Diagnostic Single Gene Analyses Beyond Sanger

Juliane Najm; Matthias Rath; Winnie Schröder; Ute Felbor

doi:10.5482/HAMO-17-01-0008

Hämostaseologie, Table of Contents

Hamostaseologie 2018; 38(03): 158-165
DOI: 10.5482/HAMO-17-01-0008

Original Article

Georg Thieme Verlag KG Stuttgart · New York

Diagnostic Single Gene Analyses Beyond Sanger

Economic high-throughput sequencing of small genes involved in congenital coagulation and platelet disorders

Juliane Najm^*

¹Department of Human Genetics, University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

,

Matthias Rath^*

¹Department of Human Genetics, University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

,

Winnie Schröder

¹Department of Human Genetics, University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

,

Ute Felbor

¹Department of Human Genetics, University Medicine Greifswald, and Interfaculty Institute of Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany

› Author Affiliations

Abstract

Summary

Molecular testing of congenital coagulation and platelet disorders offers confirmation of clinical diagnoses, supports genetic counselling, and enables predictive and prenatal diagnosis. In some cases, genotype-phenotype correlations are important for predicting the clinical course of the disease and adaptation of individualized therapy. Until recently, genotyping has been mainly performed by Sanger sequencing. While next generation sequencing (NGS) enables the parallel analysis of multiple genes, the cost-value ratio of custom-made panels can be unfavorable for analyses of specific small genes. The aim of this study was to transfer genotyping of small genes involved in congenital coagulation and platelet disorders from Sanger sequencing to an NGS-based method. A LR-PCR approach for target enrichment of the entire genomic regions of the genes F7, F10, F11, F12, GATA1, MYH9, TUBB1 and WAS was combined with high-throughput sequencing on a MiSeq platform. NGS detected all variants that had previously been identified by Sanger sequencing. Our results demonstrate that this approach is an accurate and flexible tool for molecular genetic diagnostics of single small genes.

Zusammenfassung

Die molekulare Diagnostik hereditärer Gerinnungs- und Thrombozytenstörungen ermöglicht die Bestätigung einer klinischen Verdachtsdiagnose, ist aber auch für die genetische Beratung und die prädiktive und pränatale Diagnostik notwendig. In einigen Fällen ist eine Genotyp-Phänotyp-Korrelation für die Prognose des klinischen Krankheitsverlaufs wichtig und erlaubt eine individuelle Therapieplanung. Im Gegensatz zur Sanger-Sequenzierung ermöglicht Next Generation Sequencing (NGS) die parallele Analyse mehrerer Gene. Allerdings kann das Kosten-Nutzen-Verhältnis von großen Genpanels für die Analyse spezifischer kleiner Gene ungünstig sein. Ziel dieser Studie war es, die Genotypisierung kleiner Gene für hereditäre Gerinnungs- und Thrombozytenstörungen von der Sanger-Sequenzierung auf eine NGS-basierte Methode umzustellen. Zu diesem Zweck wurde ein LR-PCR-Ansatz zur Anreicherung der gesamten genomischen Regionen der Gene F7, F10, F11, F12, GATA1, MYH9, TUBB1 und WAS mit einer Hochdurchsatzsequenzierung auf einer MiSeq-Plattform kombiniert. Mit dieser Methode konnten alle durch Sanger-Sequenzierung identifizierten Veränderungen sicher nachgewiesen werden. Unsere Ergebnisse zeigen, dass dieser Ansatz ein präzises und flexibles Werkzeug in der molekulargenetischen Diagnostik einzelner kleiner Gene sein könnte.

Keywords

congenital coagulation and platelet disorders - LR-PCR - NGS - high throughput sequencing

Schlüsselwörter

hereditäre gerinnungs- und thrombozytenstörungen - LR-PCR - NGS - hochdurchsatzsequenzierung

Full Text

References

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Supplementary Material

Supplementary Material