Eneli Oitmaa

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 ∼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.

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

Distribution of CFTR gene mutations in cystic fibrosis patients from Estonia

Editor—Cystic fibrosis (CF) is the most common recessive genetic disorder affecting about 1 in 2500 white live births with a carrier frequency of 1 in 25.1 The incidence of this disease has been found to be somewhat lower in Nordic countries,2 especially Finland.3 To date more than 800 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (http://www.genet.sickkids.on.ca/cftr/). The major mutation, ΔF508, accounts for 72.8% of the CF chromosomes in northern and central European countries,4 but the frequencies and types of mutations in different populations vary considerably depending on the ethnic and geographical origin of the population tested. In our previous report, the incidence of CF was estimated to be about 1 in 4500 live births in Estonia.5 The present study was undertaken to identify the whole spectrum of CFTR gene mutations in Estonian patients. Thirty families with CF patients were studied. We think the patients should account for most of the CF cases in Estonia, as they attend the central hospitals, but the exact number of CF patients is not known. Diagnostic criteria were based on repeated positive sweat chloride tests (>60 mmol/l) and on typical findings of pulmonary/gastrointestinal disease. In addition, 20 more patients suspected of having CF, with either undefined pancreatitis or a broad spectrum of respiratory diseases and borderline (40-60 mmol/l/l) or normal (<40 mmol/l) sweat chloride values, were analysed for mutations in the CFTR gene. First, several known mutations were tested directly by the heteroduplex analysis (HA; ΔF508, 394delTT, polyT variants in IVS8), restriction digestion (RD; G551D, R553X, 1811+1.6kbA→G, L206W, 3849+10kbC→T), and amplification refractory mutation system (ARMS, kits from Cellmark Diagnostics, UK; G542X, 621+1G→T, N1303K). As a result of this screening, only two mutations were found: ΔF508 was found in 31 (51.7%) alleles and …

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.

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.

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.

Kuulmislanguse geneetilised põhjused Eesti lastel ning leitud genotüübi ja fenotüübi omavaheline võrdlus

Eesmark. Selgitada valja kuulmislanguse (KL) geneetilised pohjused Eesti lastel ja kirjeldada nende fenotuupi. Metoodika. Uuringus osales 233 KLiga last, kellele tehti APEX-geenikiibi analuus 201 erineva mutatsiooni suhtes 8 parilikku KLi pohjustavas geenis (GJB2, GJB3, GJB6, GJA1, SLC26A4, SLC26A5, 12S-rRNA ja tRNA (Ser) geenid). Tulemused. Leidsime 115 patsiendil (49%) GJB2 mutatsiooni vahemalt uhes alleelis, neist 100 lapsel esines vahemalt uhes alleelis mutatsioon c.35delG. 5 patsiendi (2%) KL oli tingitud kaasasundinud tsutomegalovi irusinfektsioonist. Sundroomne KL kinnitati 7 uuritaval. Kogu genoomi genotupiseerimisplatformi analuus tehti 28 patsiendile, selle tulemusel leidsime 4 erinevat potentsiaalselt patogeenset deleteerunud kromosoomipiirkonda. Jareldused. Koige sagedasem lapseea KLi pohjustav mutatsioon on c.35delG, mille osakaal KLiga laste hulgas on 75% GJB2 geeni mutatsioonidest. Uuringu tulemusena selgus voi tapsustus KLi etioloogias geneetiline faktor 140 patsiendil (60%). Eesti Arst 2010; 89(12):781−789

Pärilik ehk geneetiline kuulmislangus

Kuulmislangus on koige enam levinud sensoorne haigus kogu maailmas. Varajase ehk kone-eelse kuulmislanguse esinemissagedus arvatakse olevat 1–2 juhtu 1000 lapse kohta ning pooltel juhtudest on see parilik. Geneetiline kuulmislangus jagatakse sundroomseks vormiks, mille korral kuulmislangus on seotud teiste elundite anomaaliatega, ja mittesundroomseks ehk isoleeritud vormiks. Eesti Arst 2007; 86 (4): 254–261