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Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis

European Journal of Human Genetics volume 28, pages 193–201 (2020)Cite this article

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Abstract

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder, which causes loss of renal proximal tubular function and progressive loss of glomerular function, finally leading to end stage renal failure at school age. In the course of the disease most patients will need kidney transplantation if treatment has not been started before clinical manifestation. With an effective treatment available, a newborn screening assay is highly demanded. Since newborns with cystinosis usually do not show symptoms within the first months of life and no biochemical markers are easily detectable, a DNA-based method seems to be an obvious tool for early diagnosis. Screening was performed using high-throughput nucleic acid extraction followed by 384-well qPCR and melting analysis for the three most frequent variants (57 kb deletion NC_000017.11:g.3600934_3658165del (GRCh38); c.18_21del GACT; c.926dupG) responsible for the defective lysosomal membrane protein cystinosin (CTNS). To increase sensitivity, all heterozygous samples identified in qPCR assay were verified and screened for additional variants by applying next generation sequencing. From January 2018 to July 2019 nearly 292,000 newborns were successfully screened. We identified two newborns with a homozygous 57 kb deletion and a second one with heterozygous 57 kb deletion and a G>C substitution at position c.-512 on the second allele. Cystinosis is an example for diseases caused by a limited number of high prevalence and a high number of low prevalence variants. We have shown that qPCR combined with NGS can be used as a high throughput, cost effective tool in newborn screening for such diseases.

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Acknowledgements

We highly appreciate the help of Prof. Dr Carsten Bergmann and Dr Neuber in selecting clinically relevant variants in the CTNS gene.

Funding

The screening was funded by the Cystinose Foundation (Cystinose Stifung, DSZ-Regional Office Munich; Widenmayerstr. 10; 80538 Munich; Germany) and the Dietmar Hopp Foundation (Raiffeisenstraße 51, 68789 St. Leon-Rot, Germany). The funders played no role in design, interpretation, and publication of this study.

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Authors and Affiliations

  1. Laboratory Becker & Colleagues, Führichstr. 70, 81671, Munich, Germany

    Tobias Fleige, Siegfried Burggraf, Ludwig Czibere, Julia Häring, Birgit Glück, Lisa Marie Keitel, Jürgen Durner, Wulf Röschinger & Marc Becker

  2. TIB Molbiol GmbH, Eresburgstr. 22–23, 12103, Berlin, Germany

    Olfert Landt

  3. University Hospital Münster, Albert-Schweitzer-Campus 1, 48149, Münster, Germany

    Erik Harms

  4. Pediatric Nephrology, RoMed Klinikum Rosenheim, Pettenkoferstr. 10, 83022, Rosenheim, Germany

    Katharina Hohenfellner

  5. Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig Maximilian University Munich, Goethestr. 70, 80337, Munich, Germany

    Jürgen Durner & Marc Becker

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  1. Tobias Fleige
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  2. Siegfried Burggraf
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Correspondence to Tobias Fleige.

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Fleige, T., Burggraf, S., Czibere, L. et al. Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis. Eur J Hum Genet 28, 193–201 (2020). https://doi.org/10.1038/s41431-019-0521-3

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