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The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report biallelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

TALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analysed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non cell-autonomous manner. Analysis of human gut fetal tissues with aKIAA0586mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning

BackgroundHirschsprung disease (HSCR) is characterized by absence of ganglia in the intestine. Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). HSCR is a complex genetic disease in which the loss of enteric ganglia stems from a combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Pinpointing the responsible culprits within a large CNV is challenging as often many genes are affected. We investigated if we could find deleterious CNVs and if we could identify the genes responsible for the aganglionosis.ResultsDeleterious CNVs were detected in three groups of patients: HSCR-AAM, HSCR patients with a confirmed causal genetic variant and HSCR-isolated patients without a known causal variant and controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients withoutRETcoding variants. CNV profiling proved that HSCR-AAM patients had larger copy number losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes in Copy Number Losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution ofUFD1L, TBX2, SLC8A1andMAPK8to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expressionin vivo.ConclusionRare large Copy Number losses - oftende novo- contribute to the disease in HSCR-AAM patients specifically. We proved the involvement of five genes in enteric nervous system development and Hirschsprung disease.

Aim of the Study Patients born with esophageal atresia (EA) appear to have a 30 times higher prevalence of infantile hypertrophic pyloric stenosis (IHPS). This makes sense from a developmental perspective as both the esophagus and the pyloric sphincter are foregut derived structures. We hypothesized that genetic defects, disturbing foregut morphogenesis, are responsible for the specific combination of EA and IHPS. Methods Patients with both EA and IHPS born between 1970 and 2017 and where possible their parents were included. This study was approved by the internal review board of the Erasmus MC. After parental written informed consent, we determined genetic profiles with whole exome sequencing and SNP-array-based copy number variation analysis. We focused on (1) genetic variation in known EA and IHPS disease genes, (2) pathways important for foregut morphogenesis, (3) shared rare genetic variation, and (4) ultrarare variants in variant-intolerant genes, which have a high chance of being de novo. Segregation analysis of possible candidate variants was performed. Results Twenty-seven out of 664 patients (4.1%) born with EA during the study period developed IHPS, of which 15 cases were analyzed. As none of the parents were affected, we considered dominant (de novo) or recessive and X-linked inheritance models. Unfortunately, we could neither identify rare de novo mutations, nor variants fitting a recessive model. We did however identify inherited putative deleterious heterozygous variants in genes either known to be involved in EA or IHPS (e.g., COL7A1, TNXB, WDR11, GDF6) or important in foregut morphogenesis (e.g., GLI3, NKX2.1) in all patients. Moreover, seven genes were affected by rare variation in ≥2 patients (ADAMTSL4, ANKRD26, CNTN2, HSPG2, KCNN3, LDB3, SEC16B) and expressed in the developing foregut, esophagus or pyloric sphincter in mice between E8.25 and E18.5. However, burden analysis did not show a significant difference with unaffected controls. Conclusions Although the presence of genetic variation in likely candidate genes suggests a genetic component, genetic factors alone could not explain the abnormalities seen in these patients. Therefore, we propose a multifactorial model in which the combination of high impact genetic, mechanical, and environmental factors together can shift the balance from normal to abnormal development.

The triple gene block (TGB; consisting of proteins TGB1–3) and coat protein (CP) of white clover mosaic potexvirus (WClMV) are required for cell-to-cell movement of viral RNA. Cell-to-cell spread of WClMV mutants in which the TGB open reading frames had been mutated was rescued in transgenic plants expressing specific TGB proteins (TGBPs). This indicated that there are no requirements for the synthesis in cis of viral TGBPs. These transgenic plants provided an experimental framework to explore the roles performed by the TGBPs and CP in cell-to-cell movement of WClMV RNA. Microinjection experiments established that TGB1 functions as the WClMV cell-to-cell movement protein (MP). Furthermore, combined microinjection and dual-channel confocal laser scanning microscopy provided direct evidence that infectious transcripts of WClMV move cell to cell as a ribonucleoprotein complex, consisting of single-stranded RNA, TGB1, and CP. Movement of this ribonucleoprotein complex displayed an absolute requirement for the presence of both TGB2 and TGB3. A model consistent with these findings is presented.

Traditional perspectives on the origins of Southern settlement patterns have undergone major revisions during the past two decades. In particular, scholars have focused increasingly on the transition from dispersed agrarian communities to the formation of town-based settlement systems during the eighteenth century. Such systems were first developed in the northern Shenandoah Valley in the Virginia backcountry. Three phases of settlement evolution occurred. A pioneer, open-country neighborhood system existed between 1730 and the mid-1740s. This was followed by the founding of Winchester and the initiation of a primate market-town system between the late 1740s and the mid-1770s. A well-developed central-place system was finally evident by the 1790s. Although this evolution was closely associated with the shift from local subsistence farming to rising consumerism associated with commercial livestock and grain production, current settlement theories have provided incomplete explanations of the founding of individual towns and of differential town growth. Private initiatives in response to the need for sites for local government functions and the commitment of local settlers to town residence were crucial to village formation and selective town status. While the export trade in farm commodities remained decentralized, control of the import trade in dry goods by Winchester merchants, financed by liberal extensions of credit using land as collateral, was essential to the maintenance of Winchester's position as a regional entrepot.

Sarcomas, including the malignant fibrous histiocytomas (MFHs), are not known to be part of the tumour spectrum of hereditary non-polyposis colorectal cancer (HNPCC) as epidemiologically established. Therefore, occurrence of MFH in an HNPCC family may very well be coincidental. HNPCC is associated with germline mutations in DNA mismatch repair genes, including the MSH2 gene. We analysed an MFH diagnosed in a 45-year-old male HNPCC patient carrying a germline MSH2 mutation for HNPCC-associated molecular characteristics, to investigate a possible relationship between the tumour and that mutation. DNA analysis revealed microsatellite instability and loss of one MSH2 copy, and immunohistochemistry showed absence of nuclear MSH2 protein staining. To investigate whether this is a common finding in MFH, microsatellite instability and nuclear MSH2 protein staining was tested for in 5 and 6 sporadic MFHs, respectively. None showed microsatellite instability and all stained positively for MSH2. Together, these findings show that in rare cases, MFH may be part of the HNPCC tumour spectrum.

Chapter 1 Preface Part 2 Part One Chapter 3 The Making of George Washington Chapter 4 Young Washington's Virginia: Opportunity in the "Golden Age" of a Planter Society Part 5 Part Two Chapter 6 "Over the Hills and Far Away": George Washington and the Changing Virginia Backcountry Chapter 7 "A Parcel of Barbarian's and an Uncooth Set of People": Settlers and Settlements of the Shenandoah Valley Chapter 8 Engaged in Enterprises Pregnant with Terror": George Washington's Formative Years among the Indians Part 9 Part Three Chapter 10 A Stake in the West: George Washington as Backcountry Surveyor and Landholder Chapter 11 School for Command: Young George Washington and the Virginia Regiment Chapter 12 George Washington and Revolutionary Asceticism: The Localist as Nationalist Chapter 13 Contributors Chapter 14 Index