The Arabidopsis INNER NO OUTER (INO) gene acts exclusively and quantitatively in regulation of ovule outer integument development.

The INNER NO OUTER (INO) gene is essential for formation of the outer integument of ovules in Arabidopsis thaliana. Initially described lesions in INO were missense mutations resulting in aberrant mRNA splicing. To determine the null mutant phenotype, we generated frameshift mutations and found, in confirmation of results on another recently identified frameshift mutation, that such mutants have a phenotype identical to the most severe splicing mutant (ino-1), with effects specific to outer integument development. We show that the altered protein of an ino mRNA splicing mutant with a less severe phenotype (ino-4) does not have INO activity, and the mutant is partial because it produces a small amount of correctly spliced INO mRNA. Screening for suppressors of ino-4 in a fast neutron-mutagenized population identified a translocated duplication of the ino-4 gene, leading to an increase in the amount of this mRNA. The increased expression led to a decrease in the severity of the mutant effects, indicating that the amount of INO activity quantitatively regulates outer integument growth. The results further confirm that the role of INO in Arabidopsis development is specific to the outer integument of ovules where it quantitatively affects the growth of this structure.

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation.

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed.

Parents' perceptions of personal utility of exome sequencing results.

PURPOSE: Clinical genome or exome sequencing (GS/ES) provides a diagnosis for many individuals with suspected genetic disorders, but also yields negative or uncertain results for the majority. This study examines how parents of a child with an undiagnosed condition attribute personal utility to all types of ES results. METHODS: Return of 31 exome sequencing results was observed during clinic sessions, followed by semistructured interviews with parents one month later. Observations and interviews were recorded and transcribed. Data display matrices were used for content analysis and systematic comparisons of parents' perceptions of utility. RESULTS: ES results could not provide all the answers to parents' questions, especially in cases of clinically uninformative results, but parents nonetheless attributed utility to the knowledge gained. Parents across all results categories used the genomic information to rule out possible causes, end or postpone the diagnostic odyssey, and shift focus to treatment and management of symptoms. CONCLUSION: This study suggests that parents value even uninformative ES results while expressing hope for future discoveries. As pediatric genetics moves toward GS/ES as a first-tier test, how parents perceive the personal utility of negative or uncertain results is an important topic for genetic counseling and further research.

Development and evolution of the unique ovules of flowering plants.

Ovules are the precursors to seeds and as such are critical to plant propagation and food production. Mutant studies have led to the identification of numerous genes regulating ovule development. Genes encoding transcription factors have been shown to direct ovule spacing, ovule identity and integument formation. Particular co-regulators have now been associated with activities of some of these transcription factors, and other protein families including cell surface receptors have been shown to regulate ovule development. Hormone levels and transport, especially of auxin, have also been shown to play critical roles in ovule emergence and morphogenesis and to interact with the transcriptional regulators. Ovule diversification has been studied using orthologs of regulatory genes in divergent angiosperm groups. Combining modern genetic evidence with expanding knowledge of the fossil record illuminates the possible origin of the unique bitegmic ovules of angiosperms.

A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds.

The molecular pathways that trigger the initiation of embryogenesis after fertilization in flowering plants, and prevent its occurrence without fertilization, are not well understood1. Here we show in rice (Oryza sativa) that BABY BOOM1 (BBM1), a member of the AP2 family2 of transcription factors that is expressed in sperm cells, has a key role in this process. Ectopic expression of BBM1 in the egg cell is sufficient for parthenogenesis, which indicates that a single wild-type gene can bypass the fertilization checkpoint in the female gamete. Zygotic expression of BBM1 is initially specific to the male allele but is subsequently biparental, and this is consistent with its observed auto-activation. Triple knockout of the genes BBM1, BBM2 and BBM3 causes embryo arrest and abortion, which are fully rescued by male-transmitted BBM1. These findings suggest that the requirement for fertilization in embryogenesis is mediated by male-genome transmission of pluripotency factors. When genome editing to substitute mitosis for meiosis (MiMe)3,4 is combined with the expression of BBM1 in the egg cell, clonal progeny can be obtained that retain genome-wide parental heterozygosity. The synthetic asexual-propagation trait is heritable through multiple generations of clones. Hybrid crops provide increased yields that cannot be maintained by their progeny owing to genetic segregation. This work establishes the feasibility of asexual reproduction in crops, and could enable the maintenance of hybrids clonally through seed propagation5,6.

Recent advances in understanding female gametophyte development.

The haploid female gametophyte (embryo sac) is an essential reproductive unit of flowering plants, usually comprising four specialized cell types, including the female gametes (egg cell and central cell). The differentiation of these cells relies on spatial signals which pattern the gametophyte along a proximal-distal axis, but the molecular and genetic mechanisms by which cell identities are determined in the embryo sac have long been a mystery. Recent identification of key genes for cell fate specification and their relationship to hormonal signaling pathways that act on positional cues has provided new insights into these processes. A model for differentiation can be devised with egg cell fate as a default state of the female gametophyte and with other cell types specified by the action of spatially regulated factors. Cell-to-cell communication within the gametophyte is also important for maintaining cell identity as well as facilitating fertilization of the female gametes by the male gametes (sperm cells).

"Possibly positive or certainly uncertain?": participants' responses to uncertain diagnostic results from exome sequencing.

PurposeClinical genome sequencing produces uncertain diagnostic results, raising concerns about how to communicate the method's inherent complexities in ways that reduce potential misunderstandings and harm. This study investigates clinicians' communications and patient/participant responses to uncertain diagnostic results arising from a clinical exome sequencing research study, contributing empirical data to the debate surrounding disclosure of uncertain genomic information.MethodsWe investigated the communication and impact of uncertain diagnostic results using ethnographic observations of result disclosures with 21 adults and 11 parents of child patients, followed by two semistructured interviews with these same participants.ResultsParticipants understood their uncertain results in ways that were congruent with clinical geneticists' communications. They followed recommendations for further consultation, although family testing to resolve uncertainty was not always done. Participants were prepared for learning an uncertain result and grasped the key concept that it should not be used to guide health-care or other decisions. They did not express regret for having learned the uncertain result; most regarded it as potentially valuable in the future.ConclusionThis study suggests that uncertain diagnostic results from genome sequencing can be relayed to patients in ways they can understand and consistent with providers' interpretations, without causing undue harm.

Age and perceived risks and benefits of preventive genomic screening.

PURPOSE: As genome sequencing moves from research to clinical practice, sequencing technologies focused on "medically actionable" targets are being promoted for preventive screening despite the dearth of systematic evidence of risks and benefits and of criteria for selection of screening subjects. This study investigates researchers' and research participants' perceptions of these issues within the context of a preventive genomic screening study, GeneScreen. METHODS: We recorded researcher deliberations regarding age eligibility criteria and the risks and benefits of screening, and conducted interviews with 50 GeneScreen participants about their motivations for joining and their perceptions of risks and benefits. RESULTS: Researchers made assumptions about who would want and benefit from screening based on age. After discussion, researchers opted not to have an upper age limit for enrollment. Participants of all ages perceived similar benefits, including prevention, treatment, and cascade testing, and similar risks, such as insurance discrimination and worry. CONCLUSION: While clinical benefits of preventive genomic screening for older adults are debatable, our respondents perceived a range of benefits of screening in both clinical and research settings. Researchers and clinicians should carefully consider decisions about whether to exclude older adults and whether to provide information about benefits and risks across age groups.

Integument Development in Arabidopsis Depends on Interaction of YABBY Protein INNER NO OUTER with Coactivators and Corepressors.

Arabidopsis thaliana INNER NO OUTER (INO) is a YABBY protein that is essential for the initiation and development of the outer integument of ovules. Other YABBY proteins have been shown to be involved in both negative and positive regulation of expression of putative target genes. YABBY proteins have also been shown to interact with the corepressor LEUNIG (LUG) in several systems. In support of a repressive role for INO, we confirm that INO interacts with LUG and also find that INO directly interacts with SEUSS (SEU), a known corepressive partner of LUG. Further, we find that INO can directly interact with ADA2b/PROPORZ1 (PRZ1), a transcriptional coactivator that is known to interact with the histone acetyltransferase GENERAL CONTROL NONREPRESSIBLE PROTEIN 5 (GCN5, also known as HAG1). Mutations in LUG, SEU, and ADA2b/PRZ1 all lead to pleiotropic effects including a deficiency in the extension of the outer integument. Additive and synergistic effects of ada2b/prz1 and lug mutations on outer integument formation indicate that these two genes function independently to promote outer integument growth. The ino mutation is epistatic to both lug and ada2b/prz1 in the outer integument, and all three proteins are present in the nuclei of a common set of outer integument cells. This is consistent with a model where INO utilizes these coregulator proteins to activate and repress separate sets of target genes. Other Arabidopsis YABBY proteins were shown to also form complexes with ADA2b/PRZ1, and have been previously shown to interact with SEU and LUG. Thus, interaction with these corepressors and coactivator may represent a general mechanism to explain the positive and negative activities of YABBY proteins in transcriptional regulation. The LUG, SEU, and ADA2b/PRZ1 proteins would also separately be recruited to targets of other transcription factors, consistent with their roles as general coregulators, explaining the pleiotropic effects not associated with YABBY function.

Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges.

PurposeWe investigated the diagnostic and clinical performance of exome sequencing in fetuses with sonographic abnormalities with normal karyotype and microarray and, in some cases, normal gene-specific sequencing.MethodsExome sequencing was performed on DNA from 15 anomalous fetuses and from the peripheral blood of their parents. Parents provided consent to be informed of diagnostic results in the fetus, medically actionable findings in the parents, and their identification as carrier couples for significant autosomal recessive conditions. We assessed the perceptions and understanding of exome sequencing using mixed methods in 15 mother-father dyads.ResultsIn seven (47%) of 15 fetuses, exome sequencing provided a diagnosis or possible diagnosis with identification of variants in the following genes: COL1A1, MUSK, KCTD1, RTTN, TMEM67, PIEZO1 and DYNC2H1. One additional case revealed a de novo nonsense mutation in a novel candidate gene (MAP4K4). The perceived likelihood that exome sequencing would explain the results (5.2 on a 10-point scale) was higher than the approximately 30% diagnostic yield discussed in pretest counseling.ConclusionExome sequencing had diagnostic utility in a highly select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy and variant interpretation must be addressed by highly tailored pre- and posttest genetic counseling.

The nuanced negative: Meanings of a negative diagnostic result in clinical exome sequencing.

Genomic sequencing technology is moving rapidly from the research setting into clinical medicine but significant technological and interpretive challenges remain. Exome sequencing (ES) in its recent clinical application provides a genetic diagnosis in about 25 per cent of cases (Berg 2014). While this diagnostic yield is substantial, it also indicates that in a majority of cases, patients are receiving negative results (i.e. no explanatory genetic variant found) from this technology. There are a number of uncertainties regarding the meaning of a negative result in the current context of ES. A negative result may be due to current technological limitations that hinder detection of disease-causing variants or to gaps in the knowledge base that prohibit accurate interpretation of their pathogenicity; or it may indicate that there is not a genetic etiology for the disorder. In this paper we examine the uncertainties and nuances of the negative result from genome sequencing and how both clinicians and patients make meaning of it as revealed in ethnographic observations of the clinic session where results are returned, and in interviews with patients. We find that clinicians and patients construct the meaning of a negative result in ways that are uncertain, contingent, and multivalent; but invested with optimism, promise, and potentiality.

Conservation of the role of INNER NO OUTER in development of unitegmic ovules of the Solanaceae despite a divergence in protein function.

BACKGROUND: The INNER NO OUTER (INO) gene is expressed in the outermost cell layer of the outer integument of bitegmic ovules and is essential for this organ's growth. The role and cross-species functional conservation of INO orthologs were examined in members of the Solanaceae, which have unitegmic ovules. Unitegmy has evolved several times in disparate angiosperm lineages. INO expression has been observed in the outermost cell layers of all examined unitegmic ovules, but the functional role of INO in unitegmic ovules has not previously been evaluated. RESULTS: INO orthologs were unambiguously identified in tobacco and tomato by sequence homology. Expression of the tomato INO gene was limited to the outer cell layer of the single integument indicating that this single integument has properties of the outer integument. Expression occurred only after integument initiation, later than observed in ovules of other examined angiosperms. Virus-induced knock-down of expression of the INO ortholog in tobacco inhibited growth of the outer cell layer of the integument leading to a decrease in both integument extension and curvature of the ovule. The altered ovules closely resemble those of the aberrant testa shape (ats) ino mutant combination in Arabidopsis where we see the effect of the ino mutation on a single fused integument produced by the ats mutation. Despite significant sequence identity and similar expression patterns, the tomato INO coding region was not able to complement the Arabidopsis ino mutant. CONCLUSIONS: The similarity of effects of ino mutations on the unitegmic ovules of tobacco and the fused integuments of the Arabidopsis ats mutant show that: 1) INO orthologs play the same role in promoting integument growth in ovules of tobacco and Arabidopsis; and 2) the unitegmic ovules of tobacco (and hence other solanaceous species) are most likely the result of a congenital fusion of two ancestral integuments. Our results further indicate that INO has a conserved role in growth of the outermost cell layer of integuments. The curvature of solanaceous ovules is driven by unequal growth of the outer layers of the single integument that likely correspond to an ancestral outer integument.

"Not Tied Up Neatly with a Bow": Professionals' Challenging Cases in Informed Consent for Genomic Sequencing.

As the use of genomic technology has expanded in research and clinical settings, issues surrounding informed consent for genome and exome sequencing have surfaced. Despite the importance of informed consent, little is known about the specific challenges that professionals encounter when consenting patients or research participants for genomic sequencing. We interviewed 29 genetic counselors and research coordinators with considerable experience obtaining informed consent for genomic sequencing to understand their experiences and perspectives. As part of this interview, 24 interviewees discussed an informed consent case they found particularly memorable or challenging. We analyzed these case examples to determine the primary issue or challenge represented by each case. Challenges fell into two domains: participant understanding, and facilitating decisions about testing or research participation. Challenges related to participant understanding included varying levels of general and genomic literacy, difficulty managing participant expectations, and contextual factors that impeded participant understanding. Challenges related to facilitating decision-making included complicated family dynamics such as disagreement or coercion, situations in which it was unclear whether sequencing research would be a good use of participant time or resources, and situations in which the professional experienced disagreement or discomfort with participant decisions. The issues highlighted in these case examples are instructive in preparing genetics professionals to obtain informed consent for genomic sequencing.

Experiences with obtaining informed consent for genomic sequencing.

Despite the increased utilization of genome and exome sequencing, little is known about the actual content and process of informed consent for sequencing. We addressed this by interviewing 29 genetic counselors and research coordinators experienced in obtaining informed consent for sequencing in research and clinical settings. Interviews focused on the process and content of informed consent; patients/participants' common questions, concerns and misperceptions; and challenges to obtaining informed consent. Content analysis of transcribed interviews revealed that the main challenges to obtaining consent related to the broad scope and uncertainty of results, and patient/participants' unrealistic expectations about the likely number and utility of results. Interviewees modified their approach to sessions according to contextual issues surrounding the indication for testing, type of patient, and timing of testing. With experience, most interviewees structured sessions to place less emphasis on standard elements in the consent form and technological aspects of sequencing. They instead focused on addressing misperceptions and helping patients/participants develop realistic expectations about the types and implications of possible results, including secondary findings. These findings suggest that informed consent sessions should focus on key issues that may be misunderstood by patients/participants. Future research should address the extent to which various stakeholders agree on key elements of informed consent.

Flexible positions, managed hopes: the promissory bioeconomy of a whole genome sequencing cancer study.

Genomic research has rapidly expanded its scope and ambition over the past decade, promoted by both public and private sectors as having the potential to revolutionize clinical medicine. This promissory bioeconomy of genomic research and technology is generated by, and in turn generates, the hopes and expectations shared by investors, researchers and clinicians, patients, and the general public alike. Examinations of such bioeconomies have often focused on the public discourse, media representations, and capital investments that fuel these "regimes of hope," but also crucial are the more intimate contexts of small-scale medical research, and the private hopes, dreams, and disappointments of those involved. Here we examine one local site of production in a university-based clinical research project that sought to identify novel cancer predisposition genes through whole genome sequencing in individuals at high risk for cancer. In-depth interviews with 24 adults who donated samples to the study revealed an ability to shift flexibly between positioning themselves as research participants on the one hand, and as patients or as family members of patients, on the other. Similarly, interviews with members of the research team highlighted the dual nature of their positions as researchers and as clinicians. For both parties, this dual positioning shaped their investment in the project and valuing of its possible outcomes. In their narratives, all parties shifted between these different relational positions as they managed hopes and expectations for the research project. We suggest that this flexibility facilitated study implementation and participation in the face of potential and probable disappointment on one or more fronts, and acted as a key element in the resilience of this local promissory bioeconomy. We conclude that these multiple dimensions of relationality and positionality are inherent and essential in the creation of any complex economy, "bio" or otherwise.

Symptoms of depression and their management among low-income African-American and White mothers in the rural South.

OBJECTIVE: This study examines experiences of depressive symptoms among a group of 32 low-income, African-American and White mothers of young children who resided in rural Eastern North Carolina, USA. DESIGN: Women's experiences of depressive symptoms were elicited through a series of longitudinal ethnographic interviews, including an explanatory models interview specifically designed to elicit their beliefs about the causes, symptomatology and help-seeking behavior and management of depressive symptoms. RESULTS: A content analysis of interview data indicated that most women (11 African-Americans and 15 Whites) reported having depressive symptoms currently or in the past. Both African-American and White women perceived the main causes of these symptoms as being relationship problems with a spouse, a partner, or a family member; lack of finances; and parenting stresses. There were no differences in the depressive symptoms African-American and White women reported, but there were differences in how they managed these symptoms and where they sought help. Most of the African-American women sought no formal treatment (i.e., pharmacotherapy and/or psychotherapy), but instead turned to their religious faith to deal with their feelings. White women were more likely to seek formal treatment. CONCLUSION: These findings provide insights into the ways in which women in one nonurban area in the USA explained and experienced depressive symptoms and demonstrate differences in help-seeking behaviors that can be linked to beliefs about depression and perceptions of societal responses to those who have it, as well as to perceptions of and experiences with the health-care system. Results have implications for the implementation of education, intervention, and treatment programs in more culturally sensitive ways.

Obtaining consent from both parents for pediatric research: what does "reasonably available" mean?

OBJECTIVE: Circumstances surrounding parental availability and decision-making were examined in the setting of a research protocol involving newborn screening (NBS) for fragile X syndrome, in which the institutional review board (IRB) had determined that consent (permission) was required from both parents. METHODS: A survey was conducted with 3001 families who were approached to participate in optional NBS. In addition to basic demographics, observational notes detailed the reasons why fathers were not present or deemed "not reasonably available" (per IRB regulations), and content analysis identified the factors for this lack of availability. Logistic regression models estimated the likelihood that both parents would agree to enroll their infant in the screening project. RESULTS: Fathers were not present in 589 cases, including 158 in which fathers were ultimately determined to be not reasonably available. Primary reasons for father's unavailability were deployment with the military, incarceration, living out of state, or not involved in the mother's life. In cases in which both parents were available, 64% agreed to enroll in the NBS study. Criteria to guide researchers in making required determinations were developed from consultations with IRB officials and legal counsel. CONCLUSIONS: In a large-scale population-based study, 19.6% of fathers were absent for the consent process. Scenarios encountered underscore the complexity of parental relations and their implications for obtaining consent for research involving children. The algorithm developed may serve as a useful tool for others in applying the regulatory requirements for dual parental permission.

Newborn, carrier, and early childhood screening recommendations for fragile X.

Fragile X syndrome, diagnosed by Fragile X Mental Retardation 1 (FMR1) DNA testing, is the most common single-gene cause of inherited intellectual disability. The expanded CGG mutation in the FMR1 gene, once thought to have clinical significance limited to fragile X syndrome, is now well established as the cause for other fragile X-associated disorders including fragile X-associated primary ovarian insufficiency and fragile X-associated tremor ataxia syndrome in individuals with the premutation (carriers). The importance of early diagnostic and management issues, in conjunction with the identification of family members at risk for or affected by FMR1 mutations, has led to intense discussion about the appropriate timing for early identification of FMR1 mutations. This review includes an overview of the fragile X-associated disorders and screening efforts to date, and discussion of the advantages and barriers to FMR1 screening in newborns, during childhood, and in women of reproductive age. Comparison with screening programs for other common genetic conditions is discussed to arrive at action steps to increase the identification of families affected by FMR1 mutations.

Parenting young children with and without Fragile X syndrome.

The purpose of this study was to examine maternal parenting styles across age-matched siblings using a within-family design, in which one child has Fragile X syndrome. Thirteen families participated; children were aged 16 to 71 months. Mothers completed several videotaped activities with each child separately as well as an interview. Mothers used a consistent, responsive style with both children, using the same degree of positive affect and warmth. Differences included using more behavior management strategies with the child with Fragile X and a conversational style of interaction with the sibling. Differences in approaches suggest the mothers adapted to the developmental differences between the children. The interview data supported these findings; mothers were aware of the changes made to accommodate the developmental differences.