The deficiency of natural anticoagulants-antithrombin (AT), protein C (PC), and protein S (PS)-is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected-SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.
- Publikační typ
- časopisecké články MeSH
AIMS: Turner syndrome is the only chromosome monosomy that is postnatally compatible with life. The reported incidence of TS is 1 in 2500 liveborn girls. The phenotype of these girls is highly variable, with cardiac abnormalities being life-threatening defects. The aim of the study was to reveal the possible influence of the parental origin of the X chromosome in these patients on a selected phenotype that is associated with Turner syndrome. Selected symptoms and parameters were: a bicuspid aortic valve, aortic coarctation, lymphoedema, pterygium colli, coeliac disease, thyroiditis, otitis media, diabetes mellitus 2, renal abnormalities, spontaneous puberty, and IVF. METHODS: The X chromosome haplotype was determined for a group of 45,X patients verified by native FISH. A molecular diagnostic method based on the detection of different lengths of X chromosome-linked STR markers using the Argus X-12 QS kit was used to determine the X haplotype. RESULTS: Our results, analysed by Fisher's exact (factorial) test, suggest independence between the maternal/paternal origin of the inherited X chromosome and the presence of the anomalies that were studied (P=1 to P=0.34). CONCLUSION: In the group of 45,X patients, who were precisely selected by means of the native FISH method, no correlation was demonstrated with the parental origin of the X chromosome and the observed symptom.
- MeSH
- chromozom X MeSH
- fenotyp MeSH
- haplotypy MeSH
- lidé MeSH
- Turnerův syndrom * genetika MeSH
- vrozené srdeční vady * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Trombofilní stavy představují vrozenou nebo získanou predispozici vzniku trombózy v cévním řečišti. Mezi rutinně vyšetřované vrozené trombofilie patří v populaci vysoce frekventní leidenská mutace a mutace v genu protrombinu G20210A. Vrozený deficit antikoagulačních proteinů (proteinu S, proteinu C a antitrombinu III) patří mezi vzácné trombofilie s nižší populační frekvencí, ale vyšším relativním rizikem tromboembolické příhody a je způsobený mutacemi v genech kódujících tyto proteiny. Postup molekulárně genetické analýzy závisí na skutečnosti, zda se jedná o vyhledávání známé, nebo neznámé, případně vzácné varianty. U známých kauzálních variant FV Leiden a protrombinu G20210A využíváme k jejich zachycení běžně metody PCR-RFLP, reverzní Strip Assay®, alelově-specifickou PCR, TaqMan real-time PCR a SNaPshot®. Genetickému testování vzácných variant v antikoagulačních proteinech předchází precizní selekce pacientů. Vhodné je využití metodiky masivního paralelního sekvenování doplněné vyhledáváním větších genových přestaveb metodou MLPA. Tento postup je úspěšně aplikován na našem pracovišti a ve srovnání s běžně užívanou Sangerovou sekvenační metodou nabízí rychlejší odezvu a větší analytickou kapacitu.
Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements – MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method
