Patient and multidisciplinary team perspectives on watch and wait in rectal cancer.

This article adopts a multidisciplinary approach, including surgery, oncology, radiology and patient perspectives, to discuss the key points of debate surrounding a watch and wait approach. In an era of shared decision-making, discussion of watch and wait as an option in the context of complete clinical response is appropriate, although it is not the gold standard treatment. Key challenges are the difficulty in assessing for a complete clinical response, prediction of recurrence and access to timely diagnostics for surveillance. Salvage surgery has good results if regrowth is detected early but does have imperfect outcomes, with only a 90% salvage rate. Good communication with patients about the risks and alternatives is essential. Patients undergoing watch and wait should ideally be enrolled in prospective registries or clinical trials.

Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage.

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.

Robust Genetic Analysis of the X-Linked Anophthalmic (Ie) Mouse.

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.

Right Ventricular Clot in Transit in a Patient with Coronavirus Disease 2019 (COVID-19) Pneumonia Successfully Treated with Thrombolytics.

Background: Right heart thrombus or clot in transit is a rare venous thromboembolism (VTE) with high mortality. COVID-19 infection has been associated with increased risk of such events. We present the case of a 63-year-old man with no traditional VTE risk factors who was diagnosed with a clot in transit three weeks after diagnosis of COVID-19. Clinical Case. A 63-year-old male with no significant past medical history who presented to the emergency department with shortness of breath. He tested positive for COVID-19 three weeks prior. His oxygen saturation was 60% on room air and was put on nonrebreather mask. He was still showing signs of respiratory distress including tachypnea, tachycardia, diaphoresis, and accessory muscle use. The patient was subsequently intubated and mechanically ventilated. Chest computed tomography with contrast showed acute bilateral pulmonary emboli with flattening of the interventricular septum suggestive of right heart strain. Bedside echocardiogram showed severely enlarged right ventricle with reduced systolic function and evidence of right ventricular strain and a mobile echodensity in the right ventricle attached to the tricuspid valve consistent with a clot in transit. The patient was treated with full dose systemic thrombolysis with rapid improvement in his symptoms. He was extubated the following day and a repeat echocardiogram showed resolution of the clot in transit. Conclusion: Clot in transit is rare but can occur in COVID-19 patients even in the absence of traditional thromboembolism risk factors. Management includes systemic anticoagulation, systemic thrombolysis, and surgical embolectomy. Our patient was successfully treated with systemic thrombolysis.

The Dct-/- Mouse Model to Unravel Retinogenesis Misregulation in Patients with Albinism.

We have recently identified DCT encoding dopachrome tautomerase (DCT) as the eighth gene for oculocutaneous albinism (OCA). Patients with loss of function of DCT suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in Dct-/- mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages, contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanization. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of Dct-/- newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient Tyrc/c embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in Dct-/- postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The Dct-/- mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.

Cyclin-dependent Kinase 9 as a Potential Target for Anti-TNF-resistant Inflammatory Bowel Disease.

BACKGROUND & AIMS: Resistance to single cytokine blockade, namely anti-tumor necrosis factor (TNF) therapy, is a growing concern for patients with inflammatory bowel disease (IBD). The transcription factor T-bet is a critical regulator of intestinal homeostasis, is genetically linked to mucosal inflammation and controls the expression of multiples genes such as the pro-inflammatory cytokines interferon (IFN)-γ and TNF. Inhibiting T-bet may therefore offer a more attractive prospect for treating IBD but remains challenging to target therapeutically. In this study, we evaluate the effect of targeting the transactivation function of T-bet using inhibitors of P-TEFb (CDK9-cyclin T), a transcriptional elongation factor downstream of T-bet. METHODS: Using an adaptive immune-mediated colitis model, human colonic lymphocytes from patients with IBD and multiple large clinical datasets, we investigate the effect of cyclin-dependent kinase 9 (CDK9) inhibitors on cytokine production and gene expression in colonic CD4+ T cells and link these genetic modules to clinical response in patients with IBD. RESULTS: Systemic CDK9 inhibition led to histological improvement of immune-mediated colitis and was associated with targeted suppression of colonic CD4+ T cell-derived IFN-γ and IL-17A. In colonic lymphocytes from patients with IBD, CDK9 inhibition potently repressed genes responsible for pro-inflammatory signalling, and in particular genes regulated by T-bet. Remarkably, CDK9 inhibition targeted genes that were highly expressed in anti-TNF resistant IBD and that predicted non-response to anti-TNF therapy. CONCLUSION: Collectively, our findings reveal CDK9 as a potential target for anti-TNF-resistant IBD, which has the potential for rapid translation to the clinic.

Investigating the genetic architecture of eye colour in a Canadian cohort.

Eye color is highly variable in populations with European ancestry, ranging from low to high quantities of melanin in the iris. Polymorphisms in the HERC2/OCA2 locus have the largest effect on eye color in these populations, although other genomic regions also influence eye color. We performed genome-wide association studies of eye color in a Canadian cohort of European ancestry (N = 5,641) and investigated candidate causal variants. We uncovered several candidate causal signals in the HERC2/OCA2 region, whereas other loci likely harbor a single causal signal. We observed colocalization of eye color signals with the expression or methylation profiles of cultured primary melanocytes. Genetic correlations of eye and hair color suggest high genome-wide pleiotropy, but locus-level differences in the genetic architecture of both traits. Overall, we provide a better picture of the polymorphisms underpinning eye color variation, which may be a consequence of specific molecular processes in the iris melanocytes.

Role of procalcitonin as a predictor of clinical outcomes in hospitalized patients with COVID-19.

OBJECTIVES: In this study, we aimed to determine the correlation between procalcitonin (PCT) levels and clinical outcomes including in-hospital mortality, intensive care unit (ICU) length of stay, and hospital length of stay in patients hospitalized with COVID-19. METHODS: Clinical, laboratory, and demographic data of 223 patients who met inclusion criteria were analyzed. PCT measurements of 0.25 ng/mL and 0.50 ng/mL were used to stratify patients into 2 mutually exclusive groups. RESULTS: Patients with PCT above 0.25 ng/mL on admission had significantly elevated Acute Physiology and Chronic Health Evaluation II scores (9 vs 8; P = 0.042) and C-reactive proteins levels (111 µg/mL vs 79 µg/mL; P = 0.007). A multivariable binary logistic regression model demonstrated no relationship between PCT and mortality (OR = 1.00; 95% Cl: 0.97 to 1.02; P = 0.713). Kaplan-Meier analysis revealed no statistical evidence of a difference between PCT groups and hospital length of stay (P = 0.144 for 0.25 ng/mL, P = 0.368 for 0.50 ng/mL) or intensive care unit length of stay (P = 0.986 for 0.25 ng/mL, P = 0.771 for 0.50 ng/mL). CONCLUSIONS: Elevated PCT levels were associated with severity of illness but did not correlate with in-hospital mortality, hospital length of stay, or ICU length of stay.

A population of naive-like CD4+ T cells stably polarized to the TH 1 lineage.

T-bet is the lineage-specifying transcription factor for CD4+ TH 1 cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.

Chylopericardium due to Subclavian Vein Thrombosis in the Setting of Protein S Deficiency.

BACKGROUND: Chylopericardium is the accumulation of lymphatic fluid in the pericardial cavity. It can be idiopathic or secondary to trauma, cardiothoracic surgery, neoplasm, radiation, tuberculosis, lymphatic duct dysfunction, thrombosis, or other causes. We present a case of chylopericardium due to subclavian vein thrombosis in a patient with protein S deficiency. Clinical Case. A 48-year-old man with a history of protein S deficiency presented to the emergency department with shortness of breath and a productive cough. CT of the chest showed pulmonary emboli, moderate pericardial effusion, and a large thrombus of the superior vena cava, brachiocephalic vein, and subclavian veins. He developed echocardiographic evidence of cardiac tamponade so he underwent pericardiocentesis with drainage of milky-appearing fluid. Analysis of the fluid showed elevated triglycerides consistent with chylopericardium. The pericardial effusion reaccumulated, likely secondary to lymphatic duct obstruction due to his subclavian vein thrombus. Catheter-assisted thrombolysis was performed with resolution of the patient's effusion and symptoms. CONCLUSION: Chylopericardium is a rare but important complication of subclavian vein thrombosis. Management is typically with surgical intervention, although our case represents successful treatment with catheter-assisted thrombolysis.

A mouse model of brittle cornea syndrome caused by mutation in Zfp469.

Brittle cornea syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the collagen type I/collagen type V ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of collagen type I, offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus in which the cornea thins progressively. This article has an associated First Person interview with the first author of the paper.

Hypokalemic Periodic Paralysis Precipitated by Thyrotoxicosis and Renal Tubular Acidosis.

BACKGROUND: Hypokalemic periodic paralysis is a rare neuromuscular disorder characterized by transient episodes of flaccid paralysis due to a defect in muscle ion channels. Most cases are hereditary, but it can be acquired. We present a case of acquired hypokalemic periodic paralysis associated with hyperthyroidism and renal tubular acidosis. Clinical Case. A 38-year-old female with a history of Graves' disease presented to the emergency department with generalized weakness and associated nausea, vomiting, and weight loss. Examination was significant for diffuse weakness in all extremities. Labs showed hypokalemia, hyperthyroidism, and nonanion gap metabolic acidosis with a positive urine anion gap. She was treated for hypokalemic periodic paralysis and renal tubular acidosis. Potassium replacement, propranolol, methimazole, and sodium bicarbonate were initiated. Her potassium gradually corrected with resolution of her symptoms. Further investigation revealed a history of dry eyes, dry mouth, and recurrent dental carries. She had positive ANA, SS-A, and SS-B antibodies. She was diagnosed with Sjögren's syndrome, which may have been associated with her Graves' disease and thus contributed to both her RTA and hyperthyroidism. CONCLUSION: Early recognition and treatment of thyrotoxic periodic paralysis are important to prevent cardiac complications. Management includes potassium replacement with careful monitoring to prevent rebound hyperkalemia. The definitive treatment is to achieve euthyroid status.

A custom capture sequence approach for oculocutaneous albinism identifies structural variant alleles at the OCA2 locus.

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.

A Crohn's Disease-associated IL2RA Enhancer Variant Determines the Balance of T Cell Immunity by Regulating Responsiveness to IL-2 Signalling.

BACKGROUND AND AIMS: Differential responsiveness to interleukin [IL]-2 between effector CD4+ T cells [Teff] and regulatory T cells [Treg] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn's disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. METHODS: Teff and Treg were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. Teff pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. RESULTS: Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by Teff in response to IL-2 stimulation in vitro. Teff from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. CONCLUSIONS: rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective Treg modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype.

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function.

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.

Genetic background modifies vulnerability to glaucoma-related phenotypes in Lmx1b mutant mice.

Variants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst ) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+ /J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D -induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.

Dopachrome tautomerase variants in patients with oculocutaneous albinism.

PURPOSE: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. METHODS: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. RESULTS: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. CONCLUSION: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8.

Divergent Impact of Glucose Availability on Human Virus-Specific and Generically Activated CD8 T Cells.

Upon activation T cells engage glucose metabolism to fuel the costly effector functions needed for a robust immune response. Consequently, the availability of glucose can impact on T cell function. The glucose concentrations used in conventional culture media and common metabolic assays are often artificially high, representing hyperglycaemic levels rarely present in vivo. We show here that reducing glucose concentration to physiological levels in culture differentially impacted on virus-specific compared to generically activated human CD8 T cell responses. In virus-specific T cells, limiting glucose availability significantly reduced the frequency of effector-cytokine producing T cells, but promoted the upregulation of CD69 and CD103 associated with an increased capacity for tissue retention. In contrast the functionality of generically activated T cells was largely unaffected and these showed reduced differentiation towards a residency phenotype. Furthermore, T cells being cultured at physiological glucose concentrations were more susceptible to viral infection. This setting resulted in significantly improved lentiviral transduction rates of primary cells. Our data suggest that CD8 T cells are exquisitely adapted to their niche and provide a reminder of the need to better mimic physiological conditions to study the complex nature of the human CD8 T cell immune response.