Neue Entwicklungen in der molekularbiologischen Diagnostik

Zusammenfassung

Die uns heute zur Verfügung stehenden molekularbiologischen Analysemethoden sind technisch weitgehend ausgereift und warden bereits breit eingesetzt. Daraus ergibt sich die Notwendigkeit der Einführung von Regeln zur Überprüfung der Qualität der Tests sowie der Testlabors, was im Artikel im Detail besprochen wird. Nachdem Nukleinsäuren isoliert und amp lifiziert wurden, werden sie zur Genotypisierung herangezogen, wobei zahlreiche Fragen adressiert werden können wie z.B. die Diagnose erblich bedingter Krankheiten oder erblicher Prädispositionen, forensische Aspekte, die Identifizierung und Typisierung von Krankheitserregern, aber auch die Aufklärung evolutionärer Zusammenhänge. Die Wahl des Verfahrens zur Mutationssuche ist eng mit der Art und Heterogenität der für einen Phänotyp verantwortlichen Mutationen verbunden. Derzeit werden in vielen Labors für die Diagnostik PCR-Analysen neben der klassischen Sequenzierung nach Frederick Sanger eingesetzt. Zunehmend kommt jedoch die relativ neue „next generation sequencing” (NGS) Analyse zur Anwendung. Obwohl der Einsatz der NGS-Technologie in der klinischen Diagnostik mit zahlreichen Herausforderungen verbunden ist, wird die Umstellung auf diese Methode aufgrund der Vorteile in naher Zukunft vollzogen werden. Die deutliche Preisreduktion des NGS auf ca. 1000,- USD brachte die Genomsequenzierung schon sehr nahe an klinische Anwendungen heran. Bis zum routinemäßigen Einsatz müssen jedoch noch die Daten-Prozessierung, die Speicherung der riesigen Datenmengen und die Interpretation der Ergebnisse vereinfacht werden. Es gibt dafür unterschiedliche Datenbanken, von denen einige angeführt werden. Das Verständnis verschiedener Polymorphismen in Genen von Gerinnungsfaktoren und die Bedeutung der personalisierten Medizin, die ein wichtiges Werkzeug zur Risikostratifizierung der Patienten darstellt, wurden sehr vertieft. Beide Aspekte haben heute große Bedeutung und warden im vorliegenden Beitrag diskutiert.

Summary

Today, we have access to excellent and advanced molecular methods that are already widely used. This requires rules to control the quality of the methods as well as the laboratory. Both aspects will be discussed in the article. Following the isolation of nucleic acids they are used for genotyping which allows to address several questions: diagnosis of inherited diseases, inherited predispositions, forensic analyses, identification and typing of bacteria or viruses, elucidation of evolutionary aspects. Importantly, it has to be realized that the type and heterogeneity of phenotypically relevant mutations determines the method used for testing. Today, most laboratories use either PCR analyses or Sanger sequencing for diagnostic applications. However, increasingly next generation sequencing (NGS) is applied. The clinical use of NGS is still very challenging, but we can expect that the switch to regular application of this method will be coming in the very near future. The price for NGS has gone down to approx. USD 1000,- which makes the routine diagnostic use feasible. Nevertheless, several challenges have yet to be solved, such as the processing of the large data volume as well as storage of the data. Supporting data bases exist already and some will be discussed in the article. The understanding of the clinical relevance of many polymorphisms is another issue that has yet to be solved, particularly as in the context of personalized medicine polymorphisms have become increasingly important.

Keywords

Molecular diagnostics - molecular analyses - quality issues - next generation sequencing

Schlüsselwörter

Molekulare Diagnostik - Molekulare Analysemethoden - Qualitätssicherung - Next Generation Sequencing

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