Clinical genetics in transition-a comparison of genetic services in Estonia, Finland, and the Netherlands.

Genetics has traditionally enabled the reliable diagnosis of patients with rare genetic disorders, thus empowering the key role of today's clinical geneticists in providing healthcare. With the many novel technologies that have expanded the genetic toolkit, genetics is increasingly evolving beyond rare disease diagnostics. When placed in a transition context-like we do here-clinical genetics is likely to become a fully integral part of future healthcare and clinical genetic expertise will be required increasingly outside traditional clinical genetic settings. We explore transition effects on the thinking (culture), organizing (structure), and performing (practice) in clinical genetics, taking genetic healthcare in Estonia, Finland, and the Netherlands as examples. Despite clearly distinct healthcare histories, all three countries have initially implemented genetic healthcare in a rather similar fashion: as a diagnostic tool for predominantly rare congenital diseases, with clinical geneticists as the main providers. Dynamics at different levels, such as emerging technologies, biobanks and data infrastructure, and legislative frameworks, may require development of a new system attuned with the demands and (historic) context of specific countries. Here, we provide an overview of genetic service provisions in Estonia, Finland, and the Netherlands to consider the impact of historic and recent events on prospective developments in genetic healthcare.

The number of CAG and GGN triplet repeats in the Androgen Receptor gene exert combinatorial effect on hormonal and sperm parameters in young men.

Androgen receptor (AR) is a transcription factor that is activated upon binding to testosterone (T) and is implicated in regulating the expression of reproduction-related genes. The human AR gene (Xq11-12) spans 186,588 bp and eight exons. N-terminal transactivation domain of the encoded AR protein harbours two polymorphic stretches of identical amino acids, a polyglutamine tract (encoded by 8-37 CAG-repeats) and a polyglycine tract (encoded by 10-30 GGN-repeats). We set forward to analyse independent and combinatory effects of the length of these repetitive tracts on male reproductive hormones, testicular and sperm parameters in a population-based cohort of Baltic young men (n = 974; aged 20.1 ± 2.1 years). We designed an assay to amplify and detect simultaneously the variants of both polymorphic repeats. The study revealed that elongated AR CAG tract was associated with lower FSH (linear regression: p = 0.0002, effect per repeat -0.056 IU/L). As a novel finding, the carriers of GGN-stretch with ≥24 repeats showed a trend for decreased sperm concentration (p = 0.027). Although neither of the variants exhibited an isolated effect on circulating T, their allelic combinations modulated serum T levels, as well as sperm concentration. The lowest T was measured for men carrying the AR gene with long CAG (n ≥ 25) and short GGN (n ≤ 21) repeat tracts (mean 18.8 vs. 25.5-28.6 nmol/L for the other AR variants, p = 0.017). The lowest sperm concentration was detected among individuals with both elongated repetitive stretches (CAG, n ≥ 25 and GGN, n ≥ 24; mean 49.0 vs. 68.4-72.1 mill/mL for the other variants; p = 0.00059). The innovative study design enabled to clearly demonstrate a combinatory impact of CAG and GGN repeat lengths at male reproductive parameters. As AR regulates transcription of over 900 genes in many tissues and organs, the combinatory effects of these common repeat-length variants on male physiology in the wider context and across lifetime are still to be assessed.

Hearing impairment in Estonia: an algorithm to investigate genetic causes in pediatric patients.

PURPOSE: The present study was initiated to establish the etiological causes of early onset hearing loss (HL) among Estonian children between 2000-2009. METHODS: The study group consisted of 233 probands who were first tested with an arrayed primer extension assay, which covers 199 mutations in 7 genes (GJB2, GJB6, GJB3, SLC26A4, SLC26A5 genes, and two mitochondrial genes - 12S rRNA, tRNASer(UCN)). From probands whose etiology of HL remained unknown, DNA analysis of congenital cytomegalovirus (CMV) infection and G-banded karyotype and/or chromosomal microarray analysis (CMA) were performed. RESULTS: In 110 (47%) cases, the etiology of HL was genetic and in 5 (2%) congenital CMV infection was diagnosed. We found mutations with clinical significance in GJB2 (100 children, 43%) and in 2 mitochondrial genes (2 patients, 1%). A single mutation in SLC26A4 gene was detected in 5 probands (2.2%) and was considered diagnostic. In 4 probands a heterozygous IVS2-2A>G change in the SLC26A5 gene was found. We did not find any instances of homozygosity for this splice variant in the probands. CMA identified in 4 probands chromosomal regions with the loss of one allele. In 2 of them we were able to conclude that the found abnormalities are definitely pathogenic (12q13.3-q14.2 and 17q22-23.2 microdeletion), but the pathogenity of 2 other findings (3p26.2 and 1p33 microdeletion) remained unknown. CONCLUSION: This practical diagnostic algorithm confirmed the etiology of early onset HL for 115 Estonian patients (49%). This algorithm may be generalized to other populations for clinical application.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

Unravelling genetic data by arrayed primer extension.

We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.