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Targeted High-Throughput Sequencing for Genetic Diagnostics of Hemophagocytic Lymphohistiocytosis Bianca Tesi1,2* , Kristina Lagerstedt-Robinson2,3, Samuel C

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Targeted High-Throughput Sequencing for Genetic Diagnostics of Hemophagocytic Lymphohistiocytosis Bianca Tesi1,2* , Kristina Lagerstedt-Robinson2,3, Samuel C Tesi et al. Genome Medicine (2015) 7:130 DOI 10.1186/s13073-015-0244-1 RESEARCH Open Access Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis Bianca Tesi1,2* , Kristina Lagerstedt-Robinson2,3, Samuel C. C. Chiang4, Eya Ben Bdira1,2, Miguel Abboud5, Burcu Belen6, Omer Devecioglu7, Zehra Fadoo8, Allen E. J. Yeoh9, Hans Christian Erichsen10, Merja Möttönen11, Himmet Haluk Akar12, Johanna Hästbacka13, Zuhre Kaya14, Susana Nunes15, Turkan Patiroglu12, Magnus Sabel16,17, Ebru Tugrul Saribeyoglu18, Tor Henrik Tvedt19, Ekrem Unal20, Sule Unal21, Aysegul Unuvar22, Marie Meeths1,2, Jan-Inge Henter1, Magnus Nordenskjöld2,3 and Yenan T. Bryceson4,23* Abstract Background: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. Methods: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. Results: Analyses revealed a mutation detection sensitivity of 97.3 %, an average coverage per gene of 98.0 %, and adequate coverage over 98.6 % of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38 %). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. Conclusions: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization. * Correspondence: [email protected]; [email protected] 1Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital Solna, SE-17176 Stockholm, Sweden 4Centre for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-14186 Stockholm, Sweden Full list of author information is available at the end of the article © 2015 Tesi et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tesi et al. Genome Medicine (2015) 7:130 Page 2 of 13 Background disorders, or secondary to infections, malignancies or Hemophagocytic lymphohistiocytosis (HLH) is a severe autoimmune disorders in individuals without any estab- hyperinflammatory syndrome that presents with unre- lished genetic disease susceptibility [1]. mitting fever, splenomegaly and cytopenia [1]. According Patients with defective lymphocyte cytotoxicity usually to the HLH-2004 protocol, HLH can be defined as ful- develop early-onset HLH with high penetrance and fillment of at least five of eight clinical and laboratory require the most radical immunosuppressive therapy. criteria [2]. Primary, genetic, as well as secondary forms Defective natural killer (NK) cell cytotoxic activity, as of HLH have been described. HLH is typically treated by measured by the 51Cr-release assay, is included among immunosuppression, followed by hematopoietic stem the HLH-2004 diagnostic criteria [2]. However, pathological cell transplantation in familial cases [1]. Current HLH results with this assay do not necessarily reflect functional criteria poorly discriminate underlying causes of disease. defects in lymphocyte cytotoxicity, but can also be caused Importantly, therapies tailored to different etiologies of by low NK cell numbers. Refined assays have been devel- HLH may improve treatment outcome [3]. oped for the identification of patients with defects in Several genetic disorders predispose to HLH, but vary lymphocyte cytotoxicity as well as XIAP signaling [18–20]. in their risk of developing disease. Congenital defects af- These assays require considerable technical expertise and fecting the perforin-mediated lymphocyte cytotoxicity, rely on fresh blood samples. Therefore, improved diagnostic such as autosomal recessive mutations in PRF1, procedures are required for guidance of treatment UNC13D, STX11,andSTXBP2, represent the most com- decisions. mon causes of primary HLH, termed familial HLH With current insights, patients with defective (FHL) type 2, 3, 4 and 5, respectively [1, 4]. The impaired lymphocyte cytotoxicity can also be diagnosed by DNA killing of infected as well as activated immune cells results sequencing. To influence the clinical management of in the sustained hyperinflammatory state characteristic of HLH patients, genetic diagnostics must be rapid and HLH, where animal models have postulated a critical role accurate. Due to genetic heterogeneity, achieving a mo- for CD8+ T cells and interferon (IFN)-γ [5]. Patients with lecular diagnosis by conventional Sanger sequencing is autosomal recessive mutations in RAB27A and LYST, labor-intensive and time-consuming. Technological causative of Griscelli syndrome type 2 (GS2) and Chediak- advances have increased sequencing throughput, with Higashi syndrome (CHS), respectively, also frequently decreased sequencing times and costs [21]. As more develop HLH. Besides defective lymphocyte cytotoxicity, bench-top sized machines have been pushed to the these syndromes are associated with hypopigmentation market, appealing solutions for diagnostic laboratory [6, 7]. Only one case of HLH has been reported in settings have become available [22]. Aimed at diagnos- Hermansky-Pudlak syndrome type 2, another ing a broad range of immune defects, high-throughput hypopigmentation syndrome specifically caused by assays have recently been reported for the simultan- mutations in AP3B1 and associated with impaired eous study of a number of primary immunodeficiency lymphocyte cytotoxicity [8]. Moreover, HLH has so far syndromes [23–25]. not been reported in Hermansky-Pudlak syndrome Here, we report our experience in implementing a type 9 patients, caused by mutations in BLOC1S6 and targeted resequencing approach for identification of also reported to display impaired lymphocyte cytotox- HLH patients with defective lymphocyte cytotoxicity. icity [9]. Genetic disorders displaying a more limited Moreover, we characterize the genetic variants in HLH- impairment of lymphocyte cytotoxicity may also related genes in the general population and discuss the present with HLH or related lymphoproliferative implications to interpretation of association of rare, po- diseases. Patients with hemizygous mutations in SH2D1A tentially damaging monoallelic variants with disease. or XIAP, associated with X-linked lymphoproliferative dis- ease, typically present with HLH or lymphoproliferative Methods diseases, often triggered by Epstein-Barr virus (EBV) in- Patients fection [10]. Lymphoproliferation and severe EBV infec- The study was performed using genomic DNA (gDNA) tions are also features of autosomal recessive mutations in samples from (1) 13 patients with a confirmed molecular ITK [11] and hemizygous mutations in MAGT1 [12], with diagnosis in an HLH-related gene, (2) 58 patients, pro- sporadic cases of HLH [13, 14]. Episodes of HLH have spectively recruited over a 12 months period, fulfilling also been reported in patients harboring other primary five or more HLH diagnostic criteria (n = 56) or with a immunodeficiencies [3, 15–17], providing evidence for cytotoxicity defect suggestive of primary HLH (n = 2). hyperinflammatory syndromes fulfilling current HLH Informed consent was obtained from all study partici- criteria in an immunological context of T-cell deficiency pants in accordance with the Declaration of Helsinki. or absent IFN-γ signaling. HLH may also arise in the con- The study was approved by the Regional Ethics Review text of inborn errors of metabolism and lysosomal storage Board in Stockholm, Sweden. Tesi et al. Genome Medicine (2015) 7:130 Page 3 of 13 AmpliSeq custom panel design Bioinformatics analyses A targeted resequencing panel covering 12 HLH-associated Assessment of sequencing quality control, mapping, genes was designed according to Ion AmpliSeq technology coverage analysis and variant calling
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