Mutations in CD2BP1 disrupt binding to PTP PEST
and are responsible for PAPA syndrome, an
Carol A. Wise1,*, Joseph D. Gillum1, Christine E. Seidman2, Noralane M. Lindor3, Rose Veile4,
Stavros Bashiardes4and Michael Lovett4
1Sarah M. and Charles E. Seay Center for Musculoskeletal Research, Texas Scottish Rite Hospital for Children,
Dallas, TX, USA,
Boston, MA, USA,
Genetics, Washington University School of Medicine, St. Louis, MO, USA
2Department of Medicine and Howard Hughes Medical Institute, Brigham and Women’s Hospital,
3Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA and
Received January 16, 2002; Revised and Accepted February 8, 2002
PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne, OMIM #604416) and familial
recurrent arthritis (FRA) are rare inherited disorders of early onset, primarily affecting skin and joint tissues.
Recurring inflammatory episodes lead to accumulation of sterile, pyogenic, neutrophil-rich material within
the affected joints, ultimately resulting in significant destruction. We recently localized the genes for PAPA
syndrome and FRA to chromosome 15q and suggested that they are the same disorder. We have now
established this by the identification of co-segregating disease-causing mutations in the CD2-binding protein
1 (CD2BP1; GenBank accession no XM 044569) gene in the two reported families with this disorder. E250Q or
A230T amino acid substitutions occur within a domain highly homologous to yeast cleavage furrow-
associated protein CDC15. CD2BP1 and its murine ortholog, proline–serine–threonine phosphatase
interacting protein (PSTPIP1), are adaptor proteins known to interact with PEST-type protein tyrosine
phosphatases (PTP). Yeast two-hybrid assays demonstrate severely reduced binding between PTP PEST and
both the E250Q and A230T mutant proteins. Previous evidence supports the integral role of CD2BP1 and its
interacting proteins in actin reorganization during cytoskeletal-mediated events. We hypothesize that the
disease-causing mutations that we have identified compromise physiologic signaling necessary for the
maintenance of proper inflammatory response. Accordingly we suggest classification of PAPA syndrome as
an autoinflammatory disease. This CD2BP1-mediated biochemical pathway(s) may function in common
inflammatory disorders with apparent etiological overlap, such as rheumatoid arthritis and inflammatory
Autoimmune disorders, estimated to affect 3–5% of the
population, pose a significant public health problem (1).
Although various criteria have been applied to define classic
autoimmunity, in general the destruction of the individual’s
own tissues seen in these diseases is thought to result from
proliferation of T lymphocytes and/or antibodies directed
against self antigens. These antigens may be tissue-specific or
ubiquitously expressed, and resulting destructive inflammation
may be localized, as in rheumatoid arthritis, or systemic, as in
lupus erythematosus. Many of these diseases are associated
with particular histocompatibility locus antigens (HLA),
suggesting that antigen presentation to T cells is important in
the pathogenesis of disease (2–4). A separate but potentially
related group of disorders comprises the so-called ‘autoin-
flammatory’ diseases. Like autoimmune disorders, these
conditions involve inflammation that appears to be due to
breakdown in self-tolerance; however, autoantibodies or
antigen-specific T lymphocytes are not detected (5). There
has been considerable recent progress in identifying causative
factors in autoimmune/autoinflammatory diseases. However,
effective treatments or preventatives are needed for most of
these disorders (2).
*To whom correspondence should be addressed. Tel: þ1 214-559-7861; Fax: þ1 214-559-7872; Email: firstname.lastname@example.org
# 2002 Oxford University PressHuman Molecular Genetics, 2002, Vol. 11, No. 8961–969
by guest on June 12, 2013
In humans, clinically distinct autoimmune and autoinflam-
matory disordersoftenshow familial clustering. Comparisonof
genome-wide linkage scans has revealed clustering of candi-
date loci as well, supporting the hypothesis that a common set
of susceptibility genes controls the pathogenesis of these
diseases (6). The availability of draft or finished human
genomic sequence and annotation with single nucleotide
polymorphisms (SNPs) will speedthedetectionof associations
between disease and non-HLA susceptibility genes (7). In the
meantime, genes for autoimmune/autoinflammatory diseases
displaying Mendelian inheritance may be rapidly identified by
application of straightforward positional cloning methods,
enabling entry into the study of biochemical pathways
controlling both rare and common inflammatory diseases with
etiological overlap. A recent example is the NOD2 gene, for
which distinct mutations have been shown not only to
predispose to Crohn’s disease, a common and genetically
complex inflammatory disorder, but also to cause Blau
syndrome, a raregranulomatous inflammatory disease display-
ing Mendelian inheritance (8,9).
Two families havebeen described with similar inflammatory
disorders, called PAPA syndrome and familial recurrent
arthritis (FRA) (10,11). Both PAPA syndrome and FRA are
characterized by autosomal dominant inheritance of early-
onset, destructive, recurrent inflammation of joints, skin and
muscle. Synovial tissue biopsy reveals massive polymorpho-
nuclear infiltrate without the presence of immunoglobulin or
complement deposits. Pyoderma gangrenosum-like ulcerative
lesions occur in the skin of some affected individuals, as does
severe cystic acne. Episodic inflammatory arthritis typically
does not resolvespontaneously, andmust betreated withintra-
articular steroids or surgical drainage of the infiltrate. An
associated infectious or environmental agent responsible for
triggering these flares has yet to be identified. We have
hypothesizedthat inflammatory episodes inbothdisorders may
arise via a mechanism having relevance to other diseases with
apparent etiological overlap, such as cystic acne, rheumatoid
arthritisandinflammatory bowel disease. Wethereforeinitiated
positional cloning of the gene(s) responsible for PAPA
syndrome and FRA. By linkage analysis of genome-wide
scans, we mapped both PAPA syndrome and FRA in two
extendedkindreds tooverlapping intervals of chromosome15q
and proposed that they are the same disease (10–12).
Wenow reportpositional cloning of asinglegeneresponsible
for both PAPA syndrome and FRA. Genes encoded in the 15q
critical region were assembled from available databases, and
exons weredetermined insilico and screened for heterozygous
mutations. In one gene encoding CD2-binding protein 1, two
different missensemutations wereidentified that co-segregated
with the clinical presentation of PAPA syndrome and FRA,
respectively, in the two originally-reported extended kindreds.
Yeast two-hybrid interaction trap assays revealed that the
known binding between the PEST-type protein tyrosine
phosphatase (PTP PEST) and CD2BP1 is almost completely
abolished in the presence of either disease-causing missense
mutation. These results support our original suggestion that
PAPA syndrome and FRA (now referred to collectively as
‘PAPA syndrome’) are the same disease and demonstrate
that alteration of CD2BP1 is responsible for this severe
Construction of a transcription map of thePAPA syndrome
We constructed a partial physical and transcription map of the
15q interval linked to PAPA syndromeby assembling bacterial
artificial chromosome(BAC) clones and known genes insilico
frompublic databases. A high-throughputgenomic sequenceof
BAC clones found in GenBank was searched for coding
regions by a combination of BLAST analysis (13) against
several protein and nucleotide databases and application of
exon prediction algorithms. The resulting physical map
confirmed genes identified fromtheG3 and GB4 radiation hy-
bridmaps(14,15) (datanotshown). A schematic representation
Figure1. Partial physical andtranscriptionmapof thePAPA syndromecritical
region. PAPA syndromewas previously localized to a 10 cM interval of chro-
mosome15qbetweenD15S1023andD15S979(rightvertical bar) intheorigin-
ally described family. FRA was localized to a larger region between
D15SD15S983 and D15S127 (left vertical bar) encompassing the PAPA syn-
dromelocus. Candidategenes encoded in this interval areshown: cellular reti-
noic acid-binding protein 1 (CRABP1); CD2BP1; reticulocalbin 2 (RCN2);
cathepsin H (CTSH); BCL2-related protein A1 (BCL2A1); interleukin-16
(IL16); SH3-domain GRB2-like 3 (SH3GL3). Those shown in bold were
screened for mutations in affected individuals.
962 Human Molecular Genetics, 2002, Vol. 11, No. 8
by guest on June 12, 2013
of the PAPA syndrome critical region and selected candidate
genes is shown in Figure 1.
Selection of candidate genes and mutation screening
PAPA syndrome is characterized by dominant inheritance of
severe and unusual episodic arthritis without discernible
infection. Erythematous ulcerative skin lesions (pyoderma
gangrenosum) and cystic acne also occur in some but not all
affected family members (Fig. 2A and B). These findings
prompted us to examine genes having a possible role in the
inflammatory response. Affected individuals from family
FRA1 werescreenedformutations inselectedgenes, including
interleukin-16 (IL16), cellular retinoic acid-binding protein 1
(CRABP1), and BCL2-related protein A1 (BCL2A1) by direct
DNA sequencing or denaturing high performance liquid
chromatography (DHPLC) (16). The CD2-binding protein 1
(CD2BP1) gene was considered a plausible candidate because
it encodes aphosphoprotein highly expressedin hematopoietic
tissues, and evidence suggests that the CD2BP1 protein may
regulate cytoskeletal organization in hematopoietic cells (17).
We designed PCR primers from the CD2BP1 sequence to
amplify thecDNA insix overlapping fragments (Fig. 3). These
amplimers weregenerated by reversetranscription–polymerase
chain reaction (RT–PCR) of total RNA from affected family
members FRA1-1 and FRA1-10 Epstein–Barr virus (EBV)
lymphoblastoid cell lines. Analysis by denaturing high-
performanceliquidchromatography (DHPLC) detectedhetero-
duplex formation in the fragment amplified by primers 4F and
4R, suggesting thepresenceof asequencevariantinoneallele.
Interestingly, alternative splicing of this region in activated T
cells has been reported to generate two transcripts differing by
57 nucleotides (17). However, we detected only the longer
version in amplified patient lymphoblastoid cDNA (data not
shown). The exon/intron boundaries of CD2BP1 were derived
by alignment of the cDNA sequence with a human genomic
sequence assembled from available databases. This analysis
indicated that the CD2BP1 gene contains 15 exons, with the
alternatively spliced region encompassing exon 12. Primers
were designed to amplify each exon (Fig. 3 and Table 1).
Sequenceanalysisof aportionof exon11 identifiedaG-to-C
transversionat nucleotide964 of theCD2BP1cDNA creating a
E250Q variant that co-segregates with disease in the FRA
family (Fig. 4A). Similar analyses in members of the PAPA
family detected a G-to-A transition at nucleotide 904 of
CD2BP1 predicted to create an A230T variant in affected
individuals (Fig. 4B). Sequencing of the cloned 50exon 11 or
Figure2. Clinical featuresof PAPA syndrome. PAPA syndromeischaracterized
by autoimmune destruction of joints and skin. (A) Arthritis in the proband
FRA1-1 characteristically involves primarily the elbows, knees and ankles.
Shown here is severe swelling of the knee during a flare. (B) Ulcerative skin
lesion (pyoderma gangrenosum) is shown for the FRA1-1 proband.
Figure 3. Structure of the CD2BP1 gene. Predicted exons are shown by alter-
nating gray and black shading and are numbered below each line. Primer se-
quences used for PCR amplification of cDNA are underlined; primer names
are given in bold print above the sequence for forward primers and below for
reverse. The G904A and G964C mutations are shown.
Human Molecular Genetics, 2002, Vol. 11, No. 8 963
by guest on June 12, 2013
Table 1. Exons of the CD2BP1 gene
Exon Position in cDNAForward PCR primer 50–30
Position Reverse PCR primer 50–30
Position Product bp
Thepositionof eachexoninthepublishedCD2BP1L sequenceisgiven. Positionsof oligonucleotideprimersaregivenasdistancesinnucleotidesfromthe50or30
end of each exon; for example, ‘?95’ is 95 nucleotides away from the beginning or end of the exon, and ‘þ16’ is 16 nucleotides within the exon.
Figure4. Segregation of theG964C or G904A mutations with FRA or PAPA syndrome, respectively. (A) Theproband FRA1-1 is denoted by an arrow. Reverse
sequence and DHPLC traces are shown for each individual, except FRA1-2 and FRA1-13 for which DHPLC was not performed.
964Human Molecular Genetics, 2002, Vol. 11, No. 8
by guest on June 12, 2013
exon 10 amplimers from affected individual FRA1-1 or
PAPA1-114, respectively, confirmed the presence of both
mutant and wild-type sequences. DHPLC analysis of 228
unrelated control chromosomes revealed no heterozygous
sequence variants in PCR-amplified CD2BP1 exons 10 or 11;
DNA sequencing revealed no differences from the published
sequence in 72 of these (data not shown). By our analysis,
neither the E250Q or A230T alteration has been described in
publicly available single nucleotide polymorphism (SNP)
databases. As anticipated from the original DHPLC results,
no changes from wild type were detected in the remaining 14
exons amplified from genomic DNA of individuals FRA1-1
and PAPA1-114 (data not shown).
Yeast two-hybrid interaction trap assays
Three structural features have been identified in the predicted
amino acid sequences of CD2BP1 (17,18). Amino acids 123–
288, containing both E250Q and A230T, bear 30% identity to
the Schizosaccharomyces pombe Cdc15 protein, a phospho-
protein involved in organization of the actin ring and cleavage
furrow formation during cytokinesis (19). The C-terminus
contains an SH3 domain, and between these two regions is a
sequence rich in proline, glumatic acid, serine and threonine
(PEST) residues. The SH3 domain of CD2BP1 or its murine
ortholog was previously shown to interact with CD2 (17),
c-Abl (20) and Wiskott–Aldrich syndrome (WAS) protein
(WASP) (21), whereas the PTP PEST interaction domain was
mapped within the region of CD2BP1 homologous to Cdc15
andpotentially encompassing thevariantsthatwedetected(22)
(Fig. 5). We therefore undertook a series of yeast two-hybrid
experiments using as bait a full-length wild-type CD2BP1
construct cloned in frame with the GAL4-binding domain
(GAL4BD). A yeast strain expressing this fusion protein was
transformed with human thymus cDNAs fused downstream of
the GAL4 transcriptional activation domain (GAL4AD). In a
screen of 2?105transformants, 20 isolates that co-activated
boththeHIS3 andlacZ reportergenes wereidentified, curedof
the wild-type CD2BP1–GAL4BD, and re-transformed with a
full-length E250Q mutant CD2BP1 construct cloned in the
GAL4BD plasmid. Inaseparateseriesof experiments, thewild-
type CD2BP1–GAL4DB was re-transformed into each cured
strain to confirm that the positive interaction could be re-
established. Of these, only one (clone 81 plus E250Q mutant
CD2BP1–GAL4BD) failed to activate HIS3. DNA sequencing
revealed that the GAL4AD plasmid from clone 81 contained
sequences encoding the last 32 C-terminal amino acids (plus
the 30untranslated region) of the protein tyrosine phosphatase
PTP PEST. The failure of clone 81 to grow in the absence of
histidine when in combination with the mutant construct,
versusitsstrong growthwiththewild-typeCD2BP1, suggested
that the E250Q change disrupts CD2BP1:PTP PEST binding.
However, thisinteractionwasnotcompletely abrogated. Plating
clone81:CD2BP1 E250Q–GAL4BD cotransformantsonmedia
that does not select for interactions (?Leu, ?Trp, þ His)
resulted in colonies with weak but reproducible lacZ activity,
indicating low-level interaction with the mutant. We quantita-
tively assayed the interactions between clone 81 and either
wild-type or E250Q mutant CD2BP1 by measuring b-galacto-
dase (b-GAL) activity. Triplicate measurements resulted in an
average of 100 b-GAL units for wild-type CD2BP1:clone 81
interaction, compared with an average of 1.2 b-GAL units for
the E250Q mutant CD2BP1:clone 81 interaction. It therefore
appears that a residual interaction of approximately 1.2% of
wild-typelevelsstill occurs betweenPTP PEST andtheE250Q
mutant, but is insufficient to result in histidine prototrophy in
the two-hybrid screen. In repeated experiments, when the
A230T mutant was transformed with clone 81, the level of b-
GAL activity wasreducedtolessthan10% of wild-typelevels,
confirming that this residue is also important for PTP PEST
interaction with CD2BP1. In parallel quantitative two-hybrid
experiments we found no detectable differences in the
interactions between CD2 and wild-type, E250Q or A230T
CD2BP1 mutants; however, deletion of the CD2BP1 SH3
domain completely abolished this interaction, confirming the
previous observation that this domain is essential for CD2
We conclude that the heterozygous mutations in the CD2BP1
gene cause PAPA syndrome in the two families we have
studied. Given what is currently known about CD2BP1
function combined with the clinical features of this disorder,
we have formulated two alternative models of disease
pathogenesis. The first derives from the resemblance of PAPA
syndrome to classic autoimmune disease. By definition, the
autoimmune disorders are mediated by the adaptive immune
Figure 4. (B) Sequence identification of the G904A transversion in affected
(113 and 114) and unaffected (112) siblings in the original PAPA syndrome
Figure 5. CD2BP1/PSTPIP1 structure and protein/protein interactions. The
Cdc15-like and SH3 domains are shown boxed. Clear circles are PEST-rich
sequences, and stars represent the E250Q and A230T mutations. Interacting
proteins are shown below their associated domain.
Human Molecular Genetics, 2002, Vol. 11, No. 8965
by guest on June 12, 2013
response, in which T and/or B cells respond to specific self
antigens and ultimately produce tissue damageand destruction
(4,23). According tothis model, flares seeninPAPA syndrome
would be initiated by reaction of a subset of T or B cells to a
specific componentof skin, muscleandjoints (10). CD2BP1 is
expressed in T-cells and has been proposed to be important in
regulating T-cell behavior by modulation of CD2 activity (17).
CD2 is a cell surface molecule that binds to the glycoprotein
CD58 on opposing antigen-presenting cells (APCs), thereby
improving the efficiency with which T-cell receptors find a
major histocompatibility complex (MHC) molecule together
withanantigenic peptide(24,25). Increasing levelsof CD2BP1
in transfected cells reduce CD2-mediated sheep red blood cell
(RBC) rosetting, implying that CD2BP1 downregulates CD2-
mediated T-cell adhesion activity (17). Although we have not
detected infiltration by T or B cells in affected tissues (11), we
cannotruleoutthepossibility thatdisease-causing mutationsin
CD2BP1 may alter T-cell activity in PAPA syndrome patients
and precipitatetheobserved influx of polymorphonuclear cells
(neutrophils) into inflammatory sites.
The severe neutrophil infiltration in affected tissues suggests
asecondmodel of diseasepathogenesis inwhichinflammation
results directly from dysfunction of the innate immune
response. In this scenario, disease-causing mutations in
CD2BP1 somehow exaggerate the signal for proliferation and
infiltration of inflammatory initiator cells, alter apoptotic
pathwaysand/orinhibit theirclearance, leading totheobserved
abnormal accumulationsof neutrophil-richmaterial. Therecent
report that the CD2BP1 protein binds pyrin, the protein
encoded by theMEFV generesponsiblefor familial mediterra-
nean fever (FMF), supports this model (26). FMF is a rare,
recessively inheritedautoinflammatory diseasecharacterizedby
self-limited episodes of fever with arthritis, and sterile
inflammation of peritoneal and pleural membranes (27).
Although clinically distinct from PAPA syndrome, FMF is
also characterized by neutrophil infiltration into target tissues,
and pyrin itself is expressed in neutrophils but not T or B cells
(28,29). It is interesting that of five proteins (including pyrin)
responsible for inherited autoinflammatory diseases, four
regulators of apoptosis. This has led to the hypothesis that
mutations in these genes may inhibit apoptosis, leading to
prolonged accumulation of neutrophils (30). Perhaps disease-
causing mutations inCD2BP1 alter apyrin-mediated apoptotic
pathway in neutrophils.
Foreithermodel of diseasepathogenesis, wehypothesizethat
low-level inflammation is constitutively present in PAPA
syndrome, and clinical flares may result from mild physical
trauma that induces accumulation of hyperresponsive inflam-
matory cells. Consistent with this, members of both families
described here report ulcerative lesions arising at sites of
parenteral injections (10, our unpublished observations).
Whether PAPA syndrome is mediated by lesions in adaptive
or innate immunity, or some alternative pathway, will require
detailed functional analyses in cells of affected patients.
Various experiments have supported the hypothesis that
CD2BP1 and PTP PEST function in cytoskeletal regulation. It
was originally shown by confocal microscopy that endogenous
murine CD2BP1 associated with the cortical actin cytoskele-
ton, lamellipodia and membrane-bound F-actin in the
cytokinetic cleavage furrow in dividing NIH 3T3 cells.
Overexpression of the protein caused formation of distinct
morphological changes in NIH 3T3 cells, with formation of
extended filopodia (18). In the human Jurkat J77 T cell line,
stable CD2BP1 transfectants showed normal motility and
filopodial formation in response to CD2 triggering, whereas
cells lacking CD2BP1 were non-responsive, suggesting that
CD2BP1 regulates CD2-activated integrin adhesion pathways
inT cells. PTP PEST isexpressedinhematopoietic tissues, and
(17). In fibroblasts homozygously deleted for PTP PEST,
murineCD2BP1, as well as other known PTP PEST substrates
p130Cas, paxillin and focal adhesion kinase (FAK), are
hyperphosphorylated, and this correlates with a significant
increase in the number of cells apparently blocked in a late
stage of cytokinesis (31). Further studies of morphology and
function in cells of patients affected with PAPA syndrome, as
well as cell transfection studies, will determine whether
disease-causing mutations in CD2BP1 confer cytoskeletal
Theinteractionof CD2BP1 withPTP PEST iswell described
(18). This interaction is mediated by the coiled-coil domain of
murine CD2BP1 and the terminal 20 amino acids of
hematopoietic stem cell fraction PTP, or related phosphatases
(18). Introduction of a W232A mutation within the same
coiled-coil domain abolished the interaction with the hemato-
poietic stemcell fraction PTP invitro and invivo. The mutant
protein was also hyperphosphorylated, implying that CD2BP1
is a substrate of PTPs (22). Our data confirm the strong
interaction between PTP PEST and CD2BP1 and demonstrate
loss of binding between the two mutants A230T and E250Q
that lie within the predicted coiled-coil domain. Whether the
pathophysiology of PAPA syndrome is due to loss of function
from reduction in PTP PEST binding, a dominant negative
effect due to alteration of CD2BP1 interaction with other
proteins oranalternativemechanismwill requirefurther study.
In any case, it is interesting that the changes produced by
CD2BP1 missense mutations are sufficient to confer suscept-
ibility to inflammatory episodes, suggesting that CD2BP1 is
critical in the homeostasis of inflammation. This also suggests
to us that more drastic nonsense or deletion mutations in
CD2BP1 could confer chronic, severe inflammation or
CD2BP1 or its murine ortholog was previously shown to
interact with two additional proteins, c-Abl and WASP. The
c-Abl proto-oncoprotein involved in various human leukemias
the cytoskeleton and binds actin filaments directly through a
C-terminal domain (32). The protein also binds and phosphor-
ylatesmurineCD2BP1. A model hasbeenproposedsuggesting
thatCD2BP1 functionstocouplePTP PEST toc-Abl, allowing
dephosphorylationandhenceregulationof activity of thec-Abl
protein (20). WASP is linked to actin polymerization via
interaction with the Rho family GTPase CDC42Hs (33).
Mutations in WASP are responsible for the X -linked
hematopoietic deficiencies and platelet abnormalities of WAS
as well as X-linked congenital neutropenia (34–36). WASP
binding to murine CD2BP1 appears to be phosphorylation—
dependent; mutation of tyrosine 367 within the SH3 binding
domain to aspartate or glutamate to mimic the negativecharge
966Human Molecular Genetics, 2002, Vol. 11, No. 8
by guest on June 12, 2013
of a phosphorylated tyrosine severely reduced co-immunopre-
cipitation of the two proteins (21). Although binding between
WASP (or CD2) and both E250Q and A230T mutant forms of
CD2BP1 was unaltered in yeast two-hybrid experiments (our
unpublished results), in mammalian cells, these interactions
may be affected as a result of CD2BP1 hyperphosphorylation
in the absence of PTP PEST. In particular, WASP association
with CD2BP1 is predicted to decrease in the presence of the
disease-causing mutations. In addition, the subsequent activ-
ities each of these substrates might be altered in vivo by an
inability to couple to PTP PEST.
Pyoderma gangrenosum(PG) and cystic acne are conditions
sometimes associatedwithcommoninflammatory autoimmune
diseases. PG is a painful disease of the skin in which
erythematous sterile pustules develop into ulcerative necrotic
lesions, typically on the feet and extremities. Although
successful treatments have been described, PG can be fatal
(37–39). Kindreds segregating PG have been reported (40);
however, a genetic etiology is not generally attributed to this
disease. The disease occurs most often in association with
rheumatoid arthritis or inflammatory bowel disease (Crohn’s
disease or ulcerative colitis). In children, PG is most often
associated with ulcerative colitis (41). In this regard, it is
interesting that the human PTP PEST is encoded on
chromosome 7q in a region exhibiting evidence of linkage to
inflammatory bowel disease (42). Cystic acne is common,
estimatedtooccurin10–20% of adolescents andyoung adults.
Morerarely it is associated witharthritis or PG (43–45). These
observations suggest to us some etiological overlap between
PAPA syndrome and more common and genetically complex
disorders such as rheumatoid arthritis and inflammatory bowel
disease. Functional studies of CD2BP1, PTP PEST and other
CD2BP1-binding partners will be important in elucidating
pathways involved in PAPA syndrome and related inflamma-
MATERIALS AND METHODS
Electronic transcript mapping
A high-throughput genomic sequenceof BACs identified from
public databases (primarily at the National Center for
Biotechnology Information) was obtained from GenBank.
The NIX platform (http://menu.hgmp.mrc.ac.uk/menu-bin/
Nix/Nix.pl) was used for both exon prediction and BLAST
CD2BP1 exon prediction
Thegenomic structureof CD2BP1 was derivedby aligning the
CD2BP1 cDNA sequence to a human genomic sequence
obtained from available databases by the Seqman program
(DNASTAR, Inc., Madison, WI).
Whole-cell RNA was isolated from EBV-transformed lympho-
10 by a modified guanidium thiocyanate lysis procedure
(Roche Boehringer Mannheim, Indianapolis, IN). RNA was
converted to cDNA by both random priming and dT priming
with reverse transcriptase (Roche Boehringer Mannheim).
CD2BP1 was produced in six fragments by amplification of
cDNA with the following primers: CD2BP1-1F: ATG ATG
CCC CAG CTG CA; CD2BP1-1R: CCT TCT GCC TCT CAC
GAA AC; CD2BP1-2F: CAA ATG GAG AAT GTG GGC AG;
CD2BP1-2R: TGG CTT TGT TCT GAC TCT TCT; CD2BP1-
3F: CCA AGA AGA CAT ACG AGC AGA AG; CD2BP1-3R:
CCG CAC TTC CTC GTA GAG; CD2BP1-4F: GCA GTG
TGT CAA GGA TGA TG; CD2BP1-4R: GTC TCT GTG GAC
GCA GCA G; CD2BP1-5F: TGC CCT ACC AGA ACT ATT
ACG; CD2BP1-5R: CCA CAG TCC ACC AGC CAT;
CD2BP1-6F: CAG CGC AGA ACC CAG ATG A; CD2BP1-
6R: AGA ACA GAA CGC ACT CCT TT. Amplification
conditions for all cDNA fragments were 35 cycles, 94?C 30 s,
56?C 30 s, 72?C 60 s. CD2BP1 exons were amplified from
genomic DNA by oligonucleotideprimersdescribedinTable1.
The 50exon 11 fragment was amplified by CD2BP1 exon 11
?109F: CAC AAT GGC CTG TGA GGA G and CD2BP1-
1038R: CGT GCT CTT GGC CTG GAT. All amplimers were
generated in the PCR in a GeneAmp 9600 (Perkin Elmer,
Foster City, CA) or Tetrad (MJ Research, Waltham, MA)
thermocycler. Reactions were performed in 25ml containing
1.5mM MgCl2, 500mM KCl, 0.25mM each dNTP, 1.0 pmol
each primer, 0.5 U Taq polymerase (PE Corp., Norwalk, CT),
and 100 ng patient DNA. Amplification conditions were 35
cyclesof 94?C 307s, 56?C 30 s, 72?C 30 s, except50exon11,
for which an annealing temperature of 55?C was used.
DHPLC was performed by WAVE analysis (Transgenomic,
San Jose, CA) utilizing conditions recommended by Wave-
Maker v3.3.3 software. CD2BP1 amplified fragments 1, 2, 3
and5 wereanalyzedatanoventemperatureof 65?C, fragments
4 and 50exon 11 at 64?C, and fragment 6 at 66?C. PCR
amplimers were purified from 1.2% agarose gels with the
QIAquick gel purification kit (Qiagen, Hilden, Germany) and
sequenced with the ABI PRISM BigDye Terminator Cycle
Sequencing Ready Reaction kit (Applied Biosystems, Foster
City, CA). Sequencing products were separated by electro-
phoresis 36cm 4.25% denaturing acrylamide gel at 760 V for
3–7hourswithanABI PRISM 377DNA Sequencer/Genotyper
Yeast two-hybrid analysis
The wild-type CD2BP1 cDNA was PCR amplified from a
human spleen cDNA library using primers 1 (50ATTGGATC-
CAGCTGCAGTTCAAAGATG 30) and 2 (50AATGTCGAC-
TAGCAAGGTGGGGACAGTGC 30). The CD2BP1 SH3
domain deletion construct was built using primer 1 and primer
3 (50AATGTCGACGGGTTTCCCTGTATCTCCTGT 30).
Amplification products were purified, restriction-digested with
BamHI and SalI and cloned into the corresponding sites of
pAS2-1 (Clontech, Palo Alto, CA). The E250Q and A230T
mutant cDNA clones were derived by PCR site-directed
mutagenesis of wild-type CD2BP1 and subcloned into
pAS2-1. The cytoplasmic domain of CD2 was PCR-amplified
Human Molecular Genetics, 2002, Vol. 11, No. 8967
by guest on June 12, 2013
from a human spleen cDNA library using primers 4
and 5 (50AATGTCGACTAGCAAGGTGGGGACAGTGC
30). Amplification products were purified, restriction-digested
with BamHI and SacI and cloned into the corresponding sites
of ACT2AD (Clontech). Wild-typeandmutant constructs were
purified, and the DNA sequence of the fusion proteins was
confirmed by automated fluorescence sequencing as described
above. Yeast two-hybrid screening was performed using the
strains, media, vectors and protocols from the Matchmaker
two-hybrid system (Clontech). Cells containing the wild-type
CD2BP1–GAL4BD constructweretransformedwith10 mg of a
human thymus cDNA library in pACT2 (Clontech). Approxi-
mately 2?105transformants were spread on SD/?Leu/?Trp/
?His/þ3-AT plates. An aliquot was also plated on SD/?Leu/
30?C, positive clones were patched on SD/?Leu/?Trp/?His/
þ3-AT (25 mM) plates, and assayed for b-galactosidase (b-
GAL) activity using a colony-lift filter assay. Quantitative
b-GAL assays were performed in triplicate using CPRG
(Roche Molecular Biochemicals) as a substrate. Binding
domain constructs were cured fromyeast clones by growth in
?Leu media followed by duplicate plating to ?Trp media to
identify clones that had lost the GAL4BD construct. These
cured clones were then re-transformed with the E250Q
mutant–GAL4BD construct or with the wild-type construct,
and were retested for histidine prototrophy.
We are especially indebted to the families involved in this
study. C.A.W. thanks R. Barnes and members of the
McDermott Center for Human Growth and Development,
University of Texas Southwestern Medical Center, for their
cooperation and support of this project. We also wish to thank
V. Pascual for clinical information and A.M. Bowcock for
critical reading of themanuscript. This work was supported by
the Texas Scottish Rite Hospital for Children Research Fund
(C.A.W.) and the Howard Hughes Medical Institute (C.E.S).
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