Immune regulation of intestinal-stem-cell function in Drosophila.

Intestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, we know little about contributions of stem-cell immunity to gut homeostasis. We used Drosophila to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. We showed that loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, we found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. Our results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut.

Microbial recognition regulates intestinal epithelial growth in homeostasis and disease.

The intestine is constantly exposed to a dynamic community of microbes. Intestinal epithelial cells respond to microbes through evolutionarily conserved recognition pathways, such as the immune deficiency (IMD) pathway of Drosophila, the Toll-like receptor (TLR) response of flies and vertebrates, and the vertebrate nucleotide-binding oligomerization domain (NOD) pathway. Microbial recognition pathways are tightly controlled to respond effectively to pathogens, tolerate the microbiome, and limit intestinal disease. In this review, we focus on contributions of different model organisms to our understanding of how epithelial microbe recognition impacts intestinal proliferation and differentiation in homeostasis and disease. In particular, we compare how microbes and subsequent recognition by the intestine influences barrier integrity, intestinal repair and tumorigenesis in Drosophila, zebrafish, mice, and organoids. In addition, we discuss the importance of microbial recognition in homeostatic intestinal growth and discuss how immune pathways directly impact stem cell and crypt dynamics.

Universal access to genetic counseling for women with epithelial ovarian cancer in Nova Scotia: Evaluating a new collaborative care model.

Women with pathogenic variants in BRCA1/2 have a significantly increased lifetime risk of breast and ovarian cancers. The availability of genetic testing to identify BRCA1/2 carriers is imperative to disease prevention and treatment. We evaluated the effectiveness of a new collaborative care model in Nova Scotia, involving the integration of genetic counselors into tumor board rounds, reduction in time allotted for initial genetic counseling appointments from 60 to 45 min, and a standardized dictation template, to increase referral rate for genetic counseling. We also assessed the study cohorts' preferences on timing for genetic testing. A retrospective chart review was performed on all women diagnosed with epithelial ovarian cancer (EOC) from 2012 to 2017 (N = 386). Pertinent clinical outcomes were categorized and wait times to different nodes of the clinical pathway assessed. A questionnaire was sent to this same cohort of women to identify preference for the timing of genetic testing (n = 103). The chi-square and Wilcoxon's rank-sum tests were used to compare demographic and clinical variables pre- and post-care model implementation. We identified a 48.2% (95% CI: 39.4-56.7, p < .001) increase in referral for genetic counseling following implementation of the new care model. Median time from diagnosis to referral decreased by 74.0 days (p < .001) and median time from referral to first appointment by 54.0 days (p < .001). 56.3% of women desired referral at the time of diagnosis. This care model for women newly diagnosed with EOC in Nova Scotia was successful in increasing referral rates for genetic counseling. Majority of women pursued genetic testing following and favored that referral for genetic counseling be made at the time of diagnosis, highlighting the importance for timely access.

Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine.

Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, we know little about immediate effects of commensal bacteria on stem cell division programs. In this study, we examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. We identified Lactobacillus brevis as a potent stimulator of stem cell divisions. In a wild-type midgut, L.brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L.brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, we showed that L.brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, our data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment.

Clinical predictors of a positive test result in patients undergoing genetic evaluation for a hereditary kidney cancer syndrome.

INTRODUCTION: Guidelines are available to assist providers in identifying patients with renal cell carcinoma (RCC) that may benefit from genetic counselling, however, the evidence for these recommendations lacks support from the literature and controversy remains as to who should be referred. We aimed to delineate risk factors associated with a positive genetic test in a real-life cohort of patients with RCC referred to a regional medical genetics unit for evaluation of a hereditary kidney cancer syndrome. METHODS: Patients with a diagnosis of RCC referred to Maritime Medical Genetics Service (Nova Scotia, Canada) from 2006-2017 were reviewed using retrospective data. The primary outcome was identification of clinical features that were associated with a positive test result. Logistic regression models were used for analysis. RESULTS: A total of 135 patients were referred to medical genetics for evaluation; 102 patients were evaluated, 75 underwent testing, and 74 were included in the final analysis. Five patients tested positive: three Birt-Hogg-Dubé, one Cowden syndrome, and one Von Hippel-Lindau. Presence of dermatological lesions (specifically fibrofolliculomas) and more than two high-risk features were the only predictors of a positive test result. CONCLUSIONS: The presence of dermatological lesions and more than two high-risk features are the only predictors of a positive test result in patients with a suspected hereditary kidney cancer syndrome. These findings are not reflected in current guidelines, and the clinical implementation of our results may improve the identification of high-risk patients for genetic counselling.

Vibrio cholerae-Symbiont Interactions Inhibit Intestinal Repair in Drosophila.

Pathogen-mediated damage to the intestinal epithelium activates compensatory growth and differentiation repair programs in progenitor cells. Accelerated progenitor growth replenishes damaged tissue and maintains barrier integrity. Despite the importance of epithelial renewal to intestinal homeostasis, we know little about the effects of pathogen-commensal interactions on progenitor growth. We find that the enteric pathogen Vibrio cholerae blocks critical growth and differentiation pathways in Drosophila progenitors, despite extensive damage to epithelial tissue. We show that the inhibition of epithelial repair requires interactions between the Vibrio cholerae type six secretion system and a community of common symbiotic bacteria, as elimination of the gut microbiome is sufficient to restore homeostatic growth in infected intestines. This work highlights the importance of pathogen-symbiont interactions for intestinal immune responses and outlines the impact of the type six secretion system on pathogenesis.

The Immune Deficiency Pathway Regulates Metabolic Homeostasis in Drosophila.

Immune and metabolic pathways collectively influence host responses to microbial invaders, and mutations in one pathway frequently disrupt activity in another. We used the Drosophila melanogaster model to characterize metabolic homeostasis in flies with modified immune deficiency (IMD) pathway activity. The IMD pathway is very similar to the mammalian TNF-α pathway, a key regulator of vertebrate immunity and metabolism. We found that persistent activation of IMD resulted in hyperglycemia, depleted fat reserves, and developmental delays, implicating IMD in metabolic regulation. Consistent with this hypothesis, we found that imd mutants weigh more, are hyperlipidemic, and have impaired glucose tolerance. To test the importance of metabolic regulation for host responses to bacterial infection, we challenged insulin pathway mutants with lethal doses of several Drosophila pathogens. We found that loss-of-function mutations in the insulin pathway impacted host responses to infection in a manner that depends on the route of infection and the identity of the infectious microbe. Combined, our results support a role for coordinated regulation of immune and metabolic pathways in host containment of microbial invaders.

Constitutive Immune Activity Promotes Tumorigenesis in Drosophila Intestinal Progenitor Cells.

Gut innate immune defenses control bacterial populations and protect the host interior from invasion. Although excess intestinal immune activity frequently promotes inflammatory illnesses, we know little about the consequences of chronic innate immune activity exclusively in endodermal gut cells of an otherwise normal animal. To address this question, we examined the consequences of persistent inflammatory signals in adult fly intestinal progenitor cells. We found that constitutive immune activity disrupts expression of homeostatic regulators such as Notch pathway components and induces hyperplasia throughout the gut. Consistent with these observations, we found that persistent immune signals interfere with progenitor cell differentiation and exacerbate the formation of Notch-dependent intestinal tumors. These findings uncover a link between constitutive immune activity and tumorigenesis in intestinal stem cells.

Stability of Attitudes to the Ethical Issues Raised by the Return of Incidental Genomic Research Findings in Children: A Follow-Up Study.

OBJECTIVE: We explore the stability of parental attitudes to the ethical issues raised by the return of genomic research results. METHODS: A 19-item questionnaire was mailed to participants in a large genome research consortium 18 months following a baseline survey. We describe the stability of parental attitudes to (a) sharing of genomic research results, (b) endorsement of children in genomic research, (c) responsibilities of researchers, and (d) responsibilities to extended family. We also explore their experience in receiving results. RESULTS: Of 170 original participants, 154 (91%) responded. Most participants expressed positive rights to receive incidental genomic research findings (85%), including when ameliorative therapy was unknown (85%). Only 3% found it acceptable to delegate the decision to return results to an independent committee. Researchers, either with a parent (42%) or physician (17%), were felt to be responsible to convey research results to children when they reach adulthood. Most participants (74%) indicated that results should be shared with potentially affected extended family. These results are very similar to those of the baseline survey. All participants who received genomic results would do so again and reported actions similar to their expressed attitudes. CONCLUSIONS: The opinions of parents regarding genomic research remain stable over time. Guidelines on the return of results should incorporate these findings.

Attitudes of parents toward the return of targeted and incidental genomic research findings in children.

PURPOSE: We describe parental attitudes toward the return of targeted and incidental genomic research results in the setting of high-risk pediatric cancer and inherited childhood diseases. METHODS: A validated 36-item questionnaire was mailed to participants in three large-scale genome research consortia examining attitudes toward receipt of genomic research results and the influence of certainty, severity, and onset of the condition, in addition to responsibilities to extended family and provision of results even after death of the proband. RESULTS: Of the 563 participants who were sent questionnaires, 362 (64%) responded. Most of them stated a positive right to receive results related to the target condition (97%) or to incidental findings (86%); no difference was found in results between participants with cancer and those with orphan diseases. Furthermore, 92% indicated that genomic research for childhood-onset conditions should occur. The majority wanted incidental results predicting susceptibility even to untreatable fatal conditions (83%), to multiple conditions (87%), or to those with uncertain impact (70%). Most felt sibling genomic results showing serious conditions, whether treatable (93%) or not (88%), and/or results discovered after death of the proband should be shared with family (74%). CONCLUSION: Many parents of children in pediatric genomic research indicated a strong desire to receive a broader range of results than is described in consensus recommendations. Clear delineation of what will be offered should be established at the time of consent.

Mutations in origin recognition complex gene ORC4 cause Meier-Gorlin syndrome.

Meier-Gorlin syndrome is a rare autosomal recessive genetic condition whose primary clinical hallmarks include small stature, small external ears and small or absent patellae. Using marker-assisted mapping in multiple families from a founder population and traditional coding exon sequencing of positional candidate genes, we identified three different mutations in the gene encoding ORC4, a component of the eukaryotic origin recognition complex, in five individuals with Meier-Gorlin syndrome. In two such individuals that were negative for mutations in ORC4, we found potential mutations in ORC1 and CDT1, two other genes involved in origin recognition. ORC4 is well conserved in eukaryotes, and the yeast equivalent of the human ORC4 missense mutation was shown to be pathogenic in functional assays of cell growth. This is the first report, to our knowledge, of a germline mutation in any gene of the origin recognition complex in a vertebrate organism.

Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

Mutation in pyrroline-5-carboxylate reductase 1 gene in families with cutis laxa type 2.

Autosomal-recessive cutis laxa type 2 (ARCL2) is a multisystem disorder characterized by the appearance of premature aging, wrinkled and lax skin, joint laxity, and a general developmental delay. Cutis laxa includes a family of clinically overlapping conditions with confusing nomenclature, generally requiring molecular analyses for definitive diagnosis. Six genes are currently known to mutate to yield one of these related conditions. We ascertained a cohort of typical ARCL2 patients from a subpopulation isolate within eastern Canada. Homozygosity mapping with high-density SNP genotyping excluded all six known genes, and instead identified a single homozygous region near the telomere of chromosome 17, shared identically by state by all genotyped affected individuals from the families. A putative pathogenic variant was identified by direct DNA sequencing of genes within the region. The single nucleotide change leads to a missense mutation adjacent to a splice junction in the gene encoding pyrroline-5-carboxylate reductase 1 (PYCR1). Bioinformatic analysis predicted a pathogenic effect of the variant on splice donor site function. Skipping of the associated exon was confirmed in RNA from blood lymphocytes of affected homozygotes and heterozygous mutation carriers. Exon skipping leads to deletion of the reductase functional domain-coding region and an obligatory downstream frameshift. PYCR1 plays a critical role in proline biosynthesis. Pathogenicity of the genetic variant in PYCR1 is likely, given that a similar clinical phenotype has been documented for mutation carriers of another proline biosynthetic enzyme, pyrroline-5-carboxylate synthase. Our results support a significant role for proline in normal development.

Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia.

The sideroblastic anemias are a heterogeneous group of congenital and acquired hematological disorders whose morphological hallmark is the presence of ringed sideroblasts--bone marrow erythroid precursors containing pathologic iron deposits within mitochondria. Here, by positional cloning, we define a previously unknown form of autosomal recessive nonsyndromic congenital sideroblastic anemia, associated with mutations in the gene encoding the erythroid specific mitochondrial carrier family protein SLC25A38, and demonstrate that SLC25A38 is important for the biosynthesis of heme in eukaryotes.