hybrid population of ‘wild boar’ from England. Microsatellite analysis revealed that almost all the Irish individuals belonged to the ‘domestic pig’ genetic cluster, with only three individuals being classified as hybrids. All but two individuals carried Asian mtDNA haplotypes, indicating a domestic pig origin. It is clear from this study that the individuals currently found in Ireland are not pure wild boars and this result has to be factored into any management/eradication plans.     

Implications of the human genome initiative for the primary care physician

... Despite the need for physicians to be knowledgeable about and open to advances in genetic technology, little is known about the level of preparedness of primary care physicians to offer new genetic tests. Evidence suggests that several barriers exist to physicians adopting genetic tests. These include lack of knowledge, inability to interpret probabilistic information, low tolerance for uncertainty, negative attitudes about their responsibility for genetic counseling and testing, lack of confidence in their clinical skills, and unfamiliarity with ethical issues raised by testing. This paper will explore some of these barriers in further depth, discuss the ethical impact of physician unpreparedness on both patient care and the diffusion of genetic tests, and describe a study that is currently underway to investigate some of these issues.     

The diffusion of new genetic tests for predicting disease.

This paper examines the pathways by which new genetic tests will become available to the public. In view of the scarcity of genetic specialists, the pathway is likely to involve primary care physicians. Other pathways entail state-mandated testing, community-based programs, or testing by laboratories without much involvement of primary care physicians. When testing does become available the "destination" will be either family-centered testing or population-oriented screening. The deterrent to screening will not be the inability to detect disease-causing mutations but the costs and attitudes of providers and the public. When tests are provided primarily to provide information about risks to future children, some people will oppose screening on religious or moral grounds. When there are no inexpensive treatments, some will fear that insurance companies and employers will use tests to deny them health care coverage. Some may not want to know their risks for disorders about which little can be done. For common, multifactorial disorders, genetic tests will have low predictive value. Because of these problems, the decision to be tested, regardless of the destination, requires that "testees" be fully informed and consent to testing. When acceptance rates are low, screening is less likely to be cost-effective; family-centered testing becomes the default destination.     

Individual assignment using microsatellite DNA reveals unambiguous breed identification in the domestic dog

Modern individual clustering methods utilising hypervariable nuclear microsatellite DNA polymorphisms are being increasingly applied in the field of population genetics. This study explores the efficiency of the clustering methods in identifying the breeds of origin of 250 domestic dog (Canis familiaris) individuals based on 10 microsatellite loci. An allele sharing distance (DAS) matrix and the corresponding neighbour-joining tree of individuals revealed monophyletic assemblages that corresponded perfectly with the breeds of origin of the dogs. Individual assignment tests using a Bayesian statistical approach, an allele frequency based method, and a DCE genetic distance based method were all extremely powerful. Most strikingly, the Bayesian method provided 100% assignment success of individuals into their correct breeds of origin and 100% exclusion success of individuals from all alternate reference populations with a high level of statistical confidence (P < 0.0001). A Bayesian Markov Chain Monte Carlo clustering approach revealed clear distinction of individuals into groups according to their breeds of origin, with a near-zero level of 'genetic admixture' among breeds. The results demonstrate that an FST of 0.18, mean expected gene diversity of 0.6 across 10 loci, and approximately 50 individuals per reference population suffice to provide maximum individual assignment success in C. familiaris. This refutes the traditional view that DNA based dog breed identification is not feasible at the individual level of resolution.     

Phylogenetic distinctiveness of Middle Eastern and Southeast Asian village dog Y chromosomes illuminates dog origins

Modern genetic samples are commonly used to trace dog origins, which entails untested assumptions that village dogs reflect indigenous ancestry or that breed origins can be reliably traced to particular regions. We used high-resolution Y chromosome markers (SNP and STR) and mitochondrial DNA to analyze 495 village dogs/dingoes from the Middle East and Southeast Asia, along with 138 dogs from >35 modern breeds to 1) assess genetic divergence between Middle Eastern and Southeast Asian village dogs and their phylogenetic affinities to Australian dingoes and gray wolves (Canis lupus) and 2) compare the genetic affinities of modern breeds to regional indigenous village dog populations. The Y chromosome markers indicated that village dogs in the two regions corresponded to reciprocally monophyletic clades, reflecting several to many thousand years divergence, predating the Neolithic ages, and indicating long-indigenous roots to those regions. As expected, breeds of the Middle East and East Asia clustered within the respective regional village dog clade. Australian dingoes also clustered in the Southeast Asian clade. However, the European and American breeds clustered almost entirely within the Southeast Asian clade, even sharing many haplotypes, suggesting a substantial and recent influence of East Asian dogs in the creation of European breeds. Comparison to 818 published breed dog Y STR haplotypes confirmed this conclusion and indicated that some African breeds reflect another distinct patrilineal origin. The lower-resolution mtDNA marker consistently supported Y-chromosome results. Both marker types confirmed previous findings of higher genetic diversity in dogs from Southeast Asia than the Middle East. Our findings demonstrate the importance of village dogs as windows into the past and provide a reference against which ancient DNA can be used to further elucidate origins and spread of the domestic dog.     

Ontogeny of domestic dogs and the developmental foundations of carnivoran domestication

Whereas hundreds of breeds of domestic dogs are known, only several dozen domestic cat breeds are currently recognized, and the ferret is not classified into specific breeds. We studied pre- and postnatal patterns of development and growth in the domesticated forms of these three carnivoran species. We present the most comprehensive staging system for domestic dog embryos to date and define qualitative characters for phylogenetic comparisons. For postnatal development, we present analyses of new and literature measurements of cranial and limb proportions. We analyze changes in the progress of growth among different domestic dog and domestic cat breeds. All three domesticated forms drastically differ in the relative timing of prenatal development. This is correlated with ontogenetic plasticity at birth, which enables artificial selection to act. For postnatal development, we detected a greater shape variance in domestic dog ontogeny when compared to that of the domestic cat. We conclude that ontogenetic preconditions as well as body size constrain the species’ capability for artificial selection in domestic dogs and cats. However, we speculate that the human requirements for functional performance of their domesticates might render some developmental biases substantially. Although ferrets would be preferable for artificial selection given their plastic embryonic development, they have been of less interest for domestication due to their small body size - by which they were already well adapted for hunting in burrows - and due to the fact that other relevant tasks were already assumed by domestic cats and dogs since earlier phases of human cultural evolution.     

Dermanyssus gallinae in non-avian hosts: A case report in a dog and review of the literature

Non-avian attacks of the worldwide distributed mite Dermanyssus gallinae are occasionally reported. However, it is widely accepted that their occurrence is underestimated. The present study aims to describe the first Italian case of dermanyssosis in a dog, to molecularly characterize the mites collected from the patient and the animal enclosure, where poultry and dog were confined, and to review the current literature on the non-avian attacks by D. gallinae. The dog was successfully treated with an oral sarolaner-based product, followed by a spot-on formulation of imidacloprid and moxidectin. The infestation source was likely attributable to poultry and confirmed by molecular identification of D. gallinae sensu strictu.Ten articles on non-avian D. gallinae attacks in domestic animals and wildlife were retrieved, pointing out the need for more awareness amongst practitioners. The therapeutic effect of available antiparasitic drugs, currently used off-label, should also be better explored in non-avian hosts.     

PCR multiplexed microsatellite panels to expedite canine genetic disease linkage analysis

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25 centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.     

Genetic variation in Przewalski's horses, with special focus on the last wild caught mare, 231 Orlitza III

In our continuing efforts to document genetic diversity in Przewalski's horses and relatedness with domestic horses, we report genetic variation at 22 loci of blood group and protein polymorphisms and 29 loci of DNA (microsatellite) polymorphisms. The loci have been assigned by linkage or synteny mapping to 20 autosomes and the X chromosome of the domestic horse (plus four loci unassigned to a chromosome). With cumulative data from tests of 568 Przewalski's horses using blood, hair or tooth samples, no species-defining markers were identified, however a few markers were present in the wild species but not in domestic horses. Inheritance patterns and linkage relationships reported in domestic horses appeared to be conserved in Przewalski's horses. A derived type for the last wild caught mare 231 Orlitza III provided evidence for markers apparently not found in (or not currently available by descent from) the other species founders that were captured at the end of the nineteenth century. This information has been critical to the development of parentage analyses in the studbook population of Przewalski's horses at Askania Nova, at one time the largest herd of captive animals and the source of stock for reintroduction efforts. Some horses in the study showed genetic incompatibilities with their sire or dam, contradicting published studbook information. In many cases alternative parentage could be assigned from living animals. To assist in identification of correct parentage, DNA marker types for deceased horses were established from archived materials (teeth) or derived from offspring. Genetic markers were present in pedigreed animals whose origin could not be accounted for from founders. Genetic distance analysis of erythrocyte protein, electrophoretic and microsatellite markers in Przewlaski's horses and ten breeds of domestic horse place the Przewalski's horse as an outgroup to domestic horses, introgression events from domestic horses not withstanding.     

Ancient DNA Analysis Affirms the Canid from Altai as a Primitive Dog

The origin of domestic dogs remains controversial, with genetic data indicating a separation between modern dogs and wolves in the Late Pleistocene. However, only a few dog-like fossils are found prior to the Last Glacial Maximum, and it is widely accepted that the dog domestication predates the beginning of agriculture about 10,000 years ago. In order to evaluate the genetic relationship of one of the oldest dogs, we have isolated ancient DNA from the recently described putative 33,000-year old Pleistocene dog from Altai and analysed 413 nucleotides of the mitochondrial control region. Our analyses reveal that the unique haplotype of the Altai dog is more closely related to modern dogs and prehistoric New World canids than it is to contemporary wolves. Further genetic analyses of ancient canids may reveal a more exact date and centre of domestication.     

Attitudes on DNA ancestry tests

The DNA ancestry testing industry is more than a decade old, yet details about it remain a mystery: there remain no reliable, empirical data on the number, motivations, and attitudes of customers to date, the number of products available and their characteristics, or the industry customs and standard practices that have emerged in the absence of specific governmental regulations. Here, we provide preliminary data collected in 2009 through indirect and direct participant observation, namely blog post analysis, generalized survey analysis, and targeted survey analysis. The attitudes include the first available data on attitudes of those of individuals who have and have not had their own DNA ancestry tested as well as individuals who are members of DNA ancestry-related social networking groups. In a new and fluid landscape, the results highlight the need for empirical data to guide policy discussions and should be interpreted collectively as an invitation for additional investigation of (1) the opinions of individuals purchasing these tests, individuals obtaining these tests through research participation, and individuals not obtaining these tests; (2) the psychosocial and behavioral reactions of individuals obtaining their DNA ancestry information with attention given both to expectations prior to testing and the sociotechnical architecture of the test used; and (3) the applications of DNA ancestry information in varying contexts.     

Leader of the pack: gene mapping in dogs and other model organisms

The domestic dog offers a unique opportunity to explore the genetic basis of disease, morphology and behaviour. We share many diseases with our canine companions, including cancer, diabetes and epilepsy, making the dog an ideal model organism for comparative disease genetics. Using newly developed resources, whole-genome association in dog breeds is proving to be exceptionally powerful. Here, we review the different trait-mapping strategies, some key biological findings emerging from recent studies and the implications for human health. We also discuss the development of similar resources for other vertebrate organisms.     

Lack of interest by nonpregnant couples in population-based cystic fibrosis carrier screening.

We used signs and letters to offer free cystic fibrosis (CF) carrier screening to nonpregnant adults in stable relationships who visited numerous clinical and nonclinical sites in Nashville. A total of 179 individuals (<<1% of those eligible) elected to be tested. To understand this observation, we used questionnaires to assess individuals' attitudes about genetic testing in general and about CF carrier screening in particular (n=873). Participants expressed conflicting views about carrier screening. More than 90% of people thought that genetic testing should at least be available. Most respondents said that the views of their partners and physicians were important in their decision making, and most believed that these others favored genetic testing. Yet, more than two-thirds indicated that such factors as insurability, being "at risk," what they would need to learn, abortion, and religious beliefs were important in their decision making, opinions that mitigated against genetic testing. In particular, one-third feared that carriers would lose their health insurance, one-quarter said that they would have been more interested had they been able to provide DNA by buccal swab rather than by finger stick, and less than one-sixth believed that genetic testing was meddling in God's plan. In the face of both the low level of use of free CF carrier screening by nonpregnant couples when it was not offered in person by health-care professionals and the wide variety of concerns demonstrated, we believe that clinicians should not routinely offer carrier screening to nonpregnant individuals who do not have a family history of CF.     

PCR MULTIPLEXED MICROSATELLITE PANELS TO EXPEDITE CANINE GENETIC DISEASE LINKAGE ANALYSIS

ABSTRACT

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25?centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.     

Inherited occipital hypoplasia/syringomyelia in the cavalier King Charles spaniel: experiences in setting up a worldwide DNA collection

Inherited diseases commonly emerge within pedigree dog populations, often due to use of repeatedly bred carrier sire(s) within a small gene pool. Accurate family records are usually available making linkage analysis possible. However, there are many factors that are intrinsically difficult about collecting DNA and collating pedigree information from a large canine population. The keys to a successful DNA collection program include (1) the need to establish and maintain support from the pedigree breed clubs and pet owners; (2) committed individual(s) who can devote the considerable amount of time and energy to coordinating sample collection and communicating with breeders and clubs; and (3) providing means by which genotypic and phenotypic information can be easily collected and stored. In this article we described the clinical characteristics of inherited occipital hypoplasia/syringomyelia (Chiari type I malformation) in the cavalier King Charles spaniel and our experiences in establishing a pedigree and DNA database to study the disease.     

FACIL: Fast and Accurate Genetic Code Inference and Logo

MOTIVATION: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa. RESULTS: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo. Availability and implementation: FACIL is available as a web-based service at http://www.cmbi.ru.nl/FACIL/ and as a stand-alone program.     

Pharmacogenetics in Primary Care: The Promise of Personalized Medicine and the Reality of Racial Profiling

Many anticipate that expanding knowledge of genetic variations associated with disease risk and medication response will revolutionize clinical medicine, making possible genetically based Personalized Medicine where health care can be tailored to individuals, based on their genome scans. Pharmacogenetics has received especially strong interest, with many pharmaceutical developers avidly working to identify genetic variations associated with individual differences in drug response. While clinical applications of emerging genetic knowledge are becoming increasingly available, genetic tests for drug selection are not as yet widely accessible, and many primary care clinicians are unprepared to interpret genetic information. We conducted interviews with 58 primary care clinicians, exploring how they integrate emerging pharmacogenetic concepts into their practices. We found that in their current practices, pharmacogenetic innovations have not led to individually tailored treatment, but instead have encouraged use of essentialized racial/ethnic identity as a proxy for genetic heritage. Current manifestations of Personalized Medicine appear to be reinforcing entrenched notions of inherent biological differences between racial groups, and promoting the belief that racial profiling in health care is supported by cutting-edge scientific authority. Our findings raise concern for how pharmacogenetic innovations will actually affect diverse populations, and how unbiased treatment can be assured.     

Accepting Adoption’s Uncertainty: The Limited Ethics of Pre-Adoption Genetic Testing

An increasing number of children are adopted in the United States from countries where both medical care and environmental conditions are extremely poor. In response to worries about the accuracy of medical histories, prospective adoptive parents increasingly request genetic testing of children prior to adoption. Though a general consensus on the ethics of pre-adoption genetic testing (PAGT) argues against permitting genetic testing on children available for adoption that is not also permitted for children in general, a view gaining traction argues for expanding the tests permitted. The reasoning behind this view is that the State has a duty to provide a child with parents who are the best “match,” and thus all information that advances this end should be obtained. While the matching argument aims to promote the best interests of children, I show how it rests on the claim that what is in the best interests of children available for adoption is for prospective adoptive parents to have their genetic preferences satisfied such that the “genetics” of the children they end up adopting accurately reflects those preferences. Instead of protecting a vulnerable population, I conclude, PAGT contributes to the risks of harm such children face as it encourages people with strong genetic preferences to adopt children whose genetic backgrounds will always be uncertain.