FAS Haploinsufficiency Is a Common Disease
Mechanism in the Human Autoimmune
Lymphoproliferative Syndrome
This information is current as
of June 17, 2017.
Hye Sun Kuehn, Iusta Caminha, Julie E. Niemela, V. Koneti
Rao, Joie Davis, Thomas A. Fleisher and João B. Oliveira
J Immunol 2011; 186:6035-6043; Prepublished online 13
April 2011;
doi: 10.4049/jimmunol.1100021
http://www.jimmunol.org/content/186/10/6035
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Supplementary
Material
The Journal of Immunology
FAS Haploinsufficiency Is a Common Disease Mechanism in
the Human Autoimmune Lymphoproliferative Syndrome
Hye Sun Kuehn,* Iusta Caminha,* Julie E. Niemela,* V. Koneti Rao,† Joie Davis,†
Thomas A. Fleisher,* and João B. Oliveira*
T
he autoimmune lymphoproliferative syndrome (ALPS)
is characterized by early-onset development of benign
lymphadenopathy and splenomegaly, multilineage cytopenias due to autoimmune peripheral destruction and splenic sequestration of blood cells, and increased risk for B cell lymphomas (1–3). Patients typically accumulate a hallmark population of
mature TCRab+ T cells that are CD4 and CD8 negative (4, 5).
ALPS is caused by defects in proteins involved in the FAS pathway
of lymphocyte apoptosis. Most (∼65%) patients have mutations in
the FAS (TNFRSF6/APO1/CD95) gene, whereas a minority has
mutations in the gene encoding FAS ligand (FASL) or caspase-10
(6–12). Germline mutations in CASP8 or somatic mutations in
NRAS or KRAS cause ALPS-related syndromes currently classified separately (12–15).
The FAS gene contains nine exons spanning 26 kb on chromosome 10q24.1 (16). The first five exons encode the extracellular
portion of the protein containing three cysteine-rich domains that
are involved in receptor trimerization and FASL binding required
for triggering of the apoptotic signal. Exon 6 codes for the
transmembrane domain, and the intracellular portion is encoded
*Department of Laboratory Medicine, Clinical Center, National Institutes of Health,
Bethesda, MD 20814; and †ALPS Unit, Laboratory of Clinical Infectious Diseases,
National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20814
Received for publication January 5, 2011. Accepted for publication March 17, 2011.
This work was supported by the National Institutes of Health Intramural Program.
H.S.K. performed research, analyzed data, made the figures, and wrote the paper; I.C.
performed analysis of the biomarkers; J.E.N. performed genomic DNA sequencing
analysis; V.K.R. and J.D. were responsible for patient care; T.A.F. supervised research; and J.B.O. designed and supervised the project and wrote the paper.
Address correspondence and reprint requests to Dr. João B. Oliveira, Department of
Laboratory Medicine, Clinical Center, National Institutes of Health, 10 Center Drive,
Bethesda, MD 20814. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ALPS, autoimmune lymphoproliferative syndrome; DD, death domain; DiOC6, 3,39-dihexyloxacarbocyanine iodide; DISC,
death-inducing signaling complex; EC, extracellular; FADD, Fas-associated death
domain protein; FASL, FAS ligand; NMD, nonsense-mediated mRNA decay.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100021
by exons 7–9. The FAS death domain (DD), an 85-aa-long
structure encoded by exon 9, is required for FAS-induced apoptosis of lymphocytes under physiological conditions (17).
The majority of FAS defects associated with ALPS are heterozygous missense mutations that affect the intracellular DD,
allowing for the expression of a defective protein with a dominantnegative effect on the signaling pathway (7, 18). These mutations
demonstrate high penetrance for clinical symptoms including refractory cytopenias and an increased risk for lymphoma (2, 18).
However, evaluation of a large cohort of ALPS patients at our
center has revealed a significant subpopulation harboring mutations affecting the extracellular region of the protein, and these
are commonly nonsense, or insertions, deletions, and splice site
mutations with frameshift (Fig. 1). These mutations are predicted
to abolish protein expression and hence are not expected to result
in dominant-negative interference, and the mechanism by which
this affects FAS apoptosis signaling is not clearly defined. FAS
haploinsufficiency is one possible disease mechanism in ALPS
associated with extracellular mutations, but currently there are
only very limited data supporting this mechanism (19, 20). In fact,
FAS haploinsufficiency was clearly demonstrated in only one
ALPS patient to date, such that the prevalence, long-term clinical
impact, and biochemical consequences are unknown (19).
To understand the mechanism by which extracellular region
FAS mutations disrupt the apoptotic machinery, we analyzed the
functional impact of 29 representative mutations across all regions
of FAS using patient-derived B cell lines. We demonstrate that
most (8 of the 11) mutations affecting the extracellular regions
of the FAS receptor we studied are associated with haploinsufficiency. These mutations induced less severe disruption of the
FAS-mediated apoptosis-signaling platform when compared with
missense intracellular mutations, and the apoptotic defect could
be rescued in vitro by FAS overexpression. These findings define
haploinsufficiency as an ALPS-causing disease mechanism, in
addition to the well-documented dominant-negative interference
seen with intracellular mutations.
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The autoimmune lymphoproliferative syndrome (ALPS) is characterized by early-onset lymphadenopathy, splenomegaly, immune
cytopenias, and an increased risk for B cell lymphomas. Most ALPS patients harbor mutations in the FAS gene, which
regulates lymphocyte apoptosis. These are commonly missense mutations affecting the intracellular region of the protein and
have a dominant-negative effect on the signaling pathway. However, analysis of a large cohort of ALPS patients revealed that
∼30% have mutations affecting the extracellular region of FAS, and among these, 70% are nonsense, splice site, or insertions/
deletions with frameshift for which no dominant-negative effect would be expected. We evaluated the latter patients to understand
the mechanism(s) by which these mutations disrupted the FAS pathway and resulted in clinical disease. We demonstrated that
most extracellular-region FAS mutations induce low FAS expression due to nonsense-mediated RNA decay or protein instability,
resulting in defective death-inducing signaling complex formation and impaired apoptosis, although to a lesser extent as compared
with intracellular mutations. The apoptosis defect could be corrected by FAS overexpression in vitro. Our findings define
haploinsufficiency as a common disease mechanism in ALPS patients with extracellular FAS mutations. The Journal of Immunology, 2011, 186: 6035–6043.
6036
Materials and Methods
Patient samples, cell culture, and gene sequencing
All patients were studied at the National Institutes of Health under Institutional Review Board-approved protocols (93-I-0063 and 95-I-0066).
Genomic DNA samples were PCR-amplified and sequenced as previously described (21). For mRNA sequencing, cDNA was prepared from
EBV-transformed B cell lines using the RNeasy plus mini kit (Qiagen). To
rule out the presence of contaminating DNA, RNA samples were subjected
to PCR amplification using intron-specific primers; the absence of amplified product (DNA) was confirmed by denaturing agarose gel electrophoresis (data not shown). cDNA was PCR amplified (forward: 59-GTGAGGGAAGCGGTTTACGAGTGA-39; and reverse: 59-AGTGGGGTTAGCCTGTGGATAGAC-39), and the products were subjected to sequencing
(primer sequences available upon request). To determine the ratio of mutant to wild-type FAS transcripts, cDNA was amplified and cloned into the
pcDNA 3.0-HA vector (modified from pcDNA3.0 [Invitrogen]) with EcoRI
and XhoI enzyme sites. After transformation, we picked 10–20 colonies
and performed sequencing. Three samples with DD mutations (D260N,
Q276X, L294Dfs) were used as controls. For functional studies, EBVtransformed B cell lines from the patients were cultured in RPMI 1640
media with 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin,
and 2 mM L-glutamine (Life Technologies, Invitrogen).
EBV-transformed B cells (200,000/well) were aliquoted in triplicate into
96-well plates and cultured with or without APO-1-3 (1 mg/ml) (ENZO Life
Sciences) in the presence of protein A (1 mg/ml). After 24 h, cell loss was
determined by measuring the loss of the mitochondrial transmembrane
potential using 3,39-dihexyloxacarbocyanine iodide (DiOC6) (Calbiochem,
EMC Biosciences) staining. Briefly, cells were incubated with 40 nM
DiOC6 for 15 min at 37˚C, and live cells (DiOC6 high) were counted by
flow cytometry (BD FACSCanto II; BD Biosciences) by constant time
acquisition. The percentage of cell loss was calculated according to the
following formula: (number of live cells without APO-1-3 treatment 2
number of live cells with APO-1-3 treatment/number of live cells without
APO-1-3 treatment) 3 100.
FAS cell surface expression
To determine FAS (CD95) expression, EBV-transformed B cells (0.5 3 106
cells/sample) were washed with PBS and incubated with 10 mg/ml PECD95 (BD Biosciences) or IgG control for 30 min at 4˚C (dark) in 100 ml
5% FCS in PBS. After washing with PBS two times, 10,000 live cells were
analyzed by flow cytometry. Absolute number of FAS molecules on the
cell surface was established by developing a mean equivalent soluble
fluorochrome standard curve using QuantiBRITE PE (BD Biosciences)
FIGURE 1. Schematic representation of FAS
mutations in ALPS patients. Red text indicates
mutations evaluated in this study. Blue text
indicates complex mutations. Black diamonds
represent the number of additional families
with same mutation. TM, transmembrane.
beads run in parallel for each experiment, according to the manufacturer’s
protocol.
Immunoprecipitation
EBV-transformed B cells (3 3 106 cells/sample) were cultured with or
without APO-1-3 (0.5 mg/ml) in the presence of protein A (1 mg/ml) for 20
min at 37˚C. Immunoprecipitation was performed according to the manufacturer’s instructions (Pierce Classic IP Kit; Thermo Scientific). Briefly,
after exposure, cells were washed with cold PBS, lysed, and spun down
(13,000 rpm, 4˚C, 20 min). Supernatants were incubated with anti-FAS Ab
(A-20; Santa Cruz Biotechnology) and protein A/G agarose for 2 h, after
which the immune complexes were washed, and proteins were separated
by electrophoresis on 4–12% NuPAGE Bis–Tris gels (Invitrogen), transferred to nitrocellulose membranes, and probed using the anti–Fasassociated DD protein (FADD) (BD Biosciences) or anti–caspase-8 (Cell
Signaling Technology) Abs. To quantitate changes in protein level, the
ECL films were scanned using a Quantity One scanner (Bio-Rad).
FAS transfection
EBV-transformed B cells (3 3 106 cells /sample) were transfected with
5 mg GFP (pmaxGFP) or 5 mg YFP-FAS (pEYFP-N1-FAS) using the
Amaxa Human B-cell Nucleofector kit (Program U-015; Amaxa). Twentyfour hours after transfection, medium was replaced with fresh complete
medium. After 48 h of transfection, expression of FAS on the cell surface
was determined by flow cytometry as described above, gating on live GFPor YFP-transfected cells. To evaluate apoptosis, the transfected cell lines
were treated with vehicle or APO-1-3 in the presence of protein A (as
above) and live GFP+ or YFP+ cells counted as described above.
Biomarkers
The quantification of IL-10, soluble Fas ligand, vitamin B12, IL-18, TNF-a,
and TCRab+CD42CD82 T cells on patient samples was performed as
previously described (22).
Statistical analysis
Data are represented as the mean 6 SEM, except where noted. The statistical analyses were performed by unpaired Student t test or Mann–
Whitney rank-sum test. Differences were considered significant when p ,
0.05.
Results
FAS cell-surface expression in ALPS-associated FAS mutations
We reviewed genetic data from 108 ALPS probands and identified
84 distinct mutations (27 extracellular, 5 transmembrane, 24 intra-
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Apoptosis measurement
FAS HAPLOINSUFFICIENCY IN ALPS
The Journal of Immunology
6037
Low FAS expression is associated with apoptosis dysfunction
due to impaired death-inducing signaling complex formation
We next evaluated the functional impact of the 29 selected
mutations in the FAS pathway by the treatment of EBVtransformed B cell lines with the agonistic anti-FAS Ab APO-1-3
followed by cross-linking. All cell lines with extracellular, transmembrane, or intracellular mutations showed significant resistance to FAS-mediated cell death when compared with control
cell lines (Fig. 3A–C). The apoptotic defect could also be observed
on primary cultured T cells (Supplemental Table I). Mutations in
the extracellular domains resulted in milder apoptotic defects
when compared with intracellular mutations, with median cell
losses of 62 and 29%, respectively (Fig. 3D). However, this difference did not reach statistical significance (p = 0.17), likely due
to the large data spread in the extracellular group and the limited
sample number in each group.
To further dissect the impact of FAS mutations on the downstream signaling pathways, we evaluated death-inducing signaling
complex (DISC) formation in response to FAS activation. This
complex includes FADD together with caspase-8/10 and is formed
within seconds following FAS stimulation, resulting in the processing and activation of caspase-8/10 with propagation of the
apoptotic signal. Following FAS activation with APO-1-3 and
protein A, immunoprecipitated protein complexes were examined
by Western blotting to measure the levels of coimmunoprecipitated
FADD and caspase-8 bound to the intracellular portion of the FAS
receptor. Compared to mutations affecting the intracellular region
(Fig. 4B, 4C), the extracellular mutations (Fig. 4A) showed consistent but more limited impairment in FADD (p = 0.032) and
caspase-8 (p = 0.02) recruitment to the signaling complex, based
on multiple experiments (Fig. 4D). These data demonstrate that
extracellular mutations with low FAS expression attenuate downstream signaling and impair apoptosis, although to a lesser extent
when compared with the intracellular mutations, and confirm interference of apoptosis by haploinsufficiency at the molecular
level.
Table I. Summary of FAS mutations and their effects
Identification
No.
127
220
74
153
50
111
89
180
77
205
34
175
4
45
72
149
62
98
3
110
29
31
55
197
137
33
121
30
17
cDNA
Protein
Location
Surface Fas
(% Normal)
c.20T . C
c.46_47delGC
c.53T . G
c.133G . T
c.189T . A
c.197(-1)g . a
c.273C . A
c.332A . G
c.397_398delTTinsA
c.404G . A
c.444(-1)g . c
c.528G . A
c.569(-2)a . c
c.585_595del11
c.607A . T
c.651(+2)t . a
c.676(+2)t . c
c.686delT+690_694del5
c.721A . C
c.722C-.A
c.749G . A
c.749G . C
c.748C . T
c.778G . A
c.779A . G
c.826C . T
c.879_880delAT
c.880delT
c.929T . G
p.L7P
p.A16X
p.L18X
p.E45X
p.C63X
p.G66_H111del
p.Y91X
p.H111R
p.F133IfsX54
p.C135Y
p.C149NfsX32
p.W176X
p.V190GfsX2
p.Q196TfsX12
p.R203X
p.V190GfsX11
p.E218MfsX4
p.L229X
p.T241P
p.T241K
p.R250Q
p.R250P
p.R250X
p.D260N
p.D260V
p.Q276X
p.L294DfsX2
p.L294X
p.I310S
Exon 1
Exon 2
Exon 2
Exon 2
Exon 2
Intron 2
Exon 3
Exon 3
Exon 4
Exon 4
Intron 4
Exon 6
Intron 6
Exon 7
Exon 7
Intron 7
Intron 8
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
Exon 9
54
39
33
69
48
98
36
101
45
53
37
24
64
97
112
106
109
84
63
67
69
104
44
145
100
90
115
48
94
Cell Loss
(% Normal)
FADD
Recruitment
(% Normal)
Caspase-8
Recruitment
(% Normal)
RNA Stability
(% Mutant
Clones)a
74
65
1
75
73
70
19
47
42
62
21
77
31
24
26
17
9
10
32
28
6
63
8
29
59
36
44
54
71
117
45
21
99
85
89
36
83
63
49
64
51
80
14
15
14
13
13
28
6
24
70
21
11
8
18
74
38
98
98
46
30
82
71
70
30
66
42
50
42
33
70
24
24
15
17
16
27
14
22
57
15
11
13
15
48
25
92
Stable (44.5)
Stable (15)
Stable (31)
Unstable (0)
Unstable (0)
Stable
Unstable (8)
Stable
Stable (15)
Stable (22)
Unstable (0)
Stable (6)
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Stable (37.5)
Stable (28.6)
Stable
Stable (60)
Stable (60)
Stable (28)
Stable
Stable: both wild-type and mutant forms were detected; Unstable: .90% of the clones were wild-type.
a
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cellular non-DD, and 28 intracellular DD mutations) (Fig. 1). The
majority of the non-DD mutations were either nonsense (11 of 57)
or splice site or insertions/deletions with predicted frameshifts (36
of 57). More importantly, most (19 of 27) of the mutations affecting the extracellular regions of the protein were also nonsense,
insertions/deletions, or splice site with frameshifts. We selected 29
mutations across all regions to determine their functional effect
(Fig. 1, Table I). To assess the impact of the different FAS mutations
on the expression of FAS, we determined the number of FAS
molecules on the cell surface of patient-derived EBV-transformed
B cell lines using a flow cytometry-based assay. The majority of the
mutations affecting the extracellular region (8 out of 11) demonstrated significantly reduced FAS expression (Fig. 2A, Supplemental
Fig. 1). The mutation W176X, which abolishes the entire transmembrane region, also resulted in low FAS expression (Fig. 2B).
The mutation H111R and the inframe deletion G66_H111 affect
the FASL-binding domain and were not expected to result in lower
protein expression. As predicted, most mutations affecting intracellular regions of the FAS protein did not significantly change
cell-surface FAS expression (Fig. 2B, 2C, Supplemental Fig. 1).
Exceptions included the intracellular nonsense mutations R250X
and L294X, which significantly reduced FAS surface expression
(Fig. 2B, 2C, Supplemental Fig. 1). Taken together, these data
suggested haploinsufficiency as a possible disease mechanism in
ALPS caused by FAS extracellular region mutations.
6038
FAS HAPLOINSUFFICIENCY IN ALPS
Different mechanisms mediate low FAS expression in ALPS
patients
To gain insight into the mechanism(s) responsible for low cellsurface FAS protein expression associated with extracellular
FAS mutations, we first evaluated mRNA stability of mutant and
wild-type alleles by performing FAS cDNA cloning followed
by sequencing. Although rare mRNAs harboring premature stop
codons escape the degradation pathway, most transcripts containing a premature stop codon followed by a spliceable intron
undergo nonsense-mediated mRNA decay (NMD) (23). Accordingly, we detected almost exclusively wild-type FAS cDNA in
cells with the E45X, C63X, Y91X, and C149Nfs mutations (Table
I), suggesting these mutated transcripts underwent NMD. However, the nonsense mutations A16X, L18X, W176X, R250X,
L294X, and F133Ifs, and, as expected, the missense L7P and
C135Y, allowed both wild-type and mutant allele expression at
varying ratios, suggesting that alternative mechanisms other than
NMD also lead to low FAS expression seen with specific extracellular FAS mutations.
Ectopic expression of L18X and F133Ifs in 293 T cells resulted
in small amounts of truncated proteins that did reach the cell
surface (Supplemental Fig. 2A, 2B). L16X is believed to behave in
a similar manner. W176X also resulted in a short protein missing
the transmembrane region that did not anchor on the cell surface
(Supplemental Fig. 2A, 2B). Interestingly, ectopic expression of
R250X and L294X resulted in subnormal surface expression of
the mutant FAS (Supplemental Fig. 2A), similar to that observed
in the EBV B cell lines (Fig. 2). These findings suggest that these
two latter mutations may result in a combination of haploinsufficiency and dominant-negative inhibition.
L7P is located in the signal peptide, and it is plausible that
impaired intracellular trafficking to the endoplasmic reticulum and
Golgi apparatus prevents normal surface FAS expression, as it has
been demonstrated for other proteins (24, 25). However, we could
not detect any clear accumulation of FAS in the endoplasmic reticulum or Golgi by confocal microscopy (data not shown). These
experiments were complicated by the fact that the mutation is
heterozygous, such that 50% of the proteins are expected to traffic
normally. Lastly, C135Y affects a cysteine residue within the third
cysteine-rich domain, possibly resulting in an abnormally folded
protein. Ectopic expression of this mutant did result in surface
FAS expression, but at lower levels as compared with controls
(Supplemental Fig. 2A).
Taken together, these data suggest that haploinsufficiency associated with extracellular mutations can diminish FAS expression
by inducing NMD, protein instability, and, potentially, abnormal
intracellular trafficking.
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FIGURE 2. Cell-surface expression of FAS on
EBV-transformed B cells. A–C, Cells (0.5 3 106) were
stained for FAS expression using 10 mg/ml PE-conjugated anti-CD95 or PE-conjugated isotype-matched
IgG for 30 min at 4˚C. After washing two times with
PBS, 10,000 live cells were analyzed by flow cytometry. FAS expression was quantified using QuantiBRITE PE (BD Biosciences). Data were represented
as means 6 SE of three to four separate experiments.
*p , 0.05, **p , 0.01 by Student t test for comparison with normal cell lines.
The Journal of Immunology
6039
We evaluated recently described biomarkers associated with FAS
mutations, including percentage of TCRab+CD42CD82 T cells
as well as levels of soluble FASL, IL-10, IL-18, TNF-a, and vitamin B12 (27, 28). No statistically significant differences were
noticed between patients with null extracellular or missense intracellular mutations (Supplemental Fig. 3). However, one additional clinical finding that appeared to distinguish the two groups
was the absence of lymphomas in patients with extracellular FAS
mutations. Among .189 patients from 108 families with FAS
mutations in the National Institutes of Health cohort, all 21 lymphomas documented to date occurred in patients with intracellular
mutations (29). Additionally, lymphoma occurred in one patient
with a somatic mutation in NRAS (14). This finding suggests that
the residual apoptotic function observed in haploinsufficiencyassociated extracellular mutations may translate clinically not
only into lower penetrance and expressivity but also into a lower
risk for lymphomagenesis.
Overexpression of FAS in haploinsufficient samples corrects
the apoptotic defect
Discussion
FIGURE 3. Sensitivity of EBV-transformed B cells to CD95-induced
cell death. A–C, EBV-transformed B cells from ALPS patients and three
different normal controls were stimulated with APO-1-3 (1 mg/ml) and
protein A (1 mg/ml) for 24 h. Cell death was determined by measuring the
loss of the mitochondrial transmembrane potential using DiOC6 by flow
cytometry. D, Comparison of the degree of apoptotic defect between cells
with extracellular (EC) mutations (including W176X) affecting FAS expression (n = 9) and intracellular (IC) mutations with normal FAS expression (n = 16), with numbers normalized to the value in normal cells.
The data in A–C were represented as means 6 SE of three separate
experiments. The data in D were presented as medians and interquartile
ranges, and medians were compared using Mann–Whitney U test. All
results from ALPS patients showed significant reduction in FAS-mediated
cell death compared with normal cell lines.
Clinical and laboratory findings in patients with
haploinsufficient alleles
We next analyzed the impact of the different FAS mutations on
clinical and laboratory parameters in ALPS patients. Extracellular
mutations are known to result in lower penetrance and variable
expressivity, and these were not readdressed in this study (18, 26).
Missense mutations affecting the DD of the FAS gene are the most
common genetic abnormality detected in patients with ALPS and
disrupt the apoptosis pathway by dominant-negative interference
(7, 18, 30). This occurs because FAS is present on the cell surface
as a preassociated homotrimeric receptor, such that in the presence
of a heterozygous mutation, seven out of eight trimers will contain
at least one mutant copy of the FAS protein, and this prevents
adequate signaling (31). However, analysis of our extensive cohort
of patients revealed that a significant proportion of ALPS patients
harbor null mutations in the extracellular portion of the FAS
protein. These mutations were not expected to have a dominantnegative effect, as no mutant protein expression was anticipated,
and alternative mechanisms were explored. We analyzed the impact of disease-associated, naturally occurring FAS mutations on
the apoptotic signaling pathway and associated clinical and laboratory findings in ALPS patients. This was, to our knowledge, the
first comprehensive effort to understand the functional consequences of a large group of non-DD ALPS-associated FAS mutations.
Our findings demonstrate that haploinsufficiency is associated
with nonsense or frameshift FAS mutations, primarily located in
gene regions encoding for the extracellular portion of the FAS
protein. From 11 extracellular mutations associated with a defect
in apoptosis, only 3 did not result in decreased FAS expression.
Among these is E45X, a truncation that did not reduce surface
FAS levels to statistical significance and correspondingly did not
affect FAS signaling in any measurable way. It is possible that
even a mild decrease in FAS expression may result in dysfunction
in vivo, although the underlying mechanism for disease in this
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To conclusively demonstrate haploinsufficiency as a disease
mechanism, we transfected YFP-tagged wild-type FAS or an empty
GFP-expressing plasmid into selected EBV-transformed cell lines
that showed reduced FAS expression and reduced sensitivity to
FAS-induced cell death. Transfection efficiency ranged from 10–
25% (data not shown). Forty-eight hours after transfection, we
measured cell-surface FAS expression by flow cytometry on livegated GFP+- or YFP+-transfected cells (Fig. 5). Cell-surface FAS
expression was increased after transfection with YFP-FAS, and
this corrected the apoptotic defect, as compared with control GFPtransfected cells and normal controls. These results suggest that
the level of surface FAS expression is a crucial determinant of the
sensitivity to FAS-induced cell death.
6040
FAS HAPLOINSUFFICIENCY IN ALPS
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FIGURE 4. DISC formation in EBV-transformed B cells of ALPS patients. A–C, Cells were stimulated with APO-1-3 (0.5 mg/ml) or isotype control IgG
in the presence of protein A (1 mg/ml) for 20 min. After lysis, cell lysates were incubated with anti-FAS Ab and protein A/G agarose for 2 h. The
immunocomplexes were then subjected to Western blotting using anti-FADD and anti–caspase-8 (CASP8). Caspase-8 band represents cleaved caspase-8
(∼43 kDa). Because FAS size overlaps with IgG H chain, we could not show immunoprecipitated FAS. D, Comparison of the degree of recruitment of
FADD or caspase-8 to the FAS receptor upon stimulation between cells with extracellular mutations affecting FAS expression (EC; n = 9) and intracellular
mutations with normal FAS expression (IC; n = 16). All numbers were normalized to the value in normal cells. Densitometry data (A–C) are presented as
means 6 SE of (n = 3) separate experiments and compared by Student t test. The data in D were presented as medians and interquartile ranges, and medians
were compared using Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.001.
patient remains to be fully understood. The recurrent mutation
H111R and the in-frame G66_H111 deletion also did not reduce
FAS expression but are predicted to affect the FASL binding site,
providing a possible explanation for the clinical and cellular
phenotype. Curiously, despite having documented apoptosis defects
for all mutations tested, no impairment in DISC formation could
be detected for the extracellular mutations L7P, E45X, H111R,
G66_H111del, and the intracellular I310S. This may reflect a low
sensitivity of the immunoprecipitation assay used in the study to
very mild but still clinically significant functional defects.
Previous case reports lend support to the concept that haploinsufficiency is a pathogenetic mechanism in ALPS. Vaishnaw et al.
(20) suggested that haploinsufficiency was at play in two patients
with a nonsense mutation in the extracellular domain, and Roesler
The Journal of Immunology
6041
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FIGURE 5. Overexpression of wild-type FAS into
FAS-haploinsufficient samples. A–F, Normal and patient EBV-transformed B cells (3 3 106 cells/sample)
were transfected with 5 mg GFP (pEGFP-C1) or 5 mg
YFP-FAS (pEYFP-N1-wt FAS) using Amaxa Human
B-cell Nucleofector. After 48 h of transfection, overexpressed FAS on the cell surface was determined by
flow cytometry using PE-CD95, and cells were stimulated with APO-1-3 (1 mg/ml) and protein A (1 mg/
ml) for 24 h. Cell death was determined by flow
cytometry. Analysis was performed by gating on live
GFP- or YFP-FAS–transfected cells. Data were presented as means of duplicate experiments. N, normal
control.
et al. (19) demonstrated haploinsufficiency in another ALPS patient
with a transmembrane mutation that prevented FAS expression.
Studies in lpr mice, which harbor spontaneous mutations in the Fas
gene, also indirectly support our findings. Although commonly
considered an exclusively recessive phenotype, a heterozygous lpr
mutation does induce the development of autoimmunity in certain
backgrounds, with autoantibody production, glomerulonephritis,
sialoadenitis, and lymphoid accumulation (32, 33). At odds with
human disease, lpr heterozygous mice do not appear to develop
TCRab+CD42CD82 T cell accumulation, but the mutation was
crossed to only a limited number of genetic backgrounds as compared with the outbred human population (32, 33).
6042
Acknowledgments
We thank the patients and their families for contributions to the study. We
also thank Richard Siegel for the FAS-YFP plasmid construct.
Disclosures
The authors have no financial conflicts of interest.
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