Single Passage of Human Metapneumovirus in LLC-MK2 Cells Does Not Affect Viral Protein-Coding Capacity.

Here, we report the complete genome sequences of human metapneumovirus (HMPV) prior to and after passaging in LLC-MK2 cells. Paired comparisons of the 13,335-nucleotide genomes revealed that the virus acquired the T10736C transition in its genome, which did not affect the amino acid sequences of HMPV proteins.

Cumulative Small Effect Genetic Markers and the Risk of Colorectal Cancer in Poland, Estonia, Lithuania, and Latvia.

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.

Arrayed primer extension microarray for the analysis of genes associated with congenital stationary night blindness.

Arrayed primer extension (APEX) is a microarray-based genotyping method that enables to simultaneously analyze hundreds of known mutations in the genome. APEX-based microarrays are successfully used for molecular diagnostics of various genetic disorders. Congenital stationary night blindness (CSNB) is a rare retinal disease caused by mutations in genes involved in phototransduction cascade and signaling from photoreceptors to adjacent neurons in the retina. As CSNB is clinically and genetically heterogeneous, the identification of the underlying cause of the disease can be challenging. In this chapter, we describe an APEX-based method for the analysis of genes associated with CSNB.

Arrayed primer extension technology simplifies mutation detection in Bardet-Biedl and Alström syndrome.

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.

Molecular diagnosis of Down syndrome using quantitative APEX-2 microarrays.

OBJECTIVE: To develop a new rapid and high-throughput microarray-based prenatal diagnostic test for the detection of trisomy 21 (T21). METHODS: The T21 arrayed primer extension-2 (APEX-2) assay discriminates between trisomy and euploid DNA samples by comparing the signal intensities of allelic fractions of heterozygous single nucleotide polymorphisms (SNPs) after APEX reaction. After preliminary validation using DNA samples from Down syndrome patients, we analyzed DNA samples from cultured and uncultured amniocytes and chorionic villus for 90 SNPs with high heterozygosity from the 21(q21.1q22.2) region. Differences in allelic ratios of heterozygous SNPs in normal and T21 individuals were verified by t-test. RESULTS: Analysis of the T21 APEX-2 assay results revealed that 90 SNPs were sufficient for reliable discrimination between T21 and euploid DNA samples (P≤0.05 for one or both strands). Using 134 clinical samples from cultured or uncultured fetal cells, both the sensitivity and the specificity of the assay were 100%. CONCLUSION: Our study provides a proof of principle demonstration of the use of the modified APEX-2 assay as a new, fast and reliable method for prenatal diagnosis of fetal T21.

Prevalence of c.35delG and p.M34T mutations in the GJB2 gene in Estonia.

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.

Splice variant IVS2-2A>G in the SLC26A5 (Prestin) gene in five Estonian families with hearing loss.

OBJECTIVE: The aim of our study was to identify the IVS2-2A>G sequence change in the SLC26A5 (Prestin) gene in Estonian individuals with hearing loss and in their family members. METHODS: In the years 2005-2007 we have screened 194 probands with early onset hearing loss and 68 family members with an arrayed primer extension (APEX) microarray, which covers 201 mutations in six nuclear genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5) and two mitochondrial genes encoding 12S rRNA and tRNA-Ser (UCN). RESULTS: In four probands with early onset hearing loss and in five unaffected family members from five families we identified the IVS2-2A>G change in one allele of the SLC26A5 gene. We did not find any homozygosity for this splice variant. IVS2-2A>G was identified in 2.1% of probands. One of these probands, however, is also homozygous for the 35delG mutation in the GJB2 gene and a second patient has Down syndrome, which is also associated with hearing impairment. Therefore, in those two cases the etiology of the hearing loss is probably not associated with the IVS2-2A>G sequence change in the SLC26A5 gene. CONCLUSION: Our data support the hypothesis that heterozygosity for the mutation IVS2-2A>G in SLC26A5 gene may not, by itself, be sufficient to cause hearing loss.

Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up.

OBJECTIVE: The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in approximately 70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes. DESIGN: We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]). RESULTS: The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults. CONCLUSIONS: Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.

Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.

Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.