Marasso S.L.,Polytechnic University of Turin |
Marasso S.L.,CNR Institute of Materials for Electronics and Magnetism |
Mombello D.,Polytechnic University of Turin |
Cocuzza M.,Polytechnic University of Turin |
And 10 more authors.
Biomedical Microdevices | Year: 2014
In this work a polymer lab-on-a-chip (LOC), fabricated through MEMS technology, was employed to execute a genetic protocol for the Single Nucleotide Polymorphisms (SNPs) detection. The LOC was made in Poly (methyl methacrylate) (PMMA) and has two levels: the lower one for the insertion and mixing of the reagents, the upper one for the interfacing with the DNA microarray chip. The hereditary hearing loss was chosen as case of study, since the demand for testing such a particular disorder is high and genetics behind the condition is quite clear. The Arrayed Primer EXtension (APEX) genetic protocol was implemented on the LOC to analyze the SNPs. A low density (for detection of up to 10 mutations) and a high density microarray chips (for detection of 245 mutations in 12 genes), containing the primers for the extension, were employed to carry out the APEX reaction on the LOC. Both the microarray chips provide a signal to noise ratio and efficiency comparable with a detection obtained in a conventional protocol in standard conditions. Moreover, significant reduction of the employed PCR volume (from 30 μL to 10 μL) was obtained using the low density chip. © 2014, Springer Science+Business Media New York.
Jaijo T.,Hospital Universitario La Paz |
Jaijo T.,CIBER ISCIII |
Aller E.,Hospital Universitario La Paz |
Aller E.,CIBER ISCIII |
And 20 more authors.
Investigative Ophthalmology and Visual Science | Year: 2010
The purpose of this study was to test the ability of the genotyping microarray for Usher syndrome (USH) to identify the mutations responsible for the disease in a cohort of 183 patients with USH. Methods. DNA from 183 patients with Usher syndrome from the Spanish population was analyzed using a genotyping microarray containing 429 previously identified disease-associated variants in eight USH genes. Mutations detected by the array were confirmed by direct sequencing. Haplotype analysis was also performed in families carrying common Spanish mutations. Results. The genotyping microarray identified 43 different variants, divided into 32 disease causative and 11 probably nonpathologic. Mutations were detected in 62 patients with USH (33.9%). According to the clinical classification of patients, pathologic variants were detected in 31.4% patients with USH1, 39.4% of with USH2, 22.2% with USH3 and 15.8% with unclassified Usher syndrome. Ninety-seven pathologic alleles were detected, corresponding to 26.5% of expected alleles. The USH2A mutations p.C3267R and p.T3571M were revealed as common in the Spanish population, and two major haplotypes linked to these mutations were observed. Conclusions. The genotyping microarray is a robust, low-cost, rapid technique that is effective for the genetic study of patients with USH. However, it also indicates variants of unclear pathologic nature and detection failures have also been observed. Results must be confirmed by direct sequencing to avoid misdiagnosis, and continuous updates of the microarray should be performed to increase the efficiency and rate of detection of mutations. © Association for Research in Vision and Ophthalmology.
Pergament E.,Northwestern Reproductive Genetics |
Alamillo C.,Northwestern Reproductive Genetics |
Sak K.,Asper Biotech |
Fiddler M.,DePaul University
Prenatal Diagnosis | Year: 2011
The objective of this study was to assess the first formal approach for monitoring genetic/developmental syndromes associated with the presence of an increased nuchal translucency (NT) thickness (>3 mm) in the first trimester of pregnancy. +Methods: Multiple technologies-a DNA chip using the APEX technology, qPCR, microfluidic PCR, and sequencing-were applied to assay 310 mutations across five conditions-Noonan syndrome, congenital adrenal hyperplasia, spinal muscular atrophy (SMA), DiGeorge syndrome, and Smith-Lemli Opitz syndrome. +Results: We report the results of assessing the first 120 patients in which 8 cases of Noonan syndrome were detected as well as an unusually high rate of heterozygosity for SMA. +Conclusion: While testing for Noonan syndrome in association with increased NT appears warranted, the reported association of the remaining four genetic syndromes is likely to be weak and possibly insignificant. © 2011 John Wiley & Sons, Ltd.
Serrano-Fernandez P.,Pomeranian Medical University |
Dymerska D.,Pomeranian Medical University |
Kurzawski G.,Pomeranian Medical University |
Derkacz R.,Pomeranian Medical University |
And 17 more authors.
Gastroenterology Research and Practice | Year: 2015
The continued identification of new low-penetrance genetic variants for colorectal cancer (CRC) raises the question of their potential cumulative effect among compound carriers. We focused on 6 SNPs (rs380284, rs4464148, rs4779584, rs4939827, rs6983267, and rs10795668), already described as risk markers, and tested their possible independent and combined contribution to CRC predisposition. Material and Methods. DNA was collected and genotyped from 2330 unselected consecutive CRC cases and controls from Estonia (166 cases and controls), Latvia (81 cases and controls), Lithuania (123 cases and controls), and Poland (795 cases and controls). Results. Beyond individual effects, the analysis revealed statistically significant linear cumulative effects for these 6 markers for all samples except of the Latvian one (corrected P value = 0.018 for the Estonian, corrected P value = 0.0034 for the Lithuanian, and corrected P value = 0.0076 for the Polish sample). Conclusions. The significant linear cumulative effects demonstrated here support the idea of using sets of low-risk markers for delimiting new groups with high-risk of CRC in clinical practice that are not carriers of the usual CRC high-risk markers. © 2015 Pablo Serrano-Fernandez et al.
PubMed | Pomeranian Medical University, Estonian Academy of Sciences, Vilnius Maternity Hospital, Hunter Medical Research Institute and 5 more.
Type: | Journal: Gastroenterology research and practice | Year: 2015
The continued identification of new low-penetrance genetic variants for colorectal cancer (CRC) raises the question of their potential cumulative effect among compound carriers. We focused on 6 SNPs (rs380284, rs4464148, rs4779584, rs4939827, rs6983267, and rs10795668), already described as risk markers, and tested their possible independent and combined contribution to CRC predisposition. Material and Methods. DNA was collected and genotyped from 2330 unselected consecutive CRC cases and controls from Estonia (166 cases and controls), Latvia (81 cases and controls), Lithuania (123 cases and controls), and Poland (795 cases and controls). Results. Beyond individual effects, the analysis revealed statistically significant linear cumulative effects for these 6 markers for all samples except of the Latvian one (corrected P value = 0.018 for the Estonian, corrected P value = 0.0034 for the Lithuanian, and corrected P value = 0.0076 for the Polish sample). Conclusions. The significant linear cumulative effects demonstrated here support the idea of using sets of low-risk markers for delimiting new groups with high-risk of CRC in clinical practice that are not carriers of the usual CRC high-risk markers.
Teek R.,University of Tartu |
Kruustuk K.,University of Tartu |
Zordania R.,Tallinn Childrens Hospital |
Joost K.,Tallinn Childrens Hospital |
And 7 more authors.
International Journal of Pediatric Otorhinolaryngology | Year: 2010
Objective: The purpose of this study was to determine the prevalence of c.35delG and p.M34T mutations in the GJB2 gene among children with early onset hearing loss and within a general population of Estonia. Methods: Using an arrayed primer extension assay, we screened 233 probands with early childhood onset hearing loss for 107 different mutations in the GJB2 gene. We then looked for the two most common mutations, c.35delG and p.M34T, in a population of 998 consecutively born Estonian neonates to determine the frequency of these mutations in the general population. Results: In 115 (49%) of the patients with early onset hearing loss, we found a mutation in at least one allele of the GJB2 gene. Seventy-three (31%) were homozygous for the c.35delG mutation, seven (3%) were homozygous for the p.M34T mutation, and five (2%) had c35delG/p.M34T compound heterozygosity. Other six identified mutations in GJB2 gene occurred rarely. Among the 998 anonymous newborn samples, we detected 45 who were heterozygous for c.35delG, 2 individuals homozygous for c.35delG, and 58 who were heterozygous for p.M34T. Additionally, we detected two c.35delG/p.M34T compound heterozygotes. Conclusion: The most common GJB2 gene mutations in Estonian children with early onset hearing loss were c.35delG and p.M34T, with c.35delG accounting for 75% of GJB2 alleles. The carrier frequency for c.35delG and p.M34T in a general population of Estonia was 1 in 22 and 1 in 17, respectively, and was higher than in most other countries. © 2010 Elsevier Ireland Ltd.
Vilbaste M.,University of Tartu |
Slavin G.,Asper Biotech |
Saks O.,University of Tartu |
Pihl V.,University of Tartu |
Leito I.,University of Tartu
Measurement: Journal of the International Measurement Confederation | Year: 2010
The GUM modelling, its Bayesian modification and the Monte Carlo method (MCM) to estimate the uncertainty are compared in two practical measurement situations (finding reference value of relative humidity and a generic chemical instrumental analysis procedure). The results of the three approaches agree very well when there are no dominant input quantities with type A evaluated uncertainty estimated from small number of repeated measurements. In the opposite case the GUM gives underestimated expanded uncertainties (by up to 20-25%), compared to both other approaches. Analysis of the practical measurement situations reveals that even in the case of several dominating input quantities of similar uncertainty contributions, if one of them is distributed according to the t-distribution and has a low number (3-4) of degrees of freedom, the output quantity cannot be safely assumed Normally distributed and in such a case coverage factor 2 is not an equivalent to 95% coverage level. © 2009 Elsevier Ltd. All rights reserved.
Vozzi D.,University of Trieste |
Aaspollu A.,Asper Biotech |
Athanasakis E.,University of Trieste |
Berto A.,University of Ferrara |
And 11 more authors.
Molecular Vision | Year: 2011
Purpose: Usher syndrome is an autosomal recessive disorder characterized by hearing and vision loss. Usher syndrome is divided into three clinical subclasses (type 1, type 2, and type 3), which differ in terms of the severity and progression of hearing loss and the presence or absence of vestibular symptoms. Usher syndrome is defined by significant genetic heterogeneity, with at least 12 distinct loci described and 9 genes identified. This study aims to provide a molecular epidemiology report of Usher syndrome in Italy. Methods: Molecular data have been obtained on 75 unrelated Italian patients using the most up-to date technology available for the screening of Usher syndrome gene mutations, i.e., the genotyping microarray developed by Asper Biotech (Tartu, Estonia), which simultaneously investigates 612 different marker positions using the well established arrayed primer extension methodology (APEX). Results: Using this method, we found that 12% of cases (9 out of 75) harbored homozygous or compound heterozygous mutations in the gene positions analyzed, whereas 20% (15 out of 75) of the patients were characterized by the presence of only one mutated allele based on the positions analyzed. One patient was found to be compound heterozygous for mutations in two different genes and this represents an example of possible digenic inheritance in Usher syndrome. A total of 66.6% of cases (50 out of 75) were found to be completely negative for the presence of Usher syndrome gene mutations in the detected positions. Mutations detected by the array were confirmed by direct sequencing. Conclusions: These findings highlight the efficacy of the APEX-based genotyping approach in the molecular assessment of Usher patients, suggesting the presence of alleles not yet identified and/or the involvement of additional putative genes that may account for the pathogenesis of Usher syndrome. © 2011 Molecular Vision.
Verma A.,Aravind Eye Hospital |
Perumalsamy V.,Aravind Eye Hospital |
Shetty S.,Aravind Eye Hospital |
Kulm M.,Asper Biotech |
Sundaresan P.,Aravind Eye Hospital
PLoS ONE | Year: 2013
Background:Leber congenital amaurosis (LCA) is the most severe form of inherited retinal visual impairment in children. So far, mutations in more than 20 genes have been known to cause LCA and among them, RPE65 is a suitable candidate for gene therapy. The mutational screenings of RPE65 and other LCA genes are requisite in support of emerging gene specific therapy for LCA. Therefore, we have carried out a comprehensive LCA genes screening using a combined approach of direct sequencing and DNA microarray based Asper chip analysis.Methodology/Principal Findings:Thirty clinically diagnosed index LCA cases from Southern India were screened for coding and flanking intronic regions of RPE65 through direct sequencing. Among thirty, 25 cases excluded from RPE65 mutations were subjected to Asper chip analysis, testing 784 known pathogenic variations in 15 major LCA genes. In RPE65 screening, four different pathogenic variations including two novel (c.361insT & c.939T>A) and two known (c.394G>A & c.361delT) mutations were identified in five index cases. In the chip analysis, seven known pathogenic mutations were identified in six index cases, involving genes GUCY2D, RPGRIP1, AIPL1, CRX and IQCB1. Overall, 11 out of 30 LCA cases (36.6%) revealed pathogenic variations with the involvement of RPE65 (16.6%), GUCY2D (10%), RPGRIP1 (3.3%), AIPL1 (3.3%) and CRX & IQCB1 (3.3%).Conclusions/Significance:Our study suggests that such combined screening approach is productive and cost-effective for mutation detection and can be applied in Indian LCA cohort for molecular diagnosis and genetic counselling. © 2013 Verma et al.
Pereiro I.,University of Vigo |
Hoskins B.E.,University College London |
Marshall J.D.,The Jackson Laboratory |
Collin G.B.,The Jackson Laboratory |
And 6 more authors.
European Journal of Human Genetics | Year: 2011
Bardet-Biedl syndrome (BBS; OMIM no. 209 900) and Alström syndrome (ALMS; OMIM no. 203 800) are rare, multisystem genetic disorders showing both a highly variable phenotype and considerable phenotypic overlap; they are included in the emerging group of diseases called ciliopathies. The genetic heterogeneity of BBS with 14 causal genes described to date, serves to further complicate mutational analysis. The development of the BBS-ALMS array which detects known mutations in these genes has allowed us to detect at least one mutation in 40.5% of BBS families and in 26.7% of ALMS families validating this as an efficient and cost-effective first pass screening modality. Furthermore, using this method, we found two BBS families segregating three BBS alleles further supporting oligogenicity or modifier roles for additional mutations. We did not observe more than two mutations in any ALMS family. © 2011 Macmillan Publishers Limited All rights reserved.