This document reports on a rare case of a female patient presenting with hemophagocytic lymphohistiocytosis (HLH) caused by X-linked inhibitor of apoptosis (XIAP) deficiency due to a heterozygous mutation and extremely skewed X chromosome inactivation. Genetic testing revealed a known nonsense mutation in XIAP and flow cytometric analysis showed absence of XIAP protein expression in the patient's T cells. An X chromosome inactivation assay demonstrated an extreme skewing ratio of 99:1 toward expression of the mutated XIAP allele. This case demonstrates that females can develop X-linked forms of HLH, such as XIAP deficiency, if skewed X chromosome inactivation favors expression of the disease-causing allele

1. Pediatr Blood Cancer 2015;62:1288–1290
BRIEF REPORT
Hemophagocytic Lymphohistiocytosis in a Female Patient Due to a Heterozygous
XIAP Mutation and Skewed X Chromosome Inactivation
Jennifer R. Holle, MS,1
Rebecca A. Marsh, MD,2
Anna Maria Holdcroft, MS,2
Stella M. Davies, MBBS, PhD,2
Lijun Wang, MD, PhD,1
Kejian Zhang, MD, MBA,1
and Michael B. Jordan, MD
2,3
*
INTRODUCTION
Hemophagocytic lymphohistiocytosis (HLH) is a disorder of
widespread hyperactivation and proliferation of T-lymphocytes and
macrophages. The classic presentation of HLH includes prolonged
fever, splenomegaly, cytopenias, hypertriglyceridemia, hyperferri-
tinemia, hypofibrinogenemia, elevated soluble interleukin-2 recep-
tor levels, hemophagocytosis in the bone marrow, spleen or other
tissues, and decreased or absent natural killer (NK) cell function [1].
HLH is associated with a number of known autosomal and X-
linked genes. The autosomal forms of the disease are caused by
biallelic mutations in genes affecting perforin-dependent cytotoxic
function [2]. Hemizygous genetic lesions in SH2D1A or XIAP cause
the X-linked lymphoproliferative diseases XLP1 and XLP2, which
may be complicated by HLH. X-linked inhibitor of apoptosis
(XIAP) deficiency (XLP2), in particular, is strongly associated with
HLH, with up to 90% of patients eventually developing
symptoms [3,4]. Other clinical features of XIAP deficiency include
hypogammaglobulinemia, splenomegaly, and colitis [3]. XIAP
mutations in males may occur de novo but are more often inherited
from carrier mothers.
Female carriers of X-linked recessive disorders are typically
healthy. However, disease-manifesting carriers have been reported
in several X-linked conditions, including chronic granulomatous
disease and Duchenne muscular dystrophy, due to abnormal
patterns of X chromosome inactivation [5,6].
X chromosome inactivation is the process of transcriptional
silencing of one of the two X chromosomes in mammalian female
cells in order to correct the dosage imbalance of X-linked genes
between males and females [7]. This process is random and takes
place early in embryonic development [8]. Expression from the
maternally and paternally inherited X chromosomes is expected to
follow a ratio of approximately 50:50 in normal females [9].
Abnormal patterns of X chromosome silencing can be caused by
selection against cells with X chromosome abnormalities or due to
defects in the X inactivation mechanism [9,10]. Skewed patterns of
X inactivation can cause a female carrier of an X-linked recessive
disease to express relatively more protein derived from the mutated
allele and experience partial or complete symptoms of the
disorder [11].
Here we describe the case of a female patient presenting with
hemophagocytic lymphohistiocytosis who was found to have XIAP
deficiency due to a heterozygous nonsense mutation in XIAP and
extremely skewed X-chromosome inactivation.
RESULTS
The patient presented at 18 months of life with symptoms of
HLH, including persistent fever, splenomegaly, pancytopenia,
elevated ferritin and sCD25, hypofibinogenemia, and hemophago-
cytosis in the bone marrow. She was treated with etoposide and
dexamethasone per the HLH-94 protocol and responded rapidly
[12]. She was found to have CMV infection at the time of HLH
diagnosis and was also treated with ganciclovir and IVIG,
eventually clearing her viremia. She subsequently underwent
matched unrelated hematopoietic cell transplant at 24 months
of age. Her transplant course was complicated by unusual gut
toxicity, prolonged feeding intolerance, subsequent graft versus
host disease affecting the gut, and development of mixed
chimerism.
The initial genetic work up included Sanger sequencing of the
genes commonly associated with HLH in females: PRF1,
UNC13D, STXBP2, STX11, and RAB27A. No mutations were
Genetic forms of hemophagocytic lymphohistiocytosis (HLH) are
caused by mutations in autosomal recessive genes affecting perforin-
dependent cytotoxic function and two X-linked genes affecting
distinct cell signaling pathways: SH2D1A and XIAP. HLH caused by
mutations in X-linked genes is typically found only in males. Here we
report the occurrence of HLH in a female caused by a heterozygous
mutation in XIAP. Flow cytometric studies confirmed the absence of
XIAP protein expression, while an X chromosome inactivation assay
revealed an extreme skewing ratio of 99:1. This finding demonstrates
that females are susceptible to X-linked forms of HLH through skewed
X chromosome inactivation. Pediatr Blood Cancer 2015;62:1288–
1290. # 2015 Wiley Periodicals, Inc.
Key words: hemophagocytic lymphohistiocytosis; X-linked inhibitor of apoptosis; X-linked lymphoproliferative disease; skewed X
chromosome inactivation
Abbreviations: CMV, cytomegalovirus; HLH, hemophagocytic lym-
phohistiocytosis; HUMARA, human androgen receptor; IVIG, Intra-
venous immunoglobulin; NK, natural killer; NOD, nucleotide-binding
oligomerization domain; PCR, polymerase chain reaction; WES, whole
exome sequencing; XIAP, X-linked inhibitor of apoptosis; XLP1, X-
linked lymphoproliferative disease type 1; XLP2, X-linked lympho-
proliferative disease type 2; XIST, X inactivation specific transcript
1
Division of Human Genetics, Cincinnati Children’s Hospital Medical
Center, Cincinnati, Ohio; 2
Division of Bone Marrow Transplantation
and Immune Deficiency, Cincinnati Children’s Hospital Medical
Center, Cincinnati, Ohio; 3
Division of Immunobiology, Cincinnati
Children’s Hospital Medical Center, Cincinnati, Ohio
Conflict of interest: Nothing to declare.
Ã
Correspondence to: Michael B. Jordan, 3333 Burnet Ave, ML7038,
Cincinnati, OH. Email: michael.jordan@cchmc.org
Received 14 December 2014; Accepted 12 January 2015
C 2015 Wiley Periodicals, Inc.
DOI 10.1002/pbc.25483
Published online 19 March 2015 in Wiley Online Library
(wileyonlinelibrary.com).

2. identified and, following informed consent, whole exome
sequencing (WES) was performed on the patient and her parents.
WES revealed a heterozygous nonsense mutation in XIAP,
c.664CT (p.Arg222Ã
). This mutation was previously identified
in one male patient with chronic splenomegaly and acute HLH and
was predicted to be pathogenic [13]. Although maternally inherited
in the previous case, the mutation was apparently de novo in our
patient. However, germline mosaicism for either parent could not
be ruled out.
XIAP expression was found to be absent in 90% of peripheral
blood T cells obtained after transplant (when donor chimerism was
12%) and in essentially all patient T cells grown from cryopreserved
cells obtained prior to transplant (Fig. 1). Heterozygous XIAP
mutations do not typically eliminate protein expression, raising
suspicion that a second factor not identifiable by WES rendered the
opposite allele nonfunctional. Large deletion mutations have been
reported in males with XIAP, but comparative genomic hybridiza-
tion of the XIAP locus revealed no deletions or duplications in our
patient.
We next evaluated our patient’s X-inactivation pattern
using the human androgen receptor (HUMARA) assay [14],
which utilizes highly polymorphic CAG trinucleotide repeats
located in the X-linked HUMARA locus. There are several
cleavage sites for the methylation-sensitive restriction enzyme
HpaII located near the CAG repeats. These sites are methylated
on the inactivated X-chromosome while the active X-chromo-
some remains unmethylated. The X-chromosome inactivation
(XCI) ratio was calculated as the percentage of cells expressing
the predominate allele (Ppa) on the active X-chromosome [15].
Our patient was found to have a highly skewed X-chromosome
inactivation pattern with approximately 99% of cells expressing
the paternal allele, presumably the one with the XIAP mutation
(Fig. 2).
DISCUSSION
Female carriers of XIAP deficiency have been traditionally
thought to be asymptomatic and to show no clinical manifestations
of the disorder [16]. Flow cytometric testing of peripheral blood
cells shows that carrier females typically have bimodal distribu-
tions of XIAP expression and often have significant skewing
toward cells expressing the normal protein [17]. However, Aguilar
et al. recently described two manifesting carrier females with
inflammatory bowel disease (but not HLH) due to skewed X
chromosome inactivation favoring the mutated allele [18].
Interestingly, only 50–60% of peripheral blood cells of these
Fig. 1. Absence of XIAP in patient T cells. Patient and control T cells
were stimulated by phytohemagglutanin (PHA), expanded in IL-2, then
stained for CD3, CD8 and XIAP protein and analyzed by flow
cytometry as previously described [15].
Fig. 2. Severely skewed X inactivation in proband. The HUMARA assay was performed on genomic DNA extracted from the patient’s pre-
transplant peripheral blood mononuclear cells. The DNA was digested with and without a methylation-sensitive restriction enzyme (HpaII),
amplified by PCR using FAM-labeled primers specific to the HUMARA locus, and analyzed by capillary electrophoresis. The resulting tracing
shows the X inactivation patterns of the patient, her mother, and her father, and demonstrates the patient’s preferential expression of the paternal
allele. (Note: peak height correlates with X inactivation.)
Pediatr Blood Cancer DOI 10.1002/pbc
Title Female XIAP Deficiency 1289

3. females expressed the mutated allele. However, the monocytes of
one evaluated carrier were significantly skewed and lacked XIAP,
suggesting that skewing of monocytes in particular predisposes to
the risk of inflammatory bowel disease. This may relate to the
requirement of XIAP for normal NOD signaling in monocytes or
related cells. These observations suggest that female carriers of
XIAP mutations may be at risk of extra-hematopoietic XIAP
deficiency disease manifestations, particularly if severely skewed
X chromosome inactivation occurs. The occurrence of HLH in our
case is unique and likely reflects the essential absence of XIAP
protein expression in our patient.
It is not clear why the X chromosome encoding the functional
XIAP allele was preferentially inactivated in our patient, especially
because the functional allele appears to be favored in asymptom-
atic carrier females. An X-autosome translocation or gross deletion
affecting the opposite X chromosome could potentially provide the
selective pressure necessary to cause such extreme skewing
towards a mutated (but relatively more intact) allele. We
investigated this possibility using constitutional karyotype analysis
and SNP microarray, however, neither test revealed such an
abnormality.
X chromosome inactivation is controlled by several factors,
including the non-protein coding X inactivation specific transcript
(XIST). XIST is expressed exclusively from the inactive X
chromosome, and functions in the initiation and spread of
chromosome silencing [10]. Mutations in the promoter of XIST
have been associated with familial skewed X-inactivation [10].
Such mutations may be a potential source of abnormal skewing in
our patient, for whom XIST sequencing is currently pending.
X-linked forms of HLH have been thought to affect males
exclusively. This case demonstrates the importance of considering
X-linked etiologies in female patients during the genetic work up.
Furthermore, this report highlights the value of genomic tools such
as whole exome sequencing in defining the etiology of primary
immune deficiencies.
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