Leber先天性黑蒙临床与GUCY2D基因突变分析

目的 分析Leber先天性黑蒙的临床特点及其候选基因变异情况。方法 连续收集分析27例年龄4mo-18a的Leber先天性黑蒙先证者临床资料,应用PCR-异源双链-SSCP法分析GUCY2D基因外显子2和8,寻找可能的变异结果 27例患者均在2a以内出现视力差或对光、物体无反应,最最好视力小于0.1。症状年龄21例在3mo内,27例均有眼球震颤（其中15例为眼球扫视运动）,22例有眼底异常,5例眼底未见异常,3例有家族史并呈常染色体隐性遗传,ERG锥杆反应重度下降或记录不到波,未发现GUCY2D基因突变。结论 Leber先天 性黑蒙临床表现多样,诊断有赖于ERG。本组病例可能与GUCY2D外显子2、8突变无关。     

Gucy2f zebrafish knockdown--a model for Gucy2d-related leber congenital amaurosis

Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible.     

A novel recessive GUCY2D mutation causing cone�Crod dystrophy and not Leber's congenital amaurosis

Cone–rod dystrophies are inherited retinal dystrophies that are characterized by progressive degeneration of cones and rods, causing an early decrease in central visual acuity and colour vision defects, followed by loss of peripheral vision in adolescence or early adult life. Both genetic and clinical heterogeneity are well known. In a family with autosomal recessive cone–rod dystrophy, genetic analyses comprising genome scan with microsatellite markers, fine mapping and candidate gene approach resulted in the identification of a homozygous missense GUCY2D mutation. This is the first GUCY2D mutation associated with autosomal recessive cone–rod dystrophy rather than Leber''s congenital amaurosis (LCA), a severe disease leading to childhood blindness. This study hence establishes GUCY2D, which is a common cause for both recessive LCA and dominant cone–rod dystrophy, as a good candidate for autosomal recessive cone–rod dystrophy.     

GUCY2C lysosomotropic endocytosis delivers immunotoxin therapy to metastatic colorectal cancer

The emergence of targeted cancer therapy has been limited by the paucity of determinants which are tumor-specific and generally associated with disease, and have cell dynamics which effectively deploy cytotoxic payloads. Guanylyl cyclase C (GUCY2C) may be ideal for targeting because it is normally expressed only in insulated barrier compartments, including intestine and brain, but over-expressed by systemic metastatic colorectal tumors. Here, we reveal that GUCY2C rapidly internalizes from the cell surface to lysosomes in intestinal and colorectal cancer cells. Endocytosis is independent of ligand binding and receptor activation, and is mediated by clathrin. This mechanism suggests a design for immunotoxins comprising a GUCY2C-directed monoclonal antibody conjugated through a reducible disulfide linkage to ricin A chain, which is activated to a potent cytotoxin in lysosomes. Indeed, this immunotoxin specifically killed GUCY2C-expressing colorectal cancer cells in a lysosomal- and clathrin-dependent fashion. Moreover, this immunotoxin reduced pulmonary tumors >80% (p<0.001), and improved survival 25% (p<0.001), in mice with established colorectal cancer metastases. Further, therapeutic efficacy was achieved without histologic evidence of toxicity in normal tissues. These observations support GUCY2C-targeted immunotoxins as novel therapeutics for metastatic tumors originating in the GI tract, including colorectum, stomach, esophagus, and pancreas.     

Evidence of a founder effect for the RETGC1 (GUCY2D) 2943DelG mutation in Leber congenital amaurosis pedigrees of Finnish origin

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies. It is a genetically heterogeneous condition as six disease-causing genes have been hitherto identified. Among them, RETGC1 (GUCY2D), is more frequently implicated in our series of LCA patients. Interestingly, 70 % of the families with RETGC1 mutations are originating from Mediterranean countries, the remaining families (30%) being originating from various countries across the world. Here, we report, the identification of the same homozygous RETGC1 nonsense mutation in three unrelated and non-consanguineous LCA families of Finnish origin, suggesting a founder effect. Interestingly, no linkage desequilibrium was found using polymorphic markers flanking the RETGC1 gene, supporting the view that the mutation is very ancient. Haplotype studies and Bayesian calculation point the founder mutation to 150 generations (95% credible interval 80-240 generations), i.e., 3000 years ago.     

An activating gucy2c mutation causes impaired contractility and fluid stagnation in the small bowel

Objective: Familial GUCY2C diarrhoea syndrome (FGDS) is caused by an activating mutation in the GUCY2C gene encoding the receptor guanylate cyclase C in enterocytes. Activation leads to increased secretion of fluid into the intestinal lumen. Twenty percent of the patients have increased risk of Crohn’s disease and intestinal obstruction (CD, 20%) and the condition resembles irritable bowel syndrome with diarrhoea. We aimed to describe fluid content, contractility, peristaltic activity and bowel wall thickness in the intestine in fasting FGDS patients, using ultrasound, with healthy volunteers serving as controls.

Methods: Twenty-three patients with FGDS and 22 healthy controls (HC) were examined with a Logiq E9 scanner in a fasting state. Bowel wall thickness was measured and fluid-filled small bowel loops were counted using three-dimensional (3D) magnetic positioning navigation. The HC ingested 500?ml PEG solution, an electrolyte balanced, non-absorbable solution, in order to investigate the contractions of the small bowel.

Results: The fasting 23 FGDS patients had significantly higher number of fluid-filled small bowel segments compared to 22 fasting HC, p?p?Conclusions: FGDS is characterised by multiple, fluid-filled small bowel loops with incomplete contractions and fluid stagnation in fasting state. These findings may play a role in the increased risk of bowel obstruction as well as IBS-like symptoms observed in these patients.     

Plasma levels of guanylins are reduced in patients with Crohn’s disease

Abstract

Background: Guanylin (GN) and uroguanylin (UGN) are endogenous ligands for the intestinal receptor guanylate cyclase C (GC-C), an important regulator of intestinal fluid homeostasis. Gene expression and protein levels of GN are suppressed in inflamed intestinal tissue from patients with inflammatory bowel disease (IBD), but knowledge about plasma levels of guanylins in these conditions is sparse. We aimed to investigate the fasting plasma levels of the prohormones proGN and proUGN in patients with Crohn’s Disease (CD) and relate these to levels found in persons with other diarrheal conditions, as well as persons with normal bowel habits.

Methods: Plasma from patients with CD, patients with Familial GUCY2C Diarrheal Disease (FGDS), diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls (HC) was analyzed using ELISA assays.

Results: Significantly lower fasting plasma levels of proguanylins were found in CD and FGDS patients, compared to HC. In CD patients, plasma proGN levels correlated negatively with Harvey Bradshaw Index and with number of stools/24?h.

Conclusion: Our data indicate that diarrhea may be a determinant for levels of proGN in plasma, and should be further explored in studies of different diarrheal disorders.