Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH.

Abstract

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.

Full text

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doi:10.1182/blood-2011-07-370148
Prepublished online August 31, 2011;




Joyce Villanueva, Kimberly A. Risma, Qian Wei, Peter S. Klein and Alexandra H. Filipovich
Kejian Zhang, Michael B. Jordan, Rebecca A. Marsh, Judith A. Johnson, Diane Kissell, Jarek Meller,
with adult-onset familial hemophagocytic lymphohistiocytosis
Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated
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Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated
with adult-onset familial hemophagocytic lymphohistiocytosis (HLH)


Kejian Zhang1, Michael B. Jordan2, 3, Rebecca A. Marsh 3, Judith A. Johnson1, Diane
Kissell1, Jarek Meller4,7, Joyce Villanueva5, Kimberly A. Risma6, Qian Wei8, Peter S.
Klein9, and Alexandra H. Filipovich3,5

1Division of Human Genetics, 2Division of Immunobiology, 3Division of Bone Marrow
Transplant and Immune Deficiency, 4Department of Environmental Health and
Biomedical Engineering, 5Diagnostic Immunology Laboratory, 6Division of Allergy and
Immunology, 7Division of Pediatric Informatics, Children’s Hospital Medical Center,
University of Cincinnati, College of Medicine, 8Duke University School of Medicine,
9Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania
School of Medicine



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: Mutations in adult-onset familial HLH

Blood First Edition Paper, prepublished online August 31, 2011; DOI 10.1182/blood-2011-07-370148
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Abstract
Familial hemophagocytic lymphohistiocytosis is a rare primary immunodeficiency
disorder, characterized by defects in cell-mediated cytotoxicity that results in fever,
hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but
rarely diagnosed in adults. We conducted a retrospective review of genetic and
immunological test results in patients who developed HLH in their adulthood. 1531
patients with a clinical diagnosis of HLH were included in this study; 175 patients were
18 years or older. Missense and splice site sequence variants in PRF1, MUNC13-4, and
STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was
found in 12 of these patients (48%). The preponderance of hypomorphic mutations in
FHL-causing genes correlates with the later-onset clinical symptoms and the more
indolent course in adult patients. Late-onset FHL occurs more commonly than
previously suspected.



Keywords: Familial hemophagocytic lymphohistiocytosis, HLH, adult FHL, PRF1,
MUNC13-4, STXBP2, mutation, variant, A91V, NK-cell, cytotoxicity





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Introduction
Familial hemophagocytic lymphohistiocytosis1 (familial HLH or FHL; MIM 267700) is an
inherited immune deficiency which is characterized by the overactivation and excessive
proliferation of macrophages and T-lymphocytes. This leads to infiltration and damage
of organs, including the liver and the central nervous system. Classic clinical features
include prolonged fevers, cytopenias, hepatosplenomegaly, and signs of immune
activation. Hemophagocytosis is the hallmark of HLH in general, but it may not present
at early stages of the disease. The functions of natural killer (NK) cells and cytotoxic
T-lymphocytes are reduced or absent. FHL in children is rapidly fatal without
appropriate immunosuppressive treatment followed by hematopoietic cell
transplantation.
To date, defects in at least seven genes (PRF1, MUNC13-4, STX11, RAB27A,
STXBP2, SH2D1A, and BIRC4) are known to be associated with FHL.1-7 The protein
products of the five autosomal recessive (AR) genes PRF1, MUNC13-4, STX11,
RAB27A, and STXBP2 are all involved in the perforin (PRF1)-dependent cytotoxic
pathway; thus affected patients often demonstrate abnormal function of cytotoxic
lymphocytes. Perforin is constitutively contained within the secretory granules of all
cytotoxic lymphocytes. Perforin facilitates the entry of granule contents into target cells
that are infected, spent, or dangerous to the immunologic well-being of the organism,
which results in the cytotoxic response. Mutations in the other AR genes interfere with
the delivery of the cytotoxic granules to the contact surface with target cells and their
extrusion of contents into the contact zone. The majority of reports in the literature
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related to the AR forms of FHL focus on patients who present symptoms of disease
within the first years of life.8-11 Only a few cases with later onset have been reported.12-15
In this report, we describe the genetic and immunological findings of adult patients in
North America who have been referred to the Diagnostic Center for Heritable
Immunodeficiency at Cincinnati Children’s Hospital Medical Center (CCHMC) for
testing.
Methods
This study was approved by the Cincinnati Children’s Hospital Institutional Review
Board. In a five-year study period, a total of 1531 patients were referred for genetic
testing because of a suspected diagnosis of HLH and/or associated conditions, as
determined by their referring physicians. Clinical and demographic features were
collected based on the information provided by ordering physicians on the test
requisition form.
Genomic DNA was obtained from blood, bone marrow, B-lymphoblastoid cell lines
(LCLs), and autopsy tissues, using a standard procedure. All coding exons and at least
50 base pairs of the adjacent intronic region of the PRF1, MUNC13-4, and STXBP2
genes were amplified by PCR, followed by bi-directional sequencing. Detailed
methodologies are either published2-5 or available upon request. Mutation nomenclature
is based on the recommendations of the American College of Medical Genetics and the
Human Genome Variation Society.
Several in silico analysis methods for the prediction of functional consequences of
sequence variants were utilized to provide initial classification. In particular, we used
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SIFT (http://blocks.fhcrc.org/sift/SIFT.html), which utilizes evolutionary information from
homologous proteins,16 and PolyPhen (http://www.bork.embl-heidelberg.de/PolyPhen/),
which incorporates structural information into classification rules.17 The Grantham
Scale18 was also used to evaluate the significance of amino acid substitutions.
Minor allele frequency (MAF) was found in the NCBI SNP database, as well as
identified through testing 50 unrelated individuals from a southern Ohio control
population (race: 83.4% White, 11.8% Black or African American, 1.6% Asian, 1.6%
Hispanic, and 0.2% Native American). NK cell cytotoxicity and perforin expression were
analyzed as previously described in the Diagnostic Immunology Laboratory at Cincinnati
Children’s Hospital.19-21 The results were compared to the normal ranges for age-
matched controls that have been obtained in our laboratory.
Results
A total of 1531 patients were referred for genetic testing because of a suspected
diagnosis of HLH and/or associated conditions, as determined by their referring
physicians. Of these patients, 175 were 18 years of age or older. Twenty-five of the
adult patients (14%) possessed mutations or sequence variants in PRF1, MUNC13-4,
or STXBP2 (Table 1). All of them are either missense base substitutions or splicing site
variations. In comparison, in patients with age of onset younger than 18 years, we found
mutation(s) in PRF1, MUNC13-4, and STXBP2 in approximately 30% of the patients,
which included nonsense, missense, splice site, and other type of deleterious
mutations. Table 2 shows the number of sequence variants found in different age
groups in patients with clinical suspected HLH. It should be noted that in some of these
younger patients, mutations and sequence variants were found in more than one gene.

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Twelve missense mutations and sequence variants were identified in PRF1 in 18
patients. The A91V-PRF1 genotype was found in 12 patients in both heterozygous and
homozygous states. Eight sequence variants in MUNC13-4 were found in seven
patients; two were splice site changes and five were missense mutations. Two splice
site sequence variants in STXBP2 were identified in two patients. Two patients (P18
and P21) were double-heterozygous, with the A91V-PRF1 genotype in the PRF1 locus
and a second mutation in either STXBP2 or MUNC13-4. It is also worth noting that more
than half of the patients (13 of 25) carried only one mutation in one of these genes,
which are inherited in an autosomal recessive fashion. It is possible that other types of
mutations (gross deletions, insertions, complex rearrangements, etc.) were not able to
be detected by the methodology used in this study or that these patients possess
mutations in additional genes that have not yet been discovered to be associated with
HLH.
Eleven novel sequence variants were identified in this study. In silico analyses of the
predicted structural effects of the missense variants are summarized in Table 3.13,22-24
A91V, P188L, R356W, and T450M in PRF1 and Y61C in MUNC13-4 are consistently
predicted to be pathogenic, with no presence in general healthy populations except
A91V (4-7% by multiple studies).20,24 The sequence variant A91V-PRF1 and its
relationship with FHL have been studied extensively in recent years.25,26 Numerous
clinical studies13-15,24 document variability in cytotoxic function in individuals who are
heterozygous or homozygous for the A91V substitution. Most of the evidence suggests
that A91V, while a “milder” or hypomorphic mutation, is not clinically neutral. 27,28 The
predictions for the rest of the sequence variants are largely inconclusive.
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Perforin expression was measured by flow cytometry in seven of the patients with PRF1
sequence variants. The level of perforin correlated well with the genetic findings in adult
patients. Perforin was absent or severely low in patients with bi-allelic mutations in
PRF1, while slightly decreased or at the lower normal level in patients with one mutation
(Table 1). Results of NK cell functional testing were available for 14 patients. NK cell
function was typically observed to be low in these adult patients with HLH, but it does
not correlate closely with the number of mutations identified in PRF1, STXBP2, or
MUNC13-4.
Discussion
In young children, the presenting symptomatology of FHL has been well described9,29
and forms the basis for the diagnostic criteria. In older children and adults, a broader
spectrum of clinical phenotypes (e.g., encephalitis, autoimmune lymphoproliferative
disease, acute lymphoblastic leukemia, aplastic anemia, systemic onset juvenile
idiopathic arthritis, etc.)11,14,15,30-35 have been described. In some of these reported
cases, patients were cleared from infections before the overt diseases. Therefore, a
negative clinical history of HLH does not rule out this syndrome. In patients with FHL
who carry hypomorphic genetic defects, residual NK and T-cell function, although
decreased, may be sufficient to prevent the development of clinical HLH for many years.
In this patient cohort, 12% of patients were 18 years and older at the time of diagnosis.
Two patients apparently developed the first symptoms of HLH in their mid-seventies.
The observations of double-heterozygosity in two adult patients (P18 and P21), with the
A91V-PRF1 genotype in the PRF1 locus and a second mutation in either STXBP2 or
MUNC13-4, are very interesting. Although there is no known direct interaction among
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these three proteins, additive effects are possible, given that they all play critical roles in
the perforin-dependent cytotoxic pathway. Additional research is required to further
define the functional effects of these genetic alterations. Additional work is also needed
to determine the optimal treatment for adult patients with HLH. Anecdotal information
from physicians who submitted samples for testing indicated that a range of treatments
have been employed, including steroids therapy alone, HLH-94 type therapy, and, in a
young adult with recurrent disease, allogeneic hematopoietic cell transplantation.
With increasing awareness of HLH, more adult patients and/or patients with milder
and/or relapsing clinical courses are being identified. In this study, we found that almost
all the sequence variations in PRF1, MUNC13-4, and STXBP2 carried by adult patients
were either missense or splice site changes. They represent hypomorphic mutations
that play a contributing role in the development of late-onset HLH when patients are
challenged by viral infection and other types of environmental stress. Genetic and
immunological diagnostic testing and timely HLH-directed treatment should be
considered for adult patients.

Acknowledgments
We thank the staff of the CCHMC Diagnostic Immunology and Molecular Genetics
laboratories, a FOCIS Center of Excellence. This work was supported by grants from
the Histiocytosis Association of America, the Jeffrey Modell foundation, NIH HL091769,
and the Doris Duke Charitable Foundation.


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Authorship Contribution:
K.Z. and A.H.F. designed the research, collected the data, analyzed and interpreted
data, and wrote the manuscript; K.A.R. analyzed results and wrote the manuscript;
R.M., P.S.K., and M.B.J. contributed to the patient cases and reviewed the paper; D.K.,
J.A.J., and J.V. performed the research and collected the data; J.M. analyzed the data
and wrote the manuscript; Q.W. collected the data and created the tables for
publication.

Conflict-of-interest disclosure:
The authors declare no competing financial interests.

Correspondence: Kejian Zhang, Division of Human Genetics, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH 45229; email: kejian.zhang@cchmc.org
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Patient ID Age at onset Gender Ethnicity
P1 18 yrs.
P2 18 yrs.
P318 yrs.
P418 yrs.
P518 yrs.
P6 18 yrs.
P718 yrs.
P819 yrs.
P919 yrs.
P1019 yrs.
P1119 yrs.
P1219 yrs.
P1319 yrs.
P1421 yrs.
P1523 yrs.
P1624 yrs.
P1724 yrs.
P1825 yrs.
P19 25 yrs.
P2028 yrs.
P2128 yrs.
P2230 yrs.
P2366 yrs.
P2474 yrs.
P25 75 yrs.
NL: Normal, wild type
ND: Not done
LU: Lytic units
Symptoms/Clinical diagnosis&
HLH
HLH
ALL without remission; suspected HLH
HLH
Mycosis fungoides, lymphadenopathy; suspected HLH
HLH
Neutropenia; suspected HLH
MAS in infancy, severe vasculitis with CNS involvement
Suspected HLH
Suspected HLH
HLH
EBV encephalitis and seizures; suspected HLH
HLH
Suspected HLH
HLH
Suspected HLH
HLH
Suspected HLH
HLH
Suspected HLH
HLH; male sibling died with HLH
Suspected HLH
HLH
HLH
HLH
C: Caucasian (non-Hispanic White)
A: Asian
AA: African American
PRF1 Results
10 C>T (R4C)/98 G>A (R33H)*
1066 C>T (R356W)/1349C>T (T450M)
272 C>T (A91V)/NL
272 C>T (A91V)/NL
272 C>T (A91V)/NL
NL/NL
272 C>T (A91V)/1042 G>A (R348H)*
272 C>T (A91V)/NL
1229 G>A (R410Q)*/NL
272 C>T (A91V)/563 C>T (P188L)*
NL/NL
ND
NL/NL
1310 C>T (A437V)*/NL
1066 C>T (R356W)/1066 C>T (R356W)
NL/NL
NL/NL
272 C>T (A91V)/NL
445 G>A (G149S)/695 G>A (R232H) + 272 C>T (A91V)
10 C>T (R4C)/NL
272 C>T (A91V)/NL
ND
272 C>T (A91V)/NL
272 C>T (A91V)/NL
272 C>T (A91V)/272 C>T (A91V)
* Previously unreported variants, not found in our control population.
MUNC13-4 Results
NL/NL
NL/NL
NL/NL
NL/NL
NL/NL
1579 C>T (R527W)/NL
ND
NL/NL
NL/NL
NL/NL
753+3G>A/NL
2240 G>A (S747N)*/2553+5C>G*
753+3G>A/NL
NL/NL
NL/NL
2174 A>G (E725G)*/NL
NL/NL
NL/NL
NL/NL
NL/NL
182 A>G (Y61C)*/NL
1369 C>A (L457M)*/2447+61_2447+150del 90*
NL/NL
ND
NL/NL
STXBP2 Results
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1782(*12) g>a/1782(*12) g>a
795-4C>T/ NL
ND
ND
NL
ND
ND
ND
NL
NK function Perforin % in NK cells
14.8
ND
Absent
1.6 (low)
ND
ND
ND
3.3
ND
2.2 (low)
Absent
ND
ND
Absent
ND
Absent
0.3 (low)
3.7
2.2 (low)
ND
ND
38.4
ND
3.8
# (low)
M
M
F
F
M
F
M
M
F
F
F
M
F
F
F
M
M
F
F
F
M
M
F
M
M
A
C
C
C
C
AA
C
C
H
C
C
A
C
C
C
C
ND
ND
90
ND
ND
ND
ND
81
75 (low)
39 (low)
98
ND
ND
61 (low)
7 (low)
ND
69 (low)
ND
Absent
ND
ND
97
ND
61 (low)
24 (low)
C (Arabic)
C
H
A
C
A
C
C
C
M: Male
F: Female
H: Hispanic
# Test was done in another institution, number is not available.
& Information collected was based on physician's notes on the requisition form.
Table 1. Adult patients with sequence variants in PRF1, MUNC13-4, and STXBP2

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Patients
Age group
0-1yrs
1-5yrs
5-12yrs
12-18yrs
18yrs and +
Total
* In some patients, mutations and sequence variants were found in more than one gene.
Count
415
483
241
217
175
1531
% of Total
27%
32%
16%
14%
11%
100%
2 Variants 1 Variant 2 Variants
7032
1631
016
321
711
96111
1 Variant 2 Variants 1 Variant
471
379
156
102
51
114
19
27
13
2
2
2
46
5
5
5
5
1
182 44%
11123%
4418%
43 20%
27
407
15%
27%
21
PRF1MUNC13-4
STXBP2
Sequence variants found*
Total number of
variants
Variants found
in patients
Table 2. Summary of genetic test results in 1531 patients with suspected HLH



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