Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

The Persistence of Bacterial Pathogens in Surface Water and Its Impact on Global Food Safety.

Water is vital to agriculture. It is essential that the water used for the production of fresh produce commodities be safe. Microbial pathogens are able to survive for extended periods of time in water. It is critical to understand their biology and ecology in this ecosystem in order to develop better mitigation strategies for farmers who grow these food crops. In this review the prevalence, persistence and ecology of four major foodborne pathogens, Shiga toxin-producing Escherichia coli (STEC), Salmonella, Campylobacter and closely related Arcobacter, and Listeria monocytogenes, in water are discussed. These pathogens have been linked to fresh produce outbreaks, some with devastating consequences, where, in a few cases, the contamination event has been traced to water used for crop production or post-harvest activities. In addition, antimicrobial resistance, methods improvements, including the role of genomics in aiding in the understanding of these pathogens, are discussed. Finally, global initiatives to improve our knowledge base of these pathogens around the world are touched upon.

Somatic mosaicism in the human genome.

Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation. In contrast to inherited mutations, somatic mosaic mutations may affect only a portion of the body and are not transmitted to progeny. These mutations affect varying genomic sizes ranging from single nucleotides to entire chromosomes and have been implicated in disease, most prominently cancer. The phenotypic consequences of somatic mosaicism are dependent upon many factors including the developmental time at which the mutation occurs, the areas of the body that are affected, and the pathophysiological effect(s) of the mutation. The advent of second-generation sequencing technologies has augmented existing array-based and cytogenetic approaches for the identification of somatic mutations. We outline the strengths and weaknesses of these techniques and highlight recent insights into the role of somatic mosaicism in causing cancer, neurodegenerative, monogenic, and complex disease.

Exploring patient expectations for pharmacist-provided literacy-sensitive communication.

BACKGROUND: Limited health literacy is common and associated with adverse health care outcomes. Although pharmacies and pharmacists are accessible to most patients, research has indicated that they do not routinely report efforts to target interventions for patients with suboptimal health literacy. Moreover, little is known about the use and expectation of literacy-based communication techniques in pharmacies from the patient perspective. OBJECTIVE: The intent of this pilot study was to describe the use of health literacy-based communication techniques, expectations for their use, and satisfaction with communication as reported by patients at high risk for medication misadventures who receive care at a clinic-based community pharmacy. METHODS: A cross-sectional telephonic interview of a purposive sample of patients aged 65 years or older who were prescribed at least 8 unique medications was conducted. Patients were recruited from 1 clinic-based pharmacy that serves a predominantly urban low-income population. A telephonic interview guide was derived from previous literature and included 52 questions related to respondent characteristics, perceptions of experiences regarding verbal communications with the pharmacist, expectations for communication with the pharmacist, and satisfaction with current pharmacy communication techniques. Responses were summarized and described. RESULTS: Nineteen patients completed the telephonic interviews. Patients commonly reported that the pharmacist provided the following counseling for new prescriptions: how to take their medication (88.9%), side effects (84.2%), and indication (47.4%). In contrast, only 44.4%, 55.6%, and 33.3% of patients expected the pharmacist to engage in these same counseling behaviors. A minority of patients reported the use of various recommended clear health communication techniques by the pharmacist, and an even smaller percentage expressed expectations for their use. Despite the limited use of literacy-based communication techniques, 73.7% of patients reported being very satisfied with pharmacy counseling, and 94.7% reported good to excellent understanding of their medications. CONCLUSION: Patient-pharmacist interactions consistently met or exceeded patient expectations. However, pharmacists use of literacy-based communication techniques was low as were patient expectations. Future research and training efforts should focus on not only increasing pharmacists' use of literacy-based communication techniques but also raising patients' expectations for performing these activities.

Unexpected relationships and inbreeding in HapMap phase III populations.

Correct annotation of the genetic relationships between samples is essential for population genomic studies, which could be biased by errors or omissions. To this end, we used identity-by-state (IBS) and identity-by-descent (IBD) methods to assess genetic relatedness of individuals within HapMap phase III data. We analyzed data from 1,397 individuals across 11 ethnic populations. Our results support previous studies (Pemberton et al., 2010; Kyriazopoulou-Panagiotopoulou et al., 2011) assessing unknown relatedness present within this population. Additionally, we present evidence for 1,657 novel pairwise relationships across 9 populations. Surprisingly, significant Cotterman's coefficients of relatedness K1 (IBD1) values were detected between pairs of known parents. Furthermore, significant K2 (IBD2) values were detected in 32 previously annotated parent-child relationships. Consistent with a hypothesis of inbreeding, regions of homozygosity (ROH) were identified in the offspring of related parents, of which a subset overlapped those reported in previous studies (Gibson et al. 2010; Johnson et al. 2011). In total, we inferred 28 inbred individuals with ROH that overlapped areas of relatedness between the parents and/or IBD2 sharing at a different genomic locus between a child and a parent. Finally, 8 previously annotated parent-child relationships had unexpected K0 (IBD0) values (resulting from a chromosomal abnormality or genotype error), and 10 previously annotated second-degree relationships along with 38 other novel pairwise relationships had unexpected IBD2 (indicating two separate paths of recent ancestry). These newly described types of relatedness may impact the outcome of previous studies and should inform the design of future studies relying on the HapMap Phase III resource.

Chromosomal variation in lymphoblastoid cell lines.

Tens of thousands of lymphoblastoid cell lines (LCLs) have been established by the research community, providing nearly unlimited source material from samples of interest. LCLs are used to address questions in population genomics, mechanisms of disease, and pharmacogenomics. Thus, it is of fundamental importance to define the extent of chromosomal variation in LCLs. We measured variation in genotype and copy number in multiple LCLs derived from peripheral blood mononuclear cells (PBMCs) of single individuals as well as two comparison groups: (1) three types of differentiated cell lines (DCLs) and (2) triplicate HapMap samples. We then validated and extended our findings using data from a large study consisting of samples from blood or LCLs. We observed high concordances between genotypes and copy number estimates within all sample groups. While the genotypes of LCLs tended to faithfully reflect the genotypes of PBMCs, 13.7% (4 of 29) of immortalized cell lines harbored mosaic regions greater than 20 megabases, which were not present in PBMCs, DCLs, or HapMap replicate samples. We created a list of putative LCL-specific changes (affecting regions such as immunoglobulin loci) that is available as a community resource.

Consanguinity in Centre d'Étude du Polymorphisme Humain (CEPH) pedigrees.

A set of Centre d'Étude du Polymorphisme Humain (CEPH) cell lines serves as a large reference collection that has been widely used as a benchmark for allele frequencies in the analysis of genetic variants, to create linkage maps of the human genome, to study the genetics of gene expression, to provide samples to the HapMap and 1000 Genomes projects, and for a variety of other applications. An explicit feature of the CEPH collection is that these multigenerational families represent reference panels of known relatedness, consisting mostly of three-generation pedigrees with large sibships, two parents, and grandparents. We applied identity-by-state (IBS) and identity-by-descent (IBD) methods to high-density genotype data from 186 CEPH individuals in 13 families. We identified unexpected relatedness between nominally unrelated grandparents both within and between pedigrees. For one pair, the estimated Cotterman coefficient of relatedness k1 exceeded 0.2, consistent with one-eighth sharing (eg, first-cousins). Unexpectedly, significant IBD2 values were discovered in both second-degree and parent-child relationships. These were accompanied by regions of homozygosity in the offspring, which corresponded to blocks lacking IBS0 in purportedly unrelated parents, consistent with inbreeding. Our findings support and extend a 1999 report, based on the use of short tandem-repeat polymorphisms, that several CEPH families had regions of homozygosity consistent with autozygosity. We benchmarked our IBD approach (called kcoeff) against both RELPAIR and PREST software packages. Our findings may affect the interpretation of previous studies and the design of future studies that rely on the CEPH resource.

Inference of relationships in population data using identity-by-descent and identity-by-state.

It is an assumption of large, population-based datasets that samples are annotated accurately whether they correspond to known relationships or unrelated individuals. These annotations are key for a broad range of genetics applications. While many methods are available to assess relatedness that involve estimates of identity-by-descent (IBD) and/or identity-by-state (IBS) allele-sharing proportions, we developed a novel approach that estimates IBD0, 1, and 2 based on observed IBS within windows. When combined with genome-wide IBS information, it provides an intuitive and practical graphical approach with the capacity to analyze datasets with thousands of samples without prior information about relatedness between individuals or haplotypes. We applied the method to a commonly used Human Variation Panel consisting of 400 nominally unrelated individuals. Surprisingly, we identified identical, parent-child, and full-sibling relationships and reconstructed pedigrees. In two instances non-sibling pairs of individuals in these pedigrees had unexpected IBD2 levels, as well as multiple regions of homozygosity, implying inbreeding. This combined method allowed us to distinguish related individuals from those having atypical heterozygosity rates and determine which individuals were outliers with respect to their designated population. Additionally, it becomes increasingly difficult to identify distant relatedness using genome-wide IBS methods alone. However, our IBD method further identified distant relatedness between individuals within populations, supported by the presence of megabase-scale regions lacking IBS0 across individual chromosomes. We benchmarked our approach against the hidden Markov model of a leading software package (PLINK), showing improved calling of distantly related individuals, and we validated it using a known pedigree from a clinical study. The application of this approach could improve genome-wide association, linkage, heterozygosity, and other population genomics studies that rely on SNP genotype data.

Genomic analysis of partial 21q monosomies with variable phenotypes.

Partial monosomy 21 was recently segregated into three regions associated with variable clinical severity. We describe 10 new patients, all examined by single nucleotide polymorphism (SNP) genotyping and G-banded karyotyping. Cohort A consisted of three patients seen in our medical genetics clinics with partial chromosome 21 monosomies. In two of these patients having terminal deletions (21q22.2-ter and 21q22.3-ter), the breakpoints differed by at least 812 Kb of sequence, containing seven RefSeq genes. A third patient had an interstitial hemizygous loss of 16.4 Mb (21q21.1-q22.11). All three patients had relatively mild phenotypes. Cohort B consisted of seven patients with partial chromosome 21 monosomies who had a greater number of dysmorphic features and some major malformations; SNP genotypes were obtained from the Coriell Genetic Cell Repository. We also collected data on partial monsomy 21 cases from the DECIPHER database. This report of 10 new cases of 21q deletion and review of a total of 36 confirms that deletion of the terminal region is associated with a mild phenotype, but suggests that deletion of regions 1 and 2 is compatible with life and have a variable phenotype perhaps relating more to other genetic and environmental variables than to genes in the interval.

Medication reconciliation in a community pharmacy setting.

OBJECTIVES: To describe the types and frequencies of medication discrepancies identified through medication reconciliation in a community pharmacy setting, to identify potential correlations between a patient's electronic medical record (EMR) and pharmacy medication list, and to determine the relationship between patients who use prescribers and/or pharmacies outside of the Family Medicine Center (FMC) and the occurrence of medication discrepancies. METHODS: Cross-sectional comparison of patients' EMR medication lists and pharmacy medication fill history for a sample of patients presenting to the Family Medicine Pharmacy (FMP), which is located in the FMC on the University of Oklahoma Health Sciences Center campus in Oklahoma City. Discrepancies identified were classified according to one of six categories that included therapeutic duplication, medication exclusion, medications that should be designated inactive in the EMR medication list, and differences in medication strength, dosage form, or dosing regimen. RESULTS: A total of 100 patients were included. Most patients reported having all of their medications dispensed from FMP (89%), and most patients had prescriptions prescribed by FMC physicians only (57%). Each patient had an average of six medication discrepancies. Most discrepancies belonged to the inactive medication category (41%). The correlation between patients' FMP medication lists and their EMR medication lists was 0.73. Patients with one or more non-FMC prescribers had a greater number of medication discrepancies than patients with FMC prescribers only, but this relationship was not identified for those who used pharmacies outside of FMP (P = 0.0264 and 0.2580, respectively). CONCLUSION: A variety of medication discrepancies were observed, signaling a need for medication reconciliation in the outpatient setting. Future research on this topic should focus on the implications of such discrepancies in the outpatient setting, interventions to reduce the number of discrepancies, and identifying patients at high risk for such discrepancies.

Whole-genome sequencing of a single proband together with linkage analysis identifies a Mendelian disease gene.

Although more than 2,400 genes have been shown to contain variants that cause Mendelian disease, there are still several thousand such diseases yet to be molecularly defined. The ability of new whole-genome sequencing technologies to rapidly indentify most of the genetic variants in any given genome opens an exciting opportunity to identify these disease genes. Here we sequenced the whole genome of a single patient with the dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. In a second metachondromatosis family, we confirmed our result by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations. This combination of a new technology and a classical genetic approach provides a powerful strategy to discover the genes responsible for unexplained Mendelian disorders.