Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis

1Kejian Zhang, 2Shanmuganathan Chandrakasan, 3Heather Chapman, 1C. Alexander Valencia, 1Ammar Husami, 1Diane Kissell, 1Judith A. Johnson, and 2Alexandra H. Filipovich

1Division of Human Genetics, 2Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Diseases Institute, 3Division of Developmental Biology,

Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA

Correspondence to: Kejian Zhang M.D.

Associate Professor of Clinical Pediatrics Division of Human Genetics, MLC 4006 Cincinnati Children’s Hospital Medical Center

3333 Burnet Ave, ML 4006

Cincinnati, OH 45229

Telephone 513-636-0121

Fax 513-636-2261

Email: kejian.zhang@cchmc.org

Running title: Digenic HLH

Blood First Edition Paper, prepublished online June 10, 2014; DOI 10.1182/blood-2014-05-573105

Copyright © 2014 American Society of Hematology

Key Points:

1. Synergistic effects in the granule mediated lymphocyte cytotoxicity

2. Digenic pathogenesis in the development of HLH

Keywords: Familial hemophagocytic lymphohistiocytosis, HLH, Digenic, PRF1, MUNC13-4,

STXBP2, STX11, Rab27a, mutation, variant, CD107a, NK function, cytotoxicity.

ABSTRACT

More than half a dozen molecules (LYST, AP3, Rab27a, STX11, STXBP2, MUNC13-4 and

PRF1) have been associated with the function of cytotoxic lymphocytes. Biallelic defects in all

of these molecules have been associated with Familial hemophagocytic lymphohistiocytosis

(FHL). We retrospectively reviewed the genetic and immunology test results in 2701 patients

with a clinically suspected diagnosis of HLH and found 28 patients with single heterozygous

mutations in two FHL-associated genes. Of these patients, 21 had mutations within PRF1 and a

degranulation gene, while 7 were found to have mutations within two genes involved in the

degranulation pathway. In patients with combination defects involving two genes in the

degranulation pathway, CD107a degranulation was decreased, comparable to patients with

biallelic mutations in one of the genes in degranulation pathway. This suggests a potential

digenic mode of inheritance of FHL due to synergistic function effect within genes involved in

cytotoxic lymphocyte degranulation.

INTRODUCTION

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive immune

disorder with defective lymphocyte granule-mediated cytotoxicity.1,2 Several genes have been

implicated in FHL. FHL types 2-5 and Griscelli syndrome involve PRF1, UNC13D (MUNC13-

4), STX11, STXBP2 and Rab27a respectively.3-6 All of these genes, except for PRF1, play a role

in the degranulation of cytotoxic lymphocytes, which affect the precise steps of docking, priming

and fusion of the cytotoxic lymphocytes to the target cell. 7,8 Perforin is then released from

granules to assist in the penetration and delivery of granzymes into the cytosol of the target cell,

where the latter cleave key substrates to initiate apoptotic cell death.3,7 An uncompromised and

coordinated function of all of these molecules is essential for normal lymphocyte cytotoxicity

activity. Although it is well know that FHL is inherited in an autosomal recessive manner,

symptomatic heterozygous mutation carriers have been presented in several reports.9,10 In these

cases, additional synergistic defects in the molecules of the cytotoxic pathway may contribute to

the final development of HLH, which will suggest a digenic inheritance of FHL.

In digenic inheritance, the combination of concurrent partial defects in two genes within

the same pathway gives rise to a clinical phenotype, while a heterozygous state in either of the

genes alone results in a less severe phenotype or none at all. Examples of a digenic mode of

inheritance have been described for an array of disorders, including retinitis pigmentosa,

holoprosencephaly, deafness, epidermolysis bullosa, Hirschsprung disease, insulin resistance,

and polycystic kidney disease. 11,12 To investigate potential digenic inheritance for FHL, we

reviewed patients with clinical FHL and identified those with heterozygous variants in two FHL-

associated genes and examined the age of onset, defective degranulation, perforin levels and NK

function activity compared to patients with one (Het) or two sequence variants in a specific gene.

We present data suggestive of synergistic heterozygosity in FHL between two genes involved in

cytotoxic lymphocyte degranulation.

METHODS

This was a retrospective chart review, approved by the Cincinnati Children's Hospital Medical

Center (CCHMC) institutional review board, which included 2701 patients referred by their

physicians for genetic testing due to clinically suspected FHL. This cohort included affected

individuals with homozygous or compound heterozygous mutations in a single gene; single

heterozygous individuals with a single mutation in one gene; or double heterozygous (digenic)

individuals with two heterozygous mutations in two separate genes. We analyzed patients’

clinical information, clinical immunology testing results, and/or genetic testing results that were

available for these patients.13-15

Interpretation of sequence variants was based on the algorithm described by the Human Genome

Variation Society (http://www.hgvs.org/mutnomen/recs.html) and included the usage of

predictions with Alamut 2.2.2 ( Interactive Biosoftware, Rouen, France) for SIFT,16 PolyPhen-217

and the Grantham Scale.18 Alamut also integrates databases for variant frequencies (dbSNP, 1000

Genomes Project, and Exome Variant Server) as well as splice site prediction algorithms (Human

Splicing Finder) and the Human Gene Mutation Database (HGMD) for reported mutations (data

not shown).

RESULTS AND DISCUSSION

In this study, a total of 2701 patients with clinically suspected FHL underwent genetic and

immunologic testing. Among these patients, 28 patients (P1-P28) were found to be heterozygous

for either a known mutation or a likely pathogenic variant in two distinct cytotoxic pathway

genes (PRF1, MUNC13-4, STXBP2, STX11, and Rab27a). These patients’ genetic variants and

clinical information was used to examine the possibility of digenic inheritance in FHL (Table 1).

Fifteen patients were found to have heterozygous variants in PRF1 and MUNC13-4; six in PRF1

and STXBP2; four in MUNC13-4 and STXBP2; one in MUNC13-4 and STX11; one in STXBP2

and STX11; and one in STXBP2 and Rab27A.

Patients were separated into two groups: heterozygotes with variants in two genes

involved in degranulation (MUNC13-4, STXBP2, STX11, and Rab27A; denoted as Deg/Deg; 7

total) and those heterozygotes with variants in PRF1 and one degranulation gene (denoted as

PRF1/Deg, 21 total). As controls, an “affected” group of patients was included that were

homozygous or compound heterozygous for PRF1 (120-the number in the parentheses denotes

the total number of patients identified), MUNC13-4(72), or STXBP2(33) mutations. Similarly, a

“single heterozygous” patient group was included with a single mutations within PRF1(163),

MUNC13-4(119), or STXBP2(29).

Deg/Deg patients had an earlier age of onset with 71.4% (5/7 or five out of seven

patients) having an onset of <24 months (Figure 1A). In contrast, PRF1/Deg patients were

observed to have a later age of onset, with only 14.3% (3/21) with < 24 months (Figure 1A). The

age of onset of Deg/Deg patients is similar to patients with biallelic mutations in any one of these

genes; with an onset of <24 months being 83.3% (100/120), 83.3% (60/72), 69.7% (23/33) for

PRF1, MUNC13-4 and STXBP2, respectively (Figure 1A). In contrast, the single heterozygous

patients were observed to have a later disease onset (Figure 1A). Thus early onset disease in

patients with two genetic defects within the degranulation pathway supports a potential digenic

mode of inheritance for FHL.

Upon degranulation, NK cells express CD107a on their surface. Thus decreased measure

of CD107a expression is indicative of defective degranulation.8,19 To determine which

individuals displayed defective degranulation, CD107a expression data from immunological

testing was analyzed. CD107a expression, Perforin expression and NK function results were

available for a small portion of these patients. From the limited number of patients, we found

75% (3/4) of patients with Deg/Deg double heterozygotes had defective CD107a degranulation.

This finding is similar to the defective degranulation seen in individuals with biallelic MUNC13-

4 (91.7% or 11/12) and STXBP2 (92.3% or 12/13) mutations. In contrast, PRF1/Deg double

heterozygotes had normal degranulation (0% or 0/8) (Figure 1C). As expected, defective NK

function was observed in all patient groups (data not shown).

Patients with defects in PRF1 had decreased perforin levels. Numerically, 100% (41/41)

of PRF1-affected; 63.6% (42/66) of PRF1 heterozygotes; and 72.7% (8/11) of PRF1/Deg

patients had decreased perforin expression (Figure 1D). Interestingly, only 20% (1/5) of Deg/Deg

double heterozygotes; 16% (4/25) of MUNC13-4-affected; 30.8% (4/13) of STXBP2-affected;

33.3% (14/42) of MUNC13-4 heterozygotes; and 40% (6/15) of STXBP2 heterozygotes having

low perforin in any of these categories (Figure 1D). Patients without PRF1 mutations had

increased or normal levels of perforin (data not shown).

Synergistic function was hypothesized for the genes in the degranulation pathway due to

the known highly precise and coordinated functions that lead to the secretion of granzyme B to

target cells. Findings from this retrospective study suggest that heterozygous defects in two

degranulation pathway genes have a synergistic deleterious effect, and thus imply that a digenic

mode of inheritance can lead to FHL. Patients with mutations in two degranulation genes in this

study had an early childhood presentation of FHL. Moreover, patients also displayed defective

degranulation of lymphocytes as expected, unlike PRF1/Deg patients. This is the first

retrospective study that suggests a potential digenic inheritance in FHL. However, it is worth

noting several limitations of this study. A detailed clinical data description is not available for a

number of patients with suspected FLH. Similarly, limited number of data on NK function,

CD107a degranulation and perforin expression results were available for analyses. In addition,

parental and/or sibling samples were not available in most families for segregation studies.

Ideally, future prospective studies should include a thorough clinical summary, outcome results

and collection of genetic and functional studies performed on the proband and family members.

Acknowledgments

We thank the staff of the Cincinnati Children’s Hospital Medical Center, a FOCIS Center of Excellence, and the Diagnostic Immunology and Molecular Genetics laboratories, which performed the clinical tests for this study. Authorship

Contribution:

K.Z. and A.H.F. designed the research, analyzed and interpreted data, and wrote the manuscript;

S.C., C.A.V. and H.C. analyzed and interpreted data, and wrote the manuscript; D.K., J.A.J., and

A.H. performed the research, collected and analyze the data.

Conflict-of-interest disclosure:

The authors declare no competing financial interests.

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Table 1: Double Heterozygous Patients with FHL

Patient ID Age of diagnosis(Years) PRF1 MUNC13-4 STXBP2 STX11 RAB27A

p1 0.25 c.1310 C>T (p.A437V) c.169 G>T (p.E57X) ND ND ND

p2 0.75 c.272 C>T (p.A91V) c.2709+6 G>T ND ND ND

p3 0.92 c.992 C>T (p.S331L) c.1232 G>A (p.R411Q) NMI NMI NMI

p4 2.25 c.272 C>T (p. A91V) c.227 C>T (p.T76M) ND ND ND

p5 3 c.272 C>T (p. A91V) c.869 C>T (p.S290L) ND ND ND

p6 3 c.272 C>T (p. A91V) c.2243 C>T (p.A748V) ND NMI ND

p7 5 c.1229 G>A (p.R410Q) c.1036 G>A (p.D346N) ND NMI NMI

p8 8 c.272 C>T (p. A91V) p.3160 A>G (p.I1054V) ND ND ND

p9 9 c.10 C>T (p.R4C) c.3232 G>C (p.A1078P) NMI NMI ND

p10 9 c.272 C>T (p. A91V) c.2896 C>T (p.R966W) NMI ND ND

p11 10 c.272 C>T (p. A91V) c.2896 C>T (p.R966W) NMI NMI NMI

p12 12 c.50 delT c.1579 C>T (p.R527W) NMI NMI NMI

p13 13 c.445 G>A c.2896 C>T (p.R966W) ND ND ND

p14 13 c.272 C>T (p. A91V) c.2896 C>T (p.R966W) NMI NMI NMI

p15 28 c.272 C>T (p. A91V) c.182 A>G (p.Y61C) NMI NMI NMI

p16 5 c.272 C>T (p. A91V) NMI c.1034 C>T (p.T345M) NMI NMI

p17 10 c.272 C>T (p. A91V) ND c.1034 C>T (p.T345M) ND ND

p18 16 655 T>A (Y219N) NMI c.1034 C>T (p.T345M) NMI ND

p19 21 c.272 C>T (p. A91V) NMI c.1586 G>C (p.R529P) NMI NMI

p20 24 c.50 del T NMI c.1459 G>A (p.V487M) NMI NMI

p21 24 c.272 C>T (p. A91V) NMI c.795-4 C>T NMI NMI

p22 0.167 NMI c.2896 C>T (p.R966W) c.911 C>T (p.T304M) NMI NMI

p23 0.417 NMI c.1389+1 G>A c.1782*12 G>A NMI ND

p24 0.667 NMI c.2828 A>G (p.N943S) c.1782*12 G>A NMI ND

p25 1 NMI c.2828 A>G (p.N943S) c.715 C>T (p.P239S) NMI NMI

p26 0.167 NMI c.2030 T>C (p.I677T) NMI c.221 C>T (p.T74M) ND

P27 14 NMI NMI c.568 C>T (p.R190C) c.9 C>A (p.D3E) NMI

P28 5 NMI NMI c.1034 C>T (p.T345M) NMI c.295 T>G (p.F99V)

ND: Not done

NMI: No mutation found