Alfredo Uribe

Selective Screening for Lysosomal Storage Diseases with Dried Blood Spots Collected on Filter Paper in 4,700 High-Risk Colombian Subjects

Lysosomal storage disorders (LSDs) are a very heterogeneous group of hereditary disorders. The diagnostic process usually involves complex sampling, processing, testing, and validation procedures, performed by specialized laboratories only, which causes great limitations in reaching a diagnosis for patients affected by these diseases.There are few studies about LSDs in Colombia. The diagnostic limitations often make medical practitioners disregard the possibility of these disorders while diagnosing their patients. The current study documents the results of a 7-year screening in high-risk patients, aimed to detect LSDs using dried blood spots (DBS) collected on filter paper, with a micromethodology that facilitates diagnosis even with a large number of samples.The activities of α-galactosidase A, α glucosidase, α-L-iduronidase, arylsulfatase B, β-galactosidase, β-glucosidase, total hexosaminidase, iduronate sulfatase, and chitotriosidase were analyzed in high-risk patients for lysosomal disease. The catalytic activity was evaluated with fluorometric micromethods using artificial substrates marked with 4-methylumbelliferone.The reference values for a control population were established for the enzymes listed above, and 242 patients were found to have an enzyme deficiency, guiding to the following diagnoses: Fabry disease (n = 31), Pompe disease (n = 16), Hurler Syndrome (n = 15), Maroteaux-Lamy Syndrome (n = 34), GM1 Gangliosidosis (n = 10), Morquio B (n = 1), Gaucher disease (n = 101), Sandhoff disease (n = 1), Mucolipidosis (n = 2), and Hunter Syndrome (n = 31). In conclusion, this protocol provides a comprehensive diagnostic approach which could be carried out in Colombia and made it available to medical services spread around the country, enabling the identification of a large number of patients affected by LSDs, which could potentially benefit from the therapeutic tools already available for many of these diseases.

Enzymatic analysis of biomarkers for the monitoring of Gaucher patients in Colombia

INTRODUCTION Gaucher disease is caused by a deficiency of the enzyme acid beta-glucosidase. There is treatment available, but given the wide variability in phenotypes, it is difficult to establish the adequate administration and change of doses. Chitotriosidase and angiotensin converting enzyme (ACE) have been described as reliable biomarkers for the monitoring of patients. The enzymatic evaluation of these biomarkers has been traditionally made in serum or plasma samples, making difficult the monitoring of Colombian patients who live far away from big cities. Dried blood spot samples have been proposed as a solution. The aim of the present study was to validate the chitotriosidase quantification in DBS with respect to the serum determination, and to standardize a microtechnique for the quantification of serum ACE. RESULTS Using a fluorometric method for the chitotriosidase quantification and a colorimetric one for ACE determinations, we found significant differences between control subjects and Gaucher patients in both serum and DBS samples. A positive correlation was observed between both kinds of samples. A reference value for the ACE determination was established. A positive correlation between chitotriosidase and ACE was found. CONCLUSION We could standardize two microtechniques for chitotriosidase and ACE analysis in serum samples. A close relation between DBS and serum samples for chitotriosidase analysis allowed us to validate DBS as a reliable sample that could facilitate the access of Colombian Gaucher patients to health services.

Deep Genotyping of the IDS Gene in Colombian Patients with Hunter Syndrome

BACKGROUND Mucopolysaccharidosis type II (MPSII), also known as Hunter syndrome, is an X-linked disorder caused by mutations in the iduronate 2 sulfatase (IDS) gene. This enzyme catalyzes the initial step in the catabolism of heparan sulfate and dermatan sulfate; thus, its deficiency leads to the accumulation of these glycosaminoglycans. MPS II has significant allelic heterogeneity, making the establishment of genotype-phenotype correlations difficult. This study assessed clinical features in combination with deep genotyping of a group of Colombian patients with MPS II and attempted to establish a degree of genotype-phenotype correlation by employing bioinformatic tools. METHODS Eighteen patients were included in this study, 11% of whom were non-neuronopathic, and the other 89% were neuronopathic. Samples were all analyzed using three molecular methodologies: MLPA, direct exon sequencing, and RFLP analysis. RESULTS A total of 13 mutations were identified, 6 of which were novel (c.548_564dup16, c.477insT, c.595_607del12, c. 549_562del13, c.182delC, and a complete deletion of exon 7). The frequency of common mutations (R468Q, Q465X, K347Q, K236N, S71N, R88H, and a conversion phenomenon) was 53.85%. The S71N mutation was frequent among the attenuated phenotype, while private frameshift mutations and rearrangements were seen in patients with severe phenotypes. Molecular docking was performed on the wild-type and mutant IDS proteins, which revealed changes in the enzyme-substrate interaction for the mutant IDS. CONCLUSION The frequency of novel mutations (46.15%) is similar to what has been reported elsewhere. The use of bioinformatic tools showed differences in enzyme-substrate interactions. Studies with larger groups of patients are needed.

Identification of mutations in Colombian patients affected with Fabry disease

Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions.

Toxicity evaluation and initial characterization of the venom of a Colombian Latrodectus sp.

ABSTRACT The genus Latrodectus has not been studied in Colombia even though it is medically important worldwide; there are three species for the country, this study focused on a non‐identified species found in the Tatacoa Desert in the Huila Department. This research is the first approximation to the extraction, composition analysis and toxicity evaluation of the venom of a species of the genus Latrodectus in Colombia; and aims to evaluate the toxicity by the initial characterization of its venom. The venom extraction was accomplished with electrostimulation and total protein concentration was determined by the Lowry method and BCA assays from crude venom; with these methods, high protein concentration of the samples was measured. Bioassays on mice were also made to evaluate the toxicity and compare the symptoms produced by this Colombian spider to the Latrodectism Syndrome. Finally, an SDS‐PAGE electrophoresis was used to separate the main components of high molecular weight from the samples and compared to a control of the venom of Latrodectus mactans to determine if the venom composition is different between these two species. HighlightsFirst analysis of venom from an undescribed Latrodectus sp. in Colombia.Symptomatology comparison between the Colombian species and the Latrodectism syndrome showed similarities in the models.The SDS‐PAGE confirmed the presence of high molecular weight proteins in the crude venom that could represent toxins.