Two new mutations and three novel polymorphisms in the<Emphasis Type="Italic"> RB1</Emphasis> gene in Ecuadorian patients

RB1 is the gene responsible for retinoblastoma, the most common malignant intraocular tumor of infancy and early childhood. There are no reports about this gene in Ecuadorian populations, and only a few studies have been published in Latin America about this subject. There is a spectrum of more than 370 mutations described in the RB1 gene mutation database (http://www.d-lohmann.de/Rb/mutations.html), and alterations have been found in 25 of the 27 exons. During the exon-by-exon analysis of 31 tumor and blood samples from Ecuadorian patients, we found two new mutations and three novel polymorphisms. One of the polymorphisms is located in intron 26 where no alterations of the gene have been described previously. The polymorphisms were found in all of the patients tumor samples, but not in normal population, suggesting there might be a relationship between these polymorphisms and the development of retinoblastoma in the Ecuadorian population.The nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY243567, AY260472, AY260473, AY273783     

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

A Randomized Study of Nutritional Supplementation in Patients with Unilateral Wet Age-Related Macular Degeneration

The purpose of this study is evaluate the efficacy and safety of medicinal products containing the original Age-Related Eye Disease group (AREDS) formulation at doses approved in Europe (EU, control group; n = 59) with a product that adds DHA, lutein, zeaxanthin, resveratrol and hydroxytyrosol to the formula (intervention group; n = 50). This was a multicenter, randomized, observer-blinded trial conducted in patients aged 50 years or older diagnosed with unilateral exudative Age related Macular Degeneration AMD. At month 12, the intervention did not have a significant differential effect on visual acuity compared with the control group, with an estimated treatment difference in Early Treatment Diabetic Retinopathy Study (ETDRS) of −1.63 (95% CI −0.83 to 4.09; p = 0.192). The intervention exhibited a significant and, in most cases, relevant effect in terms of a reduction in some inflammatory cytokines and a greater improvement in the fatty acid profile and serum lutein and zeaxantin concentration. In patients with unilateral wet AMD, the addition of lutein, zeaxanthin, resveratrol, hydroxytyrosol and DHA to the AREDS EU recommended doses in the short-term did not have a differential effect on visual acuity compared to a standard AREDS EU formula but, in addition to improving the fatty acid profile and increasing carotenoid serum levels, may provide a beneficial effect in improving the proinflammatory and proangiogenic profile of patients with AMD.     

Intercellular Interactions of an Adipogenic CXCL12-Expressing Stromal Cell Subset in Murine Bone Marrow

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12⁺ stromal cells (“Adipo-CAR” cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12⁺ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12⁺ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER⁺ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12⁺ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12⁺ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.     

Self-rated health and mortality in the NHANES-I Epidemiologic Follow-up Study.

The ability of self-rated health status to predict mortality was tested with data from the National Health and Nutrition Examination Survey (NHANES-I) Epidemiologic Follow-Up Study (NHEFS), conducted from 1971-84. The sample consists of adult NHANES-I respondents ages 25-74 years (N = 6,440) for whom data from a comprehensive physical examination at the initial interview and survival status at follow-up are available. Self-rated health consists of the response to the single item, "Would you say your health in general is excellent, very good, good, fair, or poor?" Proportional hazards analyses indicated that, net of its association with medical diagnoses given in the physical examination, demographic factors, and health related behaviors, self-rated health at Time 1 is associated with mortality over the 12-year follow-up period among middle-aged males, but not among elderly males or females of any age.     

Renal tubular acidosis in the Silver-Russell syndrome

Several patients with the Silver-Russell syndrome (SRS) attending our Genetics Clinic were diagnosed as having persistent metabolic acidosis. Since this abnormality has not been reported previously in the SRS, we reexamined 33 SRS patients to evaluate the frequency and type of metabolic acidosis, the clinical and laboratory findings, and the growth pattern in SRS patients with and without metabolic acidosis. Among them, 14 had a consistent decrease in HCO levels. Renal studies in acidotic patients showed urine pH of 5.8 and 24 h urine calcium of <2.4 mg/kg/24 h; serum creatinine, excretion of glucose, and aminoacids were normal, as were renal ultrasound and excretory urography findings. These data supported the diagnosis of renal tubular acidosis, probably type II; the patients were treated with oral bicarbonate and acidosis was corrected successfully. Clinical manifestations were similar in acidotic and non-acidotic patients. The nutritional indices at diagnosis and at last evaluation (at least 8 months after diagnosis) were abnormally low in all patients; however, acidotic patients, treated with bicarbonate, showed an improvement of nutritional status particularly in the weight/height index, although the difference between groups after follow-up did not reach statistical significance. We suggest that metabolic acidosis due to renal tubular acidosis, probably type II, may occur in children with the SRS and should be looked for and treated in all patients. © 1995 Wiley-Liss, Inc.