Creating basis for introducing non‐invasive prenatal testing in the Estonian public health setting

Objective The study aimed to validate a whole‐genome sequencing‐based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. Method In total, 424 maternal blood samples were included. Analysis pipeline involved cell‐free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre‐defined per‐chromosome sets of unique k‐mers from sequencing raw data. SeqFF was implemented to estimate cell‐free fetal DNA (cffDNA) fraction. Results NIPTmer identified correctly all samples of non‐mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false‐positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid. Conclusion We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination

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

Eesmärk. Selgitada välja kuulmislanguse (KL) geneetilised põhjused Eesti lastel ja kirjeldada nende fenotüüpi. Metoodika. Uuringus osales 233 KLiga last, kellele tehti APEX-geenikiibi analüüs 201 erineva mutatsiooni suhtes 8 pärilikku KLi põhjustavas geenis (GJB2, GJB3, GJB6, GJA1, SLC26A4, SLC26A5, 12S-rRNA ja tRNA (Ser) geenid). Tulemused. Leidsime 115 patsiendil (49%) GJB2 mutatsiooni vähemalt ühes alleelis, neist 100 lapsel esines vähemalt ühes alleelis mutatsioon c.35delG. 5 patsiendi (2%) KL oli tingitud kaasasündinud tsütomegalovi irusinfektsioonist. Sündroomne KL kinnitati 7 uuritaval. Kogu genoomi genotüpiseerimisplatformi analüüs tehti 28 patsiendile, selle tulemusel leidsime 4 erinevat potentsiaalselt patogeenset deleteerunud kromosoomipiirkonda. Järeldused. Kõige sagedasem lapseea KLi põhjustav mutatsioon on c.35delG, mille osakaal KLiga laste hulgas on 75% GJB2 geeni mutatsioonidest. Uuringu tulemusena selgus või täpsustus KLi etioloogias geneetiline faktor 140 patsiendil (60%). Eesti Arst 2010; 89(12):781−78

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1,2,3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages

Kromosomaalne mikrokiibianalüüs diagnostilise vahendina: Eesti kogemus

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Inimese genoomi keerulise struktuuri tõttu on see vastuvõtlik genoomsete ümberkorralduste tekkele, millest kõige sagedasemad on DNA lõikude kordistumised ja kadumised. Enamik ümberkorraldusi ei põhjusta muutust fenotüübis. Samas võivad muutused teatud genoomi piirkondades omada olulist rolli mitmete haiguste ja häirete korral (nt. füüsilise ja vaimse arenguhäired, epilepsia, kasvajad jm). Kromosomaalne mikrokiibianalüüs (KMA; tuntud ka „submikroskoopilise kromosoomianalüüsi“ nime all) on uus molekulaarne kromosoomide uurimismeetod, mis võimaldab analüüsida tervet genoomi ühe eksperimendi käigus tuvastades nii muutusi kromosoomide arvus kui ka struktuuris. Võrreldes klassikalise kromosoomianalüüsiga ehk kromosoomide vöödistusega on KMA lahutusvõime vähemalt 10-20 korda suurem, mis tõstab oluliselt diagnostilist efektiivsust. Üldiselt tuvastatakse KMA abil kromosoomide muutusi umbes 15-20% arenguprobleemide ja/või intellektipuudega patsientidest. KMA on alates 2011. aastast Eesti Haigekassa hinnakirjas ning on näidustatud järgmistel juhtudel: 1) ebaselge etioloogiaga vaimse arengu mahajäämus; 2) autism või autismilaadsed käitumishäired; 3) kaasasündinud hulgiväärarengud. Mõnel juhul teostatakse ka sünnieelseid analüüse, kuigi praegusel ajal jääb Eestis esmaseks tsütogeneetiliseks testiks sünnieelse diagnostika puhul siiski kromosoomide vöödistus. Haiguspõhjuslik KMA tulemus välistab edasiste kulukate uuringute tegemise. Samas võimaldab KMA haigusliku leiuta vastus edaspidises diagnostikas keskenduda harva esinevatele monogeensetele haigustele ja ainevahetushäiretele. Käesolevas töös esitatakse Eesti kogemus KMA rakendamise kohta kliinilises praktikas. Kriitiliseks analüüsi etapiks, eriti sünnieelse diagnostika puhul, jääb tulemuste interpretatsioon, mis mõnel juhul võib osutuda suhteliselt keeruliseks. Selleks et tõlgendada maksimaalset arvu KMA leidudest ning tagada patsientidele korralikku geneetilist nõustamist, on vajalik tihe koostöö tsütogeneetikute ja kliiniliste geneetikute vahel. Lisaks on vaja meeles pidada, et KMA tulemuste interpretatsioon põhineb meie praegustel teadmistel, mis võivad aja jooksul muutuda, mistõttu on tulevikus tulemuste korduv ülevaatamine väga oluline.  The higher-order architecture of the human genome has been shown to predispose to structural rearrangements, including losses and gains of DNA segments. Most rearrangements have no effect on human phenotype. However, changes in specific genomic regions can cause various pathological conditions, such as abnormal mental and physical development, neurological disorders, cancer etc. Chromosomal microarray analysis (CMA) is a novel chromosome analysis method that offers the capacity to examine the entire human genome in a single experiment to detect changes in chromosome number and structure. Compared to conventional chromosomal analysis – G-banding – the resolution of CMA is at least 10-20-fold higher, which provides a higher diagnostic yield. In general, CMA is able to detect chromosomal abnormalities in 15-20% of patients with developmental problems and/or intellectual disability. Since 2011, CMA is on the official service list of the Estonian Health Insurance Fund and is performed as the first-tier cytogenetic diagnostic test for patients with 1) developmental delay/intellectual disability, 2) autism spectrum disorders, and/or 3) multiple congenital anomalies. CMA can also be performed in some prenatal cases. However, in Estonia, G-banding remains the primary cytogenetic test in prenatal diagnostics. Detection of pathogenic aberration by CMA excludes further expensive analyses. In case of a normal result, in further diagnostic procedures it is reasonable to concentrate on rare monogenic disorders and metabolic diseases. This study summarizes the Estonian experience of using CMA in routine clinical practice. The critical step, especially in prenatal diagnostics, is interpretation of CMA findings, which can be quite difficult in some cases. Close collaboration between cytogeneticists and clinical geneticists is important in terms of interpretation and providing proper genetic counseling. In addition, it should be kept in mind that the interpretation of CMA findings is based on current knowledge and may evolve over time. Therefore, it is important to re-evaluate CMA results over the tim

Additional file 1: Table S1. of Somatic mosaicism for copy-neutral loss of heterozygosity and DNA copy number variations in the human genome

General characteristics of the four subjects examined in the study. Table S2. Studied tissues and sample naming. Table S3. Summary of the non-mosaic (germ-line) CNV regions identified in all tissues of the body from four individuals studied. Table S4. Summary of tissue-specific CNVs observed in one of the four individuals studied (KT538). Table S5. Summary of tissue-specific cn-LOH events (>5 Mb) observed in three out of the four individuals studied. Table S6. DNA concentrations (ng/μl) and quality parameters (260/280 and 260/230 nm ratios) for each tissue type and subject. (PDF 548 kb

NIPTmer: rapid k-mer-based software package for detection of fetal aneuploidies

Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool - NIPTmer - is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/ .status: publishe