A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.     

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm^Flox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm^Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.     

Rapid degradation of dominant-negative Rab27 proteins <Emphasis Type="Italic">in vivo</Emphasis> precludes their use in transgenic mouse models

Background

Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.

Results

To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.

Conclusions

We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.

     

Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease.     

Novel <Emphasis Type="Italic">CHM</Emphasis> mutations in Polish patients with choroideremia – an orphan disease with close perspective of treatment

Background

Choroideremia (CHM) is a rare X-linked recessive retinal dystrophy characterized by progressive chorioretinal degeneration in the males affected. The symptoms include night blindness in childhood, progressive peripheral vision loss and total blindness in the late stages. The disease is caused by mutations in the CHM gene encoding Rab Escort Protein 1 (REP-1). The aim of the study was to identify the molecular basis of choroideremia in five families of Polish origin.

Methods

Six male patients from five unrelated families of Polish ethnicity, who were clinically diagnosed with choroideremia, were examined in this study. An ophthalmologic examination performed in all the probands included: best-corrected visual acuity, slit-lamp examination, funduscopy, fluorescein angiography and perimetry. The entire coding region encompassing 15 exons and the flanking intronic sequences of the CHM gene were amplified with PCR and directly sequenced in all the patients.

Results

Five variants in the CHM gene were identified in the five families examined. Two of the variants were new: c.1175dupT and c.83C?>?G, while three had been previously reported.

Conclusions

This study provides the first molecular genetic characteristics of patients with choroideremia from the previously unexplored Polish population.

     

Cloning, mapping and characterization of the human RAB27A gene

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.     

Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.     

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.¹ Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.² These include RPE65 gene replacement therapy in patients with Leber''s congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt''s disease.^3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.^5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.     

Biological and biochemical consequences of the human ERCC-1 repair gene after transfection into a repair-deficient CHO cell line

The consequences of the presence of the human gene ERCC1 in repair-deficient 43-3B cells were examined. The gene restores the sensitivity of this mutant not only to UV but also to 4NQO, N-Ac-AAF and alkylating agents to the normal level. Also, the frequency of mutation induction by UV at the Na⁺/K⁺-ATPase locus returns to the level of CHO wild-type cells. Additionally, the rate of cyclobutane pyrimidine dimer removal approaches that in wild-type CHO cells. The results obtained indicate that the human gene ERCC-1 restores the impaired functions in 43-3B, and that the gene is probably functionally homologous to the defective one in the 43-3B cell line. Some evidence was found for a difference between the human gene product and its rodent counterpart, as the restoration of normal sensitivity to 4NQO, ENU and N-Ac-AAF was complete whereas it was not for UV.     

Molecular basis for Rab prenylation

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Delta null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Delta null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6(ts)) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.     

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.     

Cycloheximide resistance in Chinese hamster cells I. Spontaneous mutagenesis

Resistance to cycloheximide (CHM) was studied in cultured Chinese hamster cells. Concentrations of CHM above 5.10⁻⁷M were toxic for the cells. At concentrations above 9.10⁻⁷M no colonies were recovered in selective medium. 15 resistant clones of independent origin were isolated in selective medium containing 7.10⁻⁷M CHM. Resistance was stable when the cells were cultured under non-selective conditions. The spontaneously mutation rate was determined by the fluctuation test. Mutations to CHM resistance arose spontaneously. The spontaneous mutation rate to CHM resistance was about 10⁻⁵.     

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8⁺ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8⁺ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8⁺ T cells, on the yield, phenotype, and functional activity of expanded CD8⁺ T cells during the REP. We found that CD8⁺ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG₄ (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8⁺ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8⁺ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8⁺ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.     

Can Chinese Herbal Medicine Improve Outcomes of In Vitro Fertilization? A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background

A large number of infertile couples are choosing Chinese herbal medicine (CHM) as an adjuvant therapy to improve their success when undergoing in vitro fertilization (IVF). There is no systematic review to evaluate the impact of CHM on the IVF outcomes.

Objective

To evaluate the effectiveness of CHM with concurrent IVF versus IVF alone on the outcomes of IVF and its safety.

Methods

The protocol of this study is registered at PROSPERO. Eligible RCTs searched from 8 databases which compared a combination of CHM and IVF with IVF alone were included. Two authors independently selected studies, extracted data and assessed methodological quality. Meta-analysis of RCTs was conducted if there was non-significant heterogeneity (evaluated by I² test) among trials. All statistical analysis was performed using RevMan 5.1 software.

Results

Twenty trials involving 1721 women were included in the meta-analysis. Three trials were evaluated as having an unclear risk of bias. The remaining trials were evaluated as having a high risk of bias. Combination of CHM and IVF significantly increases clinical pregnancy rates (OR 2.04, 95%CI 1.67 to 2.49, p<0.00001) and ongoing pregnancy rates (OR 1.91, 95%CI 1.17 to 3.10, p = 0.009). Use of CHM after embryo transfer had no better outcome in reducing the rate of ovarian hyper stimulation syndrome (OR 0.39, 95%CI 0.14 to 1.11, p = 0.08).

Conclusions

This meta-analysis showed that combination of IVF and CHM used in the included trials improve IVF success, however due to the high risk of bias observed with the trials, the significant differences found with the meta-analysis are unlikely to be accurate. No conclusion could be drawn with respect to the reproductive toxicity of CHM. Further large randomized placebo controlled trials are warranted to confirm these findings before recommending women to take CHM to improve their IVF success.     

Effect and safety of Chinese herbal medicine granules in patients with severe coronavirus disease 2019 in Wuhan,China: a retrospective,single-center study with propensity score matching

BackgroundChinese herbal medicine (CHM) has been used for severe illness caused by coronavirus disease 2019 (COVID-19), but its treatment effects and safety are unclear.PurposeThis study reviews the effect and safety of CHM granules in the treatment of patients with severe COVID-19.MethodsWe conducteda single-center, retrospective study on patients with severe COVID-19 in a designated hospital in Wuhan from January 15, 2020 to March 30, 2020. The propensity score matching (PSM) was used to assess the effect and safety of the treatment using CHM granules. The ratio of patients who received treatment with CHM granules combined with usual care and those who received usual care alone was 1:1. The primary outcome was the time to clinical improvement within 28 days, defined as the time taken for the patients’ health to show improvement by decline of two categories (from the baseline) on a modified six-category ordinal scale, or to be dischargedfrom the hospital before Day 28.ResultsUsing PSM, 43 patients (45% male) aged 65.6 (57–70) yearsfrom each group were exactly matched. No significant difference was observed in clinical improvement of patients treated with CHM granules compared with those who received usual (p = 0.851). However, the use of CHM granules reduced the 28-day mortality (p = 0.049) and shortened the duration of fever (4 days vs. 7 days, p = 0.002). The differences in the duration of cough and dyspnea and the difference in lung lesion ratio on computerized tomography scans were not significant.Commonly,patients in the CHM group had an increased D-dimer level (p = 0.036).ConclusionForpatients with severe COVID-19, CHM granules, combined with usual care, showed no improvement beyond usual care alone. However, the use of CHM granules reduced the 28-day mortality rate and the time to fever alleviation. Nevertheless, CHM granules may be associated with high risk of fibrinolysis.     

Cloning of a retinally abundant regulator of G-protein signaling (RGS-r/RGS16): genomic structure and chromosomal localization of the human gene

Regulators of G-protein signaling (RGS) constitute a family of GTPase-activating proteins with varying tissue-specific expression patterns and G-protein alpha subunit specificities. Here, we describe the molecular cloning of the human RGS-r/RGS16 cDNA, encoding a predicted polypeptide of 23 kDa that shows 86% identity to mouse RGS-r. Northern blot analysis shows that, like the mouse Rgs-r message, hRGS-r mRNA is abundantly expressed in retina, with lower levels of expression in most other tissues examined. Characterization of the genomic organization of the hRGS-r gene shows that it consists of five exons and four introns. We have also mapped the human RGS-r /RGS16 gene to chromosome 1q25–1q31 by fluorescence in situ hybridzation. Analysis of human ESTs reveals that at least five members of the RGS gene family map to chromosome 1q, suggesting that at least part of the RGS family arose through gene duplication. The chromosomal location, retinal abundance, and presumed function of the human RGS-r protein in desensitizing photoreceptor signaling make the RGS-r/RGS16 locus a candidate for mutations responsible for retinitis pigmentosa with para-arteriolar preservation of retinal pigment epithelium (RP-PPRE or RP12), an autosomal recessive disorder previously mapped to 1q31.     

Cycloheximide reduces PGD2 or Δ12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD₂ or Δ¹²-PGJ₂ (a biological active metabolite of PGD₂), we examined the effect of various compounds on PGD₂ or Δ¹²-PGJ₂ cytottoxic, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD₂ cytotoxicity on NCG cells. When Δ¹²-PGJ₂ was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and α-amanitin did not affect PGD₂ or Δ¹²-PGJ₂ cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD₂ or Δ¹²-PGJ₂ exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD₂ or Δ¹²-PGJ₂.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.