Diagnosis of HLH: two siblings, two distinct genetic causes

Abstract

This report highlights case of two siblings who developed haemophagocytic lymphohystiocytosis due to distinct genetic abnormalities. Though their presentation was clinically similar, the cases demonstrate that a shared genetic diagnosis among siblings cannot be assumed.

This report highlights case of two siblings who developed haemophagocytic lymphohystiocytosis (HLH) due to distinct genetic abnormalities, in this case an X-linked condition (XLP1), and an autosomal recessive condition (FHL5). The cases demonstrate that suspicion of a primary immune deficiency (PID) must be investigated rapidly, and though their presentation was clinically similar, a shared genetic diagnosis among siblings cannot be assumed.

Graphical Abstract

Graphical Abstract.

Background

Haemophagocytic lymphohystiocytosis (HLH) is an inflammatory condition characterized by prolonged fevers, splenomegaly, and cytopenias [1]. It can be caused by genetic abnormalities, or—in the absence of detectable genetic defect—by a background of autoimmunity, viral infection, or malignancy. We report on two siblings from a not knowingly consanguineous family who developed HLH diagnosed according to HLH-2004 study criteria [1] but in whom, unexpectedly, two different genetic causes of HLH were identified.

The genetically defined subtypes of familial HLH (FHL2–5) are due to defects in the Perforin [2], Munc13-4 [3], Syntaxin-11 [4], and Munc18-2 [5] proteins, respectively. In addition, X-linked lymphoproliferative syndromes (XLP1 and XLP2) are deficient in SLAM-associated protein (SAP) or X-linked inhibitor of apoptosis protein (XIAP), respectively, and can present clinically with HLH [6]. Some patients with RAB27A mutation may also develop HLH, with or without pigment deficiency [7].

A diagnosis of HLH can be made if at least five of the eight HLH-2004 criteria are fulfilled or upon identification of a genetic mutation associated with HLH [1].

Presentation

Case 1 presented with failure to thrive at 5 weeks. By 9 weeks of age, her condition had deteriorated, and she developed pancytopenia and hepatosplenomegaly. Histiocytes were observed on a bone marrow aspirate but no haemophagocytosis was apparent. She was reported to rapidly improve, with complete resolution of her hepatosplenomegaly. However, 1 month later, she was readmitted with fever, hepatosplenomegaly, and pancytopenia. In this instance, a second bone marrow aspirate was suggestive of HLH. Her clinical phenotype fulfilled seven of eight HLH-2004 diagnostic criteria (Fig. 1A).

Fig. 1.

(A) Presentation. (B and C) Diagnosis of FHL5 in case 1. GRA using CD107a expression to assess cytotoxic function. Munc 18-2 protein expression by immunoblot of peripheral blood mononuclear cells in control and case 1: carrier 3 is the mother (II3). (D) Diagnosis of XLP1 in case 2. SAP expression in CD8⁺ T cells in control and case 2.

Case 2 presented at 4 years of age, with prolonged fever, a generalized erythematous rash, lymphadenopathy, hepatosplenomegaly, and pancytopenia. He also became jaundiced and developed neurological symptoms including drowsiness, irritability, and multiple generalized tonic-clonic seizures. Prior to this he was well, with no significant infections. Epstein–Barr virus (EBV) serology and polymerase chain reaction were consistent with acute EBV infection. Bone marrow trephine biopsy showed active haemophagocytosis. Cerebrospinal fluid findings were consistent with HLH central nervous system involvement. Clinically, he met six of eight HLH-2004 diagnostic criteria (Fig. 1A).

Investigations and outcomes

The HLH diagnostic panel includes protein analysis of Perforin (FHL2), SAP, and XIAP (XLP1 and XLP2), and assessment of cytotoxic function (FHL3–5) using the granule release assay (GRA) which measures CD107a expression post stimulation (degranulation) [1–8].

Lymphocytes isolated from case 1 had normal perforin expression (confirmed by genetics, data not shown) but an impaired cytotoxic function by GRA (Fig. 1B). Identification of a homozygous missense mutation c.1621G>A in the STXBP2 gene confirmed a diagnosis of FHL5 [6]. The sequence variation resulted in an amino acid change p.Gly541Ser and abrogated Munc18-2 protein expression (Fig. 1C, NM_006949). Although on gnomad this mutation is relatively common and has been described in a number of FHL5 patients in a compound heterozygous state [9], homozygous patients are rarely described suggesting that this may be embryonic lethal in most cases. Case 1 was treated according to the HLH-2004 protocol, and 6 months later, received a reduced intensity conditioning haematopoietic stem cell transplant from a matched unrelated donor. She is currently alive and well.

Case 2 had normal perforin expression and cytotoxic function (GRA) (data not shown). Flow cytometry showed that expression of SAP was absent (Fig. 1D). Gene sequencing defined a mutation in the SH2D1A gene, c.301C>A (p.Pro101Thr). This mutation had not previously been reported; however, the proline at codon 101 is highly conserved, and the identified mutation and lack of SAP expression were consistent with a clinical diagnosis of XLP1. Treatment on the HLH-2004 protocol was initiated, but 3 days later his condition suddenly further deteriorated, and he died shortly thereafter.

STXBP2 and SH2D1A genetic analysis demonstrated the inheritance of FHL5 and XLP1 within the family. The family pedigree in Fig. 2 shows the pathogenic STXBP2 gene variation in the maternal (II3, I1) and paternal lineage (II4). The maternal aunts were not carriers of the STXBP2 c.1621G>A mutation. The SH2D1A c.301C>A mutation of case 2 was present in the maternal (II3) lineage and was identified in the maternal aunts (II1, II2). The maternal grandmother (I1) was not a carrier of the SH2D1A mutation. One uncle and the maternal grandfather (I2) were never tested. It was noted that the maternal grandfather (I2) had died at the age of 46 years from a haematological malignancy and is a suspected but not confirmed carrier of the SH2D1A mutation. No pathological material from the malignancy of the grandfather was available for testing to confirm whether he carried the mutation in SH2D1A. Linkage analysis demonstrated that case 2 inherited the grandpaternal X chromosome (I2) predicted to have contained the SH2D1A mutation. Prenatal diagnosis and predictive testing became available for at-risk family members. Predictive testing for a first cousin (III1) did not detect the familial SH2D1A mutation.

Fig. 2.

Genetic pedigree. STXBP2 mutation carrier status is indicated in grey. SH2D1A mutation status is indicated in black. White indicates a wild-type genotype. The speckled pattern indicates that genotype was not determined.

Discussion

Case 1 with defined FHL5, exhibited seven out of eight HLH-2004 criteria. She presented in the first year of life, as is commonly seen with FLH. Her brother (case 2) presented at 4 years of age with XLP1 and EBV-driven HLH and had six of the HLH defining features. In both instances, the HLH-2004 study criteria provided an effective funnel for focusing clinical management appropriately towards treating the acute HLH presentation.

The maternal grandfather (Fig. 2, I2) died at the age of 46 years due to a haematological malignancy, presumably associated with XLP1. In contrast, his grandson with genetically confirmed XLP1 only lived until 4 years of age. The heterogeneity of age of presentation challenges the assumption that primary immunodeficiency (PID) is restricted to children. Nor did genotype correlate with a specific XLP1 phenotype. Predictive testing was offered to the family and was used to predict that a first cousin at 50% risk for XLP1 did not have the familial gene mutation (Fig. 2, III1), demonstrating that gene mutation analysis can inform family and clinician decisions.

The siblings demonstrate that suspicion of a PID must be investigated rapidly and that PID of different genetic basis can present in the same family; in this case an X-linked condition (XLP1), and an autosomal recessive condition (FHL5). Dual inheritance of recombination activating gene 1-severe combined immunodeficiency and FHL2 (personal communication), as well as SCID with non-PID diseases (SCID/Thalassaemia [10]) have also been identified. The diagnosis of PID in a family must not preclude the thorough investigation of other rare PID in siblings.

Glossary

Abbreviations

HLH

haemophagocytic lymphohystiocytosis

XLP

X-linked lymphoproliferative syndrome

SAP

SLAM-associated protein

XIAP

X-linked inhibitor of apoptosis protein

EBV

Epstein–Barr virus

PCR

polymerase chain reaction

GRA

Granule release assay

PID

primary immune deficiency

RAG1-SCID

recombination activating gene 1-severe combined immunodeficiency

Funding

E.R. and K.G. are supported by the NIHR Great Ormond Street Biomedical Research Centre. The project was carried out during an NHS Health Education England-funded Clinical Scientist pre-registration training position (C.E.). The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

Conflict of interest

None declared.

Author contributions

C.E. and E.R. performed and analysed some of the immunology investigations and wrote the draft of the paper, J.V. and P.V. identified and supervised care of the patients and provided samples, S.B. and M.A. undertook the genetic analysis, K.G. performed/analysed some, and oversaw all, the immunology investigations. All authors reviewed the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.