A sensitive and specific diagnostic test for hearing loss using a microdroplet PCR‐based approach and next generation sequencing

Abstract

Implementing DNA diagnostics in clinical practice for extremely heterogeneous diseases such as hearing loss is challenging, especially when attempting to reach high sensitivity and specificity in a cost-effective fashion. Next generation sequencing has enabled the development of such a test, but the most commonly used genomic target enrichment methods such as hybridization-based capture suffer from restrictions. In this study, we have adopted a new flexible approach using microdroplet PCR-based technology for target enrichment, in combination with massive parallel sequencing to develop a DNA diagnostic test for autosomal recessive hereditary hearing loss. This approach enabled us to identify the genetic basis of hearing loss in 9 of 24 patients, a success rate of 37.5%. Our method also proved to have high sensitivity and specificity. Currently, routine molecular genetic diagnostic testing for deafness is in most cases only performed for the GJB2 gene and a positive result is typically only obtained in 10-20% of deaf children. Individuals with mutations in GJB2 had already been excluded in our selected set of 24 patients. Therefore, we anticipate that our deafness test may lead to a genetic diagnosis in roughly 50% of unscreened autosomal recessive deafness cases. We propose that this diagnostic testing approach represents a significant improvement in clinical practice as a standard diagnostic tool for children with hearing loss.

Diagnóstico etiológico de la sordera infantil: Recomendaciones CODEPEH 2015

“…Las alteraciones en estos genes justifican entre el 10 % y el 50 % de las sorderas de origen genético, dependiendo este porcentaje de la población objeto del estudio y de las características clínicas de los pacientes evaluados (Kenneson et al, 2002) (Schrauwen et al, 2013). Por lo tanto, el análisis de los genes GJB2 y GJB6 es un elemento esencial del proceso diagnóstico de las hipoacusias infantiles (Alford et al, 2014).…”

“…Por lo tanto, el análisis de los genes GJB2 y GJB6 es un elemento esencial del proceso diagnóstico de las hipoacusias infantiles (Alford et al, 2014). El resto de casos son consecuencia de mutaciones en decenas de genes diferentes, responsables cada uno de ellos de un pequeño porcentaje de familias (Schrauwen et al, 2013) (Vona et al, 2014). Estos casos serían tributarios de técnicas más amplias como los paneles de genes realizados con estudios de secuenciación de nueva generación (NGS).…”

“…Mientras que la sensibilidad y la especificad de la secuenciación Sanger es excelente y se considera como patrón de referencia, la de la NGS debe ser contrastada para cada panel, siendo posible alcanzar cifras >99 %. Por lo tanto, el panel seleccionado debe garantizar valores de sensibilidad y especificidad equivalentes a los de la secuenciación (Schrauwen et al, 2013) (Shearer et al, 2013) (Shearer et al, 2010).…”

“…En este punto, con la intención de minimizar los costes del proceso, el primer paso recomendado es analizar la presencia de mutaciones en el gen GJB2 y de deleciones en GJB6, dada su elevada prevalencia en nuestro medio (Kenneson et al, 2002) (Schrauwen et al, 2013). Si no es posible identificar la causa de la sordera tras el análisis de estos genes, el siguiente paso debe ser la secuenciación de un panel de genes adecuadamente seleccionado, mediante NGS (Alford et al, 2014) (Shearer y Smith, 2015).…”

Targeted Next-Generation Sequencing: the Clinician’s Stethoscope for Genetic Disorders

“…At present, the targeted DNA sequencing platform from RainDance Technologies offers the possibility to successfully use up to 20,000 primer pairs in one experiment [17]. Excellent results using microdroplet-PCR followed by NGS have been achieved for the diagnostics of several heterogeneous disorders, such as congenital muscular dystrophies [53], hearing loss [54] or mitochondrial disorders [55]. Furthermore, compartmentalization of a highly multiplexed PCR can also be achieved by performing several thousands of individual reactions in separate chambers inside a microfluidic chip [48] (e.g., Access Array System from Fluidigm [47]).…”