How do stochastic processes and genetic threshold effects explain incomplete penetrance and inform causal disease mechanisms?

Incomplete penetrance is the rule rather than the exception in Mendelian disease. In syndromic monogenic disorders, phenotypic variability can be viewed as the combination of incomplete penetrance for each of multiple independent clinical features. Within genetically identical individuals, such as isogenic model organisms, stochastic variation at molecular and cellular levels is the primary cause of incomplete penetrance according to a genetic threshold model. By defining specific probability distributions of causal biological readouts and genetic liability values, stochasticity and incomplete penetrance provide information about threshold values in biological systems. Ascertainment of threshold values has been achieved by simultaneous scoring of relatively simple phenotypes and quantitation of molecular readouts at the level of single cells. However, this is much more challenging for complex morphological phenotypes using experimental and reductionist approaches alone, where cause and effect are separated temporally and across multiple biological modes and scales. Here I consider how causal inference, which integrates observational data with high confidence causal models, might be used to quantify the relative contribution of different sources of stochastic variation to phenotypic diversity. Collectively, these approaches could inform disease mechanisms, improve predictions of clinical outcomes and prioritize gene therapy targets across modes and scales of gene function. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'

Locating a Tree in a Phylogenetic Network in Quadratic Time

International audienceA fundamental problem in the study of phylogenetic networks is to determine whether or not a given phylogenetic network contains a given phylogenetic tree. We develop a quadratic-time algorithm for this problem for binary nearly-stable phylogenetic networks. We also show that the number of reticulations in a reticulation visible or nearly stable phylogenetic network is bounded from above by a function linear in the number of taxa

Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions

Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124-2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions

The decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy-directed biopsy showing high-grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high-risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124-2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124-2 methylation status in cytology. E4-positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124-2. We also examined whether these biomarkers can predict the histological outcome of the LE

Mutational analyses of UPIIIA, SHH, EFNB2, and HNF1β in persistent cloaca and associated kidney malformations

OBJECTIVES: ‘Persistent cloaca’ is a severe malformation affecting females in which the urinary, genital and alimentary tracts share a single conduit. Previously, a Uroplakin IIIA (UPIIIA) mutation was reported in one individual with persistent cloaca, and UPIIIA, Sonic Hedgehog (SHH), Ephrin B2 (EFNB2) and Hepatocyte Nuclear Factor 1β (HNF1β) are expressed during the normal development of organs that are affected in this condition. HNF1β mutations have been associated with uterine malformations in humans, and mutations of genes homologous to human SHH or EFNB2 cause persistent cloaca in mice. PATIENTS AND METHODS: We sought mutations of coding regions of UPIIIA, SHH, EFNB2 and HNF1β genes by direct sequencing in a group of 20 patients with persistent cloaca. Most had associated malformations of the upper renal tract and over half had impaired renal excretory function. The majority of patients had congenital anomalies outside the renal/genital tracts and two had the VACTERL association. RESULTS: Apart from a previously described index case, we failed to find UPIIIA mutations, and no patient had a SHH, EFNB2 or HNF1β mutation. CONCLUSION: Persistent cloaca is only rarely associated with UPIIIA mutation. Despite the fact that SHH and EFNB2 are appealing candidate genes, based on their expression patterns and mutant mice phenotypes, they were not mutated in these humans with persistent cloaca. Although HNF1β mutations can perturb paramesonephric duct fusion in humans, HNF1β was not mutated in persistent cloaca

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3–95.2) and 31.8% (95%CI 27.7–36.1) for hrHPV, 77.8% (95%CI 66.9–85.8) and 69.3% (95%CI 65.0–73.3) for methylation biomarkers and 93.1% (95%CI 84.8–97.0) and 49.4% (95%CI 44.8–53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6–95.8), methylation positivity in 75.8% (95%CI 64.2–84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5–96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6–99.7) with a specificity of 46.3% (95%CI 40.8–51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions