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Clinical Vignette

Familial Hemophagocytic Lymphohistiocytosis (FHL): An Illustrative Case and Review of Literature

Savitha H, Sankar V H
Department of Paediatrics, SAT Hospital, Government Medical College, Thiruvananthapuram, India
Correspondence to: Dr Sankar V H      Email: sankarvh@gmail.com

1 Abstract

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory disorder which occurs due to a genetic defect in the cytolytic pathway of natural killer cells and cytotoxic T cells. We present here the case of a female infant who presented with the typical features of high-grade fever, hepatosplenomegaly and pancytopenia, and was diagnosed to have FHL due to a homozygous variant in the STXBP2 gene.

Keywords: Familial hemophagocytic lymphohistiocytosis, STXBP2

2 Introduction

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder resulting from prolonged and excessive activation of antigen presenting cells (macrophages and histiocytes) and CD8+ T cells. The two forms of HLH are primary (genetic) and secondary (acquired). Primary HLH occurs due to an underlying genetic defect in the cytolytic pathway of natural killer (NK) cells and cytotoxic T cells. The disease usually presents in infancy and early childhood but the first clinically significant episode can present throughout life, ranging from prenatal presentation to the seventh decade. Secondary HLH may be triggered by an infection [Epstein-Barr virus (EBV), herpes simplex virus (HSV), cytomegalovirus (CMV), adenovirus, dengue, ebola virus etc.], malignancy, or autoimmune diseases (such as systemic onset juvenile idiopathic arthritis). Though HLH is classified as primary and secondary, there is a considerable overlap between the two and often secondary HLH is found to have an underlying genetic defect. The estimated incidence of primary HLH is 0.12/100,000 children per year in a Swedish study and 0.34/100,000 children per year in studies from Japan. The prevalence of all cases of HLH under 18 years of age has been estimated as 1.07/100,000 (Sen et al., 2017).

3 Patient details

Baby X, a 1-month-25-days-old female child, second born to a non-consanguineous couple, was admitted with history of high-grade intermittent fever, breathlessness and occasional cough for 3 days. She was a born by Caesarean section at term, with a birth weight of 3.06 kg and cried soon after birth. The postnatal period was uneventful. She was partially immunised for age (only birth dose vaccines were taken). There was family history of neonatal death; her elder sibling had expired in the newborn period due to multiorgan dysfunction.

On examination, the infant was irritable and did not have any significant facial dysmorphism except for low anterior hair line and small anterior fontanelle. Weight at admission was 4 kg, length 52 cm and head circumference 35 cm. She had mild respiratory distress but her chest was clear. Cardiovascular system was clinically normal. Abdomen was distended and soft to palpation. She had an enlarged liver which was palpable 3 cm below the right costal margin and firm in consistency. The spleen was palpable 2 cm below the left costal margin. Hernial orifices were normal and she had normal female genitalia. There was no skin rash or pigmentation. Neurological examination was normal.

She was initially managed with oxygen by continuous positive airway pressure, intravenous antibiotics (cefotaxime and amikacin) and other supportive measures. On evaluation she was found to have acute liver failure, cholestatic jaundice and multiple electrolyte abnormalities (Table 1). She was managed with N-acetyl cysteine infusion and other supportive measures. All hepatotoxic drugs were avoided. Electrolyte abnormalities were corrected with intravenous and oral supplements. But the blood counts and liver function progressively worsened (Table 1). There were no episodes of hypoglycaemia. She developed diarrhoea which was managed with oral rehydration solution, probiotics and zinc supplements. In view of high spiking fever and worsening general condition of the baby, antibiotics were upgraded to meropenem and vancomycin, and acyclovir was added suspecting a viral etiology.


Table 1: Table showing the baseline investigations and the progression in the patient









Date

DA1

DA3

DA5

DA6

DA7

DA8

DA10

Normal range










Hb (g/dl)

9.5

9.4

9.0

7.2

6.3

6

6.1

9-14










TLC (cells/mm3)

4,850

3,280

4,800

4,000

5000-19,500










DLC

P34L66

ANC 1649

P31L61

ANC 992

P12L83

ANC 576

P10L90

ANC 400

P54-62L25-33










Platelet count (cells/mm3)

65,000

35,000

31,000

10,000

1.5-4 X 105










CRP (mg/dl)

1.39

Negative

0-0.6










BU/S Cr (mg/dL)

19/0.5

13/0.4

18/0.4

21/0.5

24/0.4

25/0.3

5-20/

0.3-0.7










SGOT (IU/L)

368

241

707

1761

395

184

128

<40










SGPT (IU/L)

207

205

378

873

509

419

223

<40










Serum Na (mM/L)

128

147

132

131

136

132

132

135-145










Serum K (mM/L)

2.4

2.5

3.3

2.9

3.8

3.3

5.5

3.5-5










Serum Ca/P (mg%)

4.4/1.8 (corrected Ca- 5)

4.8/2.4

8.5/1.6

9/1.6

8.1/4.3

9-10.6/ 2.5-4.5










Serum Mg (mg%)

1

1.6

1

1.6

1.5-2.5










Serum Bil T/D (mg%)

0.9/0.5

0.8/0.2

0.5

0.5

1.7/0.3

0.2-1/

0-0.2










Serum ALP (IU/L)

720

472

293

238

210-810










Serum Pr/Alb (gm%)

5.4/3.5

4.3/2.7

4.6/2.4

6.2-7.8/ 3.5-5










PT

(sec)/INR

31/ 2.5

31.9/ 2.3

19.7/ 1.42

20.6/ 1.49

27.7/ 2.03

11-15/

0.8-1.1










APTT (sec)

27.7

28.1

39.5

32










DA – Day of admission; Hb – Hemoglobin; TLC- Total leucocyte count; DLC – Differential leucocyte count;
P – Polymorphs; L- Lymphocytes; ANC – Absolute neutrophil count; CRP- C-reactive protein; BU – Blood urea; S Cr – Serum creatinine; SGOT - Serum glutamic-oxaloacetic transaminase; SGPT - Serum glutamic-pyruvic transaminase; Serum Na – Serum sodium; Serum K – Serum potassium; Serum Ca/ P – Serum calcium/ phosphorus; Serum Mg – Serum magnesium; Serum Bil T/D – Serum bilirubin total/ direct; Serum ALP – Serum alkaline phosphatase; Serum Pr/ Alb – Serum total protein/ albumin; PT – Prothrombin time; APPT – Activated partial thromboplastin time

Hepatitis B surface antigen (HBsAg), and antibody against hepatitis C virus and hepatitis A virus (anti HCV and anti HAV) were negative. Dengue IgM, Lepto IgM and Scrub IgM serology, test for infectious mononucleosis, and Enterocheck WB test for typhoid were negative. The nasopharyngeal swab viral panel was negative. Real-time reverse transcription polymerase chain reaction (rRT-PCR) test for coronavirus disease (COVID-19) was also negative. Blood PCR for CMV, and HSV 1 and 2 IgM serology were also negative. Serum alpha fetoprotein was 46.88 mg/ml (reference range 0-9.5 mg/ml) and serum lactate dehydrogenase was 971 IU/L (reference range 230-400 IU/L). Urine metabolic screening was negative. Urine examination showed albumin 3+, pus cells 1-3/ high power field, sugar nil, and acetone nil. The other investigations were as follows: urine pH 5.5, serum sodium 113 mEq/L (130-140 mEq/L), serum osmolality 463 mOsm/kg (800-1300 mOsm/kg), and serum chloride 131 mEq/L (80-209 mEq/L).

Serum ferritin was 1473 ng/ml and serum triglycerides were 647 mg/dl. The clinical and biochemical parameters satisfied the criteria of HLH. Hence treatment with intravenous immunoglobulin (IVIG) 2 g/kg and dexamethasone regimen were started. Bone marrow aspirate showed hemophagocytes (macrophage with engulfed erythroblasts) suggestive of HLH (Figure 1). But the child failed to respond to IVIG or steroids. Pancytopenia, liver dysfunction and coagulopathy progressively worsened. Multiple platelet concentrate, fresh frozen plasma and packed red cell transfusions were given. But she developed haemorrhage and shock not responding to inotropes and succumbed to the illness.

PIC

Figure 1: Bone marrow aspiration smear showing hemophagocytosis

The peripheral blood sample was sent for clinical exome sequencing to look for mutations in genes involved in familial HLH. A homozygous missense variant (c.1730G>A; p.Gly577Asp) was identified in the STXBP2 gene (transcript ID ENST00000441779) in the proband. This variant was previously reported in a patient affected with familial HLH 5 (Pagel et al., 2012). This variant has not been reported in the 1000 genomes database (https://www.internationalgenome.org/1000-genomes-browsers) and has a minor allele frequency of 0.0004% and 0.027% in the gnomAD (https://gnomad.broadinstitute.org/) and internal databases, respectively. The in silico predictions of the variant are probably damaging by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and damaging by SIFT (https://sift.bii.a-star.edu.sg/), LRT (http://www.genetics.wustl.edu/jflab/lrt_query.html) and MutationTaster2 (http://www.mutationtaster.org/). The reference codon is conserved across mammals. The variant is classified as ‘likely pathogenic’ as per the American College of Medical Genetics and Genomics/ Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015). The child was thus diagnosed to have familial hemophagocytic lymphohistiocytosis type 5 (FHL5) (OMIM #613101). Both parents were heterozygous for this variant. Genetic study of the deceased sibling could not be done due to non-availability of the blood or DNA sample.

4 Discussion

Critical to the diagnosis of HLH is the awareness about the disease and high degree of suspicion in children with some of the clinical features. There is considerable overlap between HLH and the symptoms and signs of many other diseases and the combination of clinical and laboratory signs, their severity and the changes over time help in diagnosis. It closely resembles severe systemic sepsis and some consider HLH and severe sepsis to be phenotypes of a spectrum of hyperinflammatory reactions. The cardinal features of HLH are high grade fever, hepatosplenomegaly and cytopenias, with a failure to respond to initial anti-infective treatments. The inflammatory reaction results in elevation of levels of tumour necrosis factor (TNF) α, interleukin 6 (IL-6), interleukin 1 (IL-1) and other interleukins, which causes high fever and infiltration of tissues with activated macrophages and lymphocytes. This leads to multi-organ inflammation and damage.

The HLH-2004 study developed diagnostic criteria for the clinical diagnosis of HLH (Henter et al., 2007). In a child with appropriate clinical presentation, identification of mutation in one of the genes involved or fulfilment of five of the eight criteria, confirms the diagnosis of HLH. Fever and splenomegaly are consistent features in children other than neonates, seen in 90-100% of cases. Hyperferritinemia is a crucial marker of active HLH/macrophage activation syndrome (MAS) and values more than 10,000 g/L in children were found to be 90% sensitive and 96% specific for HLH. In case of other investigations like blood counts, erythrocyte sedimentation rate (ESR) and transaminases, change in parameters over time rather than absolute values are important for the diagnosis. A paradoxical drop in ESR in spite of active systemic inflammation in the proband is suggestive of HLH. This is due to the fall in fibrinogen level due to fibrinogen consumption and liver dysfunction. A dropping ESR in conjunction with elevated c-reactive protein (CRP) is an important sign of HLH.

While the presence of hemophagocytosis in the bone marrow can help to confirm the diagnosis of HLH, it is frequently absent especially in the early stages of the disease. Moreover, bone marrow aspiration/biopsy is an invasive procedure which may be difficult to do in a sick child. Two other biomarkers, measurement of NK cell function and soluble interleukin 2 receptor α chain (sIL-2Ra, CD25) are available only in specialised immunology or research laboratories.

HLH can be either primary due to an autosomal recessive monogenic disorder or secondary to infections, malignancy or autoimmune disorders. Genetic forms of HLH can be further classified into familial HLH and lymphoproliferative syndromes. Familial HLH can be subdivided into conditions without skin hypopigmentation and with skin hypopigmentation (Table 2) (Al-Herz et al., 2014). Genetic testing by DNA analysis of the genes involved will help to confirm the diagnosis. However, most of the patients have severe systemic symptoms at diagnosis, and timely appropriate treatment for HLH is needed before genetic testing to distinguish primary from secondary HLH.


Table 2: Classification of genetic forms of HLH (Al-Herz et al., 2014).




Disease name

Gene

Protein

Function





Familial HLH without skin hypopigmentation




FHL1

Unknown

-

-





FHL2

PRF1 (first gene reported, in 1999)

Perforin

Pore formation





FHL3

UNC13D (second gene reported, in 2003)

Munc 13-4

Vesicle priming





FHL4

STX11

Syntaxin 11

Vesicle fusion





FHL5

STXBP2 (reported in 2009)

Munc 18-2

Vesicle fusion





Familial HLH with skin hypopigmentation




Griscelli syndrome type 2

RAB27A

Rab27a

Vesicle docking





Chediak-Higashi syndrome

LYST

Lyst

Vesicle trafficking





Hermansky-Pudlak syndrome type 2

AP3B1

AP3B1

Vesicle trafficking





Lymphoproliferative disorders




XLP1

SH2D1A

SAP

Signalling in T, NK and NK-T cells





XLP2

XIAP

XIAP

Signalling pathways via NF-κB





ITK deficiency

ITK

ITK

Signalling in T cells





CD27 deficiency

CD27

CD27

Lymphocyte co-stimulatory molecule





XMEN syndrome

MAGT1

Magnesium transporter 1

T cell activation via T cell receptor





FHL - familial hemophagocytic lymphohistiocytosis; HLH - hemophagocytic lymphohistiocytosis; ITK - interleukin-2-inducible T cell kinase; NF-κB - nuclear factor kappa-light-chain-enhancer of activated B cells; NK - natural killer; SAP- signalling lymphocyte activation molecule (SLAM)-associated protein; XIAP - X-linked inhibitor of apoptosis protein; XLP- X-linked lymphoproliferative syndrome; XMEN- X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia.

HLH is characterized by multisystem inflammation due to prolonged and excessive activation of antigen-presenting cells (macrophages and histiocytes) and CD8+ T cells, and excessive proliferation and ectopic migration of T cells. NK cells modulate the initial responses of antigen-presenting cells to incoming pathogens like viruses (likely through cytokine signalling) and thus attenuate the subsequent activation of antigen-specific T cells. The perforin/granzymes, Fas/Fas ligand, membrane-bound TNF-α, membrane-bound lymphotoxin, and TNF-related apoptosis-inducing ligand (TRAIL), are the various mechanisms implicated in NK/cytotoxic T lymphocyte (CTL)-mediated cytotoxicity (Madkaikar et al., 2016; Filipovich & Chandrakasan, 2015). Among these, the perforin/granzyme and Fas/Fas ligand interactions are the two most important mechanisms. Various proteins involved in this pathway are lysosomal trafficking regulator (LYST) protein, adaptor related protein complex 3 subunit beta 1 (AP3B1), syntaxin 11, Rab27a, Munc13-4, and Munc18-2 (Figure 2). All genetic forms of HLH are due to variations in one of the genes coding for these proteins (Table 2).

PIC

Figure 2: Granule release mechanism in natural killer (NK) cells and cytotoxic T cells showing various molecules and their functions.

The initial treatment options of HLH consist of combinations of proapoptotic chemotherapy and immunosuppressive drugs targeting the hyperactivated T cells [such as steroids, cyclosporine A, antithymocyte globulins, 2-chlorodeoxyadenosine, and Alemtuzumab (Campath-1H)] and macrophages/histiocytes [etoposide, steroids, and high-dose intravenous immunoglobulin (IVIG)] (Madkaikar et al., 2016; Ishii, 2016). Recently, Alemtuzumab (Campath-1H), a monoclonal antibody to CD52, was found to have a significant response against refractory HLH, but CMV reactivation and adenoviremia were frequent complications of this therapy. Currently, definitive treatment and potential cure of FHL is only achieved by hematopoietic cell transplantation (HCT). Supportive care including prophylaxis for pneumocystis jirovecii, and other fungal or opportunistic infections by empiric broad-spectrum antibiotics or antifungal therapy also plays a major role in successful treatment. Granulocyte-colony stimulating factor (G-CSF) can also be used to increase neutrophil counts in myelosuppression.

The proband described above had all the clinical features to suspect HLH and molecular testing confirmed the diagnosis. Since there is 25% recurrence risk in each future pregnancy, prenatal diagnosis can be offered in the next pregnancy. A high index of suspicion and proper diagnostic workup including molecular testing will help in confirmation of the diagnosis and appropriate genetic counseling.

References

1.    Al-Herz W, et al. Primary immunodeficiency diseases: an update on the classification from the international union of immunological societies expert committee for primary immunodeficiency. Front Immunol 2014; 5: 162.

2.    Filipovich AH, Chandrakasan S. Pathogenesis of Hemophagocytic Lymphohistiocytosis. Hematol Oncol Clin North Am 2015; 29: 895–902.

3.    Henter JI, et al. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007; 48: 124–131.

4.    Ishii E. Hemophagocytic Lymphohistiocytosis in Children: Pathogenesis and Treatment. Front Pediatr 2016; 4: 47.

5.    Madkaikar M, et al. Current Updates on Classification, Diagnosis and Treatment of Hemophagocytic Lymphohistiocytosis (HLH). Indian J Pediatr 2016; 83: 434–443.

6.    Pagel J, et al. Distinct mutations in STXBP2 are associated with variable clinical presentations in patients with familial hemophagocytic lymphohistiocytosis type 5 (FHL5). Blood 2012; 119: 6016–6024.

7.    Richards S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17: 405–424.

8.    Sen ES, et al. Diagnosing haemophagocytic syndrome. Arch Dis Child 2017; 102: 279–284.

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