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Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis

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Abstract

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with “full” HLH and 79/111 patients with “incomplete/atypical” HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

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Abbreviations

CHS:

Chediak-Higashi syndrome

CTL:

cytotoxic T cells

FHL:

familial hemophagocytic lymphohistiocytosis

GPOH:

group of the Society for Pediatric Oncology and Hematology

GS2:

Griscelli syndrome type 2

HLH:

hemophagocytic lymphohistiocytosis

HPS2:

Hermansky-Pudlak syndrome type 2

HSCT:

hematopoietic stem cell transplantation

LCH:

Langerhans cell histiocytosis

MAS-HLH:

Macrophage-activation syndrome

NGS:

next-generation sequencing

NK cell:

natural killer cell

PID:

primary immunodeficiency

WES:

whole exome sequencing

XLP:

X-linked lymphoproliferate syndrome

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Acknowledgements

We thank the patients and their families as well as all physicians participating in the GPOH HLH study. We are grateful to the team of the CCI Advanced Diagnostic Unit for their excellent work. We thank Manuela Adao for excellent technical assistance and Marcus Tetzlaff for reliable documentation, and Martin Mynarek for performing competing event analysis with R (all Hamburg, Germany).

The following physicians contributed substantially to the HLH study of the GPOH (Germany, Austria, Switzerland):

Martina Ahlmann (Münster), Roland Ammann (Bern, Switzerland), Uta Behrends (Munich), Rita Beier (Essen), Horst von Bernuth (Berlin), Karin Beutel (Munich), Birgit Burkhardt (Münster), Gunnar Cario (Kiel), Carl-Friedrich Classen (Rostock), Matthias Dürken (Mannheim), Martin Ebinger (Tübingen), Johann Greil (Heidelberg), Ute Groß-Wieltsch (Stuttgart), Bernd Gruhn (Jena), Wolfgang Holter (Vienna, Austria), Patrick Hundsdörfer (Berlin), Ingrid Kühnle (Göttingen), Norbert Jorch (Bielefeld), Reinhard Kolb (Oldenburg), Jörn-Sven Kühl (Berlin), Britta Maecker (Hannover), Roland Meisel (Düsseldorf), Milen Minkov (Wien), Ingo Müller (Hamburg), Tim Niehuis (Krefeld), Jana Pachlopnik-Schmid (Zurich, Switzerland), Arnulf Pekrun (Bremen), Aram Prokop (Cologne), Johannes Rischewski (Luzern, Switzerland), Irene Schmid (Munich), Ansgar Schulz (Ulm), Paul-Gerhardt Schlegel (Würzburg), Michael Schündeln (Essen), Markus Seidel (Graz, Austria), Thorsten Simon (Köln), Jan Sörensen (Frankfurt), Martin Chada (Erlangen), Meinolf Suttorp (Dresden), Wilhelm Woessmann (Giessen). Centers in Germany, unless otherwise specified.

Authorʼs Contributions

All authors gave input and approved the final version of the manuscript.

Sandra Ammann: Collected patient data, supervised routine immunological testing, performed additional confirmatory tests, drafted manuscript.

Kai Lehmberg: Coordinates the GPOH HLH study, collected patient data, drafted manuscript.

Udo zur Stadt: Performed sequencing and provided genetic data.

Christian Klemann: Collected patient data.

Sebastian Bode: Collected patient data.

Carsten Speckmann: Collected patient data.

Gritta Janka: Initiated the GPOH study.

Katharina Wustrau: collected patient data for transplantation study.

Mirzokhid Rakhmanov: Coordinated diagnostic testing.

Ilka Fuchs: Coordinated diagnostic testing.

Hans Christian Hennies: Performed whole exome sequencing.

Stephan Ehl: designed the study, coordinates the GPOH HLH study, drafted the manuscript.

Funding

This work was supported by grants from the DFG (EH 145/5-1 and SFB1160, TP1) and BMBF (01 EO 0803) to S.E., from the DFG (HE 3119/10-1) and the Köln Fortune Program of the Faculty of Medicine, University of Cologne to H.C.H.

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Author notes

  1. Sandra Ammann, Kai Lehmberg contributed equally to this work.

Authors and Affiliations

  1. Center for Chronic Immunodeficiency, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Sandra Ammann, Christian Klemann, Sebastian F. N. Bode, Carsten Speckmann, Mirzokhid Rakhmanov, Ilka Fuchs & Stephan Ehl

  2. Faculty of Biology, University Freiburg, Freiburg, Germany

    Sandra Ammann

  3. Division of Pediatric Stem Cell Transplantation and Immunology, University Medical Center Hamburg Eppendorf, Hamburg, Germany

    Kai Lehmberg, Gritta Janka & Katharina Wustrau

  4. Center for Diagnostic, University Medical Center Hamburg Eppendorf, Hamburg, Germany

    Udo zur Stadt

  5. Center for Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg, Germany

    Sebastian F. N. Bode, Carsten Speckmann & Stephan Ehl

  6. Center for Human Genetics and Laboratory Diagnostics (AHC), Martinried, Germany

    Mirzokhid Rakhmanov

  7. Cologne Center for Genomics, University of Cologne, Cologne, Germany

    Hans C. Hennies

  8. Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

    Hans C. Hennies

  9. Department of Biological Sciences, University of Huddersfield, Huddersfield, UK

    Hans C. Hennies

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Consortia

the HLH study of the GPOH

  • Martina Ahlmann
  • , Roland Ammann
  • , Uta Behrends
  • , Rita Beier
  • , Horst von Bernuth
  • , Karin Beutel
  • , Birgit Burkhardt
  • , Gunnar Cario
  • , Carl-Friedrich Classen
  • , Matthias Dürken
  • , Martin Ebinger
  • , Johann Greil
  • , Ute Groß-Wieltsch
  • , Bernd Gruhn
  • , Wolfgang Holter
  • , Patrick Hundsdörfer
  • , Ingrid Kühnle
  • , Norbert Jorch
  • , Reinhard Kolb
  • , Jörn-Sven Kühl
  • , Britta Maecker
  • , Roland Meisel
  • , Milen Minkov
  • , Ingo Müller
  • , Tim Niehuis
  • , Jana Pachlopnik-Schmid
  • , Arnulf Pekrun
  • , Aram Prokop
  • , Johannes Rischewski
  • , Irene Schmid
  • , Ansgar Schulz
  • , Paul-Gerhardt Schlegel
  • , Michael Schündeln
  • , Markus Seidel
  • , Thorsten Simon
  • , Jan Sörensen
  • , Martin Chada
  • , Meinolf Suttorp
  •  & Wilhelm Woessmann

Corresponding author

Correspondence to Stephan Ehl.

Ethics declarations

Informed consent was obtained according to the institutional review board approval (University of Freiburg ethics committee’s protocol numbers 143/12 and 40/08).

Conflicts of Interest

SE has received consulting fees from UCB and Novartis, but not in relation to this study.

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Ammann, S., Lehmberg, K., zur Stadt, U. et al. Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis. J Clin Immunol 37, 770–780 (2017). https://doi.org/10.1007/s10875-017-0443-1

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