Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Article (PDF Available)inThe American Journal of Human Genetics 90(6):1108-15 · May 2012with87 Reads
DOI: 10.1016/j.ajhg.2012.05.006 · Source: PubMed
Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.
Somatic Mosaic Activating Mutations
in PIK3CA Cause CLOVES Syndrome
Kyle C. Kurek,
Valerie L. Luks,
Ugur M. Ayturk,
Ahmad I. Alomari,
Steven J. Fishman,
Samantha A. Spencer,
John B. Mulliken,
Margot E. Bowen,
Guilherme L. Yamamoto,
Harry P.W. Kozakewich,
and Matthew L. Warman
Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonheredi-
tary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES
syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively
parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We
identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from
multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoi-
nositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar
sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.
Syndromes can be genetic in origin but not necessarily
heritable. Happle postulated that such disorders arise as
the result of somatic rather than germline mutations, i.e.,
because complete heterozygosity for a causative mutation
would either be lethal to the affected individual or be inca-
pable of transmission through egg or sperm.
hypothesis was confirmed by the discovery of somatic
mosaic mutations in several diseases, including McCune-
Albright syndrome (MIM 174800) and Proteus syndrome
(MIM 176920).
Isolated malformations and nonhereditary syndromes
with malformation as a component feature could also be
caused by a somatic mutation. One such candidate is the
recently described disorder CLOVES (congenital lipoma-
tous asymmetric overgrowth of the trunk with lymphatic,
capillary, venous, and combined-type vascular malforma-
tions, epidermal nevi, and skeletal anomalies [MIM
612918]) (Figure 1).
Therefore, we sought to identify
postzygotic mutations in individuals with CLOVES by
employing massively parallel sequencing of DNA and
RNA from affected tissue to look for mutations that are
present at low frequencies. The study was approved
by the institutional review board at Boston Children’s
Hospital. Study participants provided written informed
consent to participate in the study and to authorize the
publication of clinical images.
We used fresh or frozen affected tissue from four
CLOVES-affected individuals, each of whom had under-
gone resection of a lipomatous overgrowth or a vascular
malformation with lipomatous overgrowth (Tables 1
and 2), and we produced barcoded DNA sequencing
libraries as described previously.
We assumed that lipoma-
tous tissue would contain mutant cells given that incom-
pletely resected lesions often regrow.
We also recovered
mRNA and generated barcoded cDNA sequencing
from these same specimens. For two of these
individuals, we prepared DNA sequencing libraries from
white blood cell (blood or saliva) DNA. We assumed that
this unaffected tissue would not contain a CLOVES causa-
tive mutation. Formalin-fixed paraffin-embedded tissue
blocks were available from two other individuals (Table
2) from whom we recovered DNA by using thin sections
to produce additional barcoded sequencing libraries (see
Figure S1, available online, for the experimental design).
Saliva DNA was available from one of these individuals
and was used for the preparation of a library.
We enriched each DNA library generated from the fresh
or frozen samples for exonic sequences by using the Sure-
Select human exome kit (Agilent Technologies, Santa
Clara, CA, USA). For DNA libraries generated from the
paraffin tissue blocks, we employed a custom-designed
enrichment array that contained exonic sequences from
77 genes involved in signaling pathways for several growth
factors. We had previously employed this targeted array to
screen individuals with metachondromatosis.
genes included in this array, e.g., PTEN (MIM 601728),
AKT1 (MIM 164730), AKT2 (MIM 164731), and AKT3
(MIM 611223), have been implicated in other overgrowth
We performed RNA sequencing in case
the gene responsible for CLOVES was abundantly
Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
Department of Orthopedic Surgery, Boston
Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
Department of Vascular and Interventional Radiology, Boston Children’s
Hospital and Harvard Medical School, Boston, MA 02115, USA;
Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston,
MA 02115, USA;
Department of Plastic Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
Vascular Anomalies
Center, Boston Children’s Hospital, Boston, MA 02115, USA;
Department of Genetics, Faculdade de Medicina da Universidade de Sa
˜o Paulo, 01246-
903 Sa
˜o Paulo, Brazil;
Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA 02115, USA;
Department of Genetics, Harvard Medical
School, Boston, MA 02115, USA
DOI 10.1016/j.ajhg.2012.05.006.Ó2012 by The American Society of Human Genetics. All rights reserved.
1108 The American Journal of Human Genetics 90, 1108–1115, June 8, 2012
transcribed in affected tissue. We reasoned that detecting
low-level mosaicism would be easier in an abundantly ex-
pressed transcript for which read depth can be greater than
2003as compared to only 203for a whole-exome
After we employed massively parallel sequencing and
filtering to remove PCR duplicates, we found that the
whole-exome capture data provided >203coverage for
85% of the exome for three samples and for 50% of the
exome for one sample. The 77 gene capture array sequence
yielded >203coverage for 95% of the array for
both samples. RNA-seq provided >203coverage for
the ~2,500 most abundantly expressed transcripts. We
next filtered the data to remove SNPs that were present
in dbSNP build 132, the 1000 Genomes Project, or the
National Heart, Lung, and Blood Institute (NHBLI)
whole-exome database (Table S1). We then filtered for vari-
ants present in greater than 5% of reads in affected tissue
and ranked these variants with respect to the fold coverage
for that nucleotide. For example, we ranked a variant that
was present in 3 of 50 reads (6%) higher than a variant
present in one of five reads (20%) by assuming that the
former was more likely to be a true positive and that the
latter was more likely to be a false-positive sequencing
error. Finally, we focused on highly ranked variants that
either were solely observed in the affected tissue or were
more abundant in affected tissue than in unaffected tissue
(blood or saliva).
Each CLOVES-affected individual for whom DNA from
fresh or frozen affected tissue was sequenced had
a missense PIK3CA (MIM 171834) mutation that was not
present in the blood or saliva DNA sequence (when avail-
able). Participants CL3 and CL4 had a c.1624G>A
(p.Glu542Lys) mutation, and participants CL5 and CL6
had a c.1258T>C (p.Cys420Arg) mutation based on RefSeq
NM_006218.2 (Figure 2 and Table 2). PIK3CA was among
the 77 genes included in the targeted-capture array. Indi-
viduals CL1 and CL2, whose paraffin DNA samples were
used in that array, both had a c.3140A>G (p.His1047Arg)
missense mutation in PIK3CA (Table 2). The PIK3CA
sequence was poorly represented in the RNA sequence
data (<23coverage), and missense mutations were not
found (Table S2). Nevertheless, we detected in three partic-
ipants the same mutations observed in the whole-exome
sequence data when we performed gene-specific RT-PCR
of PIK3CA by using total RNA from frozen affected tissue
as the template. We confirmed that all mutations detected
by massively parallel sequencing were present in the
participants by reanalyzing the original tissue samples
and by PCR amplifying, subcloning, and sequencing indi-
vidual amplimers (Figure 2,Table 2, and Table S3).
One individual with CLOVES syndrome required lower-
extremity amputation. Thus, we collected fresh lipoma-
tous tissue from which we separated adipocytes from
fibroblasts and vascular endothelial cells. We also recov-
ered DNA from several affected tissues, including a cuta-
neous lymphatic malformation and a marginal vein, in
the amputated limb. We found that the purified adipocytes
from the lipomatous tissue and each of the affected tissue
samples were mosaic for the same mutant allele (Figure 3).
The PIK3CA mutations we discovered have been previ-
ously identified as somatic alterations in several types of
Cell biologic studies of these mutations indicate
that they activate phosphoinositide-3-kinase (PI3K)
activity in the absence of growth-factor signaling.
Because PI3K activity normally leads to phosphorylation
of AKT family members, we extracted protein from two
participants’ lipomatous overgrowths to compare its level
of AKT phosphorylation to that of protein extracted from
normal adipose tissue and from an adipose containing
PTEN hamartoma of soft tissue (PHOST)
from an indi-
vidual with a PTEN mutation. Affected tissue from the
participants with CLOVES syndrome had higher levels of
AKT phosphorylation than did the other samples
(Figure 2).
Massively-parallel-sequencing technologies have facili-
tated the identification of causes of heritable diseases.
Challenges in applying these technologies to sporadically
Figure 1. Clinical Features of CLOVES Syndrome
(A) Participant CL5 at age 15 years. Note large, bilateral, posterior
thoracic fatty masses with overlying capillary malformation on
the right side.
(B) Participant CL2 at age 18 months. She has overgrowth of her
lower extremities, polydactyly, and wide feet with an expanded
first interdigital space.
(C) A sagittal T1-MRI (magnetic resonance image) of participant
CL5 demonstrates cervicothoracic lipomatous overgrowth
(straight arrows) extending into the posterior mediastinum and
paraspinal region (bent arrow) and scoliosis.
(D) A coronal postcontrast T1-MRI of participant CL3 shows bilat-
eral truncal (short arrows) and mediastinal (bent arrows) fatty
overgrowth, phlebectasia (long arrow), scoliosis, and asymmetrical
kidneys due to right renal hypoplasia (notched arrows).
The American Journal of Human Genetics 90, 1108–1115, June 8, 2012 1109
occurring somatic malformations include having sufficient
numbers of individuals with the same phenotype and
procuring and distinguishing affected from unaffected
tissue from these individuals. We successfully identified
somatic mutations (for which mutant allele frequencies
ranged from 8% to 30% in lesional tissue) in individuals
who have CLOVES syndrome by performing exome
sequencing of fresh or frozen lesional tissue with paired
blood and saliva samples and by performing targeted
genomic sequencing of archival paraffin tissue samples.
We did not detect mutations in blood or saliva DNA
from participants who had mutations in their anomalous
tissue. This finding is consistent with earlier studies that
demonstrated that constitutive activation of the PI3K-
AKT pathway is detrimental to hematopoiesis.
against somatic mutations in hematopoietic stem cells or
their derivatives has been described for other disorders.
Therefore, discovering the cause of other somatic disorders
might require sequencing DNA recovered from the lesional
tissue, not deep sequencing of blood-derived DNA.
PIK3CA encodes the 110-kD catalytic alpha subunit of
PI3K, which in response to tyrosine kinase receptor ligand
binding is activated and converts phosphatidylinositol
(3,4)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-
triphosphate (PIP3) (Figure 2). This leads to the transloca-
tion and phosphorylation of PDK1 (PDPK1 [MIM
605213]) at the cell membrane. PDK1 then phosphory-
lates AKT to initiate downstream cellular effects. Regula-
tion of this pathway is partly achieved by PTEN, which
catalyzes the conversion of PIP3 to PIP2 (Figure 2). Each
of the missense mutations we observed in participants
with CLOVES syndrome has previously been identified
in several types of adult-onset cancer, including cancers
of the gastrointestinal tract, brain, breast, bladder, lung,
and thyroid.
Rather than directly causing transforma-
tion in these cancers, the PIK3CA missense mutations
are thought to enhance the tumor’s growth or aggressive-
ness. The fact that conditional activation of PIK3CA
missense mutant alleles in two different mouse cancer
models increased the incidence and severity of cancer is
Table 1. Summary of Participants with CLOVES Syndrome
Age (years)
2 1 14 1 15 18
Sex male female female male male female
PIK3CA mutation c.3140A>G
Lipomatous overgrowth
Trunk þþ þþþþ
Limb(s) þþþþ
Vascular anomalies
Lymphatic malformation þþþNA þ
Capillary malformation NA NA þþþþ
Venous malformation þþ þþþþ
Fast-flow malformation NA NA þ
Wide hands or feet þþþþþ
Foot ‘‘sandal-gap’
þþ þþ
Macrodactyly – þþþþþ
Limb asymmetry NA þþþþþ
Paraspinal mass NA NA NA þNA
Scoliosis þþþþ
Renal NA hypoplastic left kidney
and Wilms tumor
right kidney
Other findings rib anomalies and
pterygium coli
NA NA NA NA abnormal marginal
venous system and
The following abbreviation is used: NA, not available.
At the time the tissue sample was obtained.
1110 The American Journal of Human Genetics 90, 1108–1115, June 8, 2012
consistent with this theory.
At the cellular level, the
missense mutations we observed in participants with
CLOVES syndrome have been shown to increase the
activity of PI3K and lead to an abundance of phosphory-
lated AKT.
The fact that we observed increased AKT
phosphorylation in affected tissues from participants
who have CLOVES syndrome is consistent with these
findings (Figure 2C). Interestingly, we did not observe
increased AKT phosphorylation in the affected tissue
from the individual with the PTEN mutation; explana-
tions for the lack of increased AKT phosphorylation in
the PTEN lesion compared to the PIK3CA lesions might
include a stoichiometric difference between heterozygous
PTEN inactivation and PIK3CA activation, a biologic
difference between a vascular malformation with
adiposity and a lipomatous overgrowth, or a difference
in how the different samples had been processed and
stored prior to protein extraction.
When overexpressed, PIK3CA missense mutations iden-
tified in participants with CLOVES syndrome have the
ability to transform cells.
We have detected Wilms
tumor (MIM 194070) in two CLOVES-affected individuals,
including participant CL2 in this study. Consequently,
endogenous expression of missense PIK3CA mutants
could be transformative in some human cell types. We
hypothesize that the low rate of malignant transformation
in individuals with CLOVES syndrome is due to the low
level of endogenous PIK3CA expression in most cells. For
example, among the four frozen samples in which we per-
formed RNA sequencing, PIK3CA was the 19,000
abundant RNA transcript. This explains why we did not
find PIK3CA mutations in our RNA sequence data and
also probably explains the difference in the rate of cell
transformation that occurs when mutant PIK3CA levels
are overexpressed in vitro versus endogenously expressed
in vivo.
An individual in whom we extracted DNA from several
different lesions was mosaic for the same PIK3CA muta-
tion at each site. Different cell types were used as the
source of DNA from the various locations. For example,
we sequenced DNA from adipocytes that had been recov-
ered after dissociation and density centrifugation from
a lipomatous lesion and from endothelial, smooth-
muscle, and fibrocytic cells from the malformed embry-
onic marginal venous system (Figure 3). The fact that
the same mutant allele was detected at each location
suggests that the mutation arose early enough in develop-
ment so as to affect several cell lineages. Interestingly, the
mutant allele frequency was 51/165 (31%) in adipocyte
DNA purified from a lipomatous overgrowth. This
frequency is significantly lower than the 50% (p <
0.005) expected if all adipocytes within the lesion con-
tained the mutation, suggesting that lipomatous over-
growth can result from paracrine signaling from mutant
to wild-type cells. The low mutant allelic frequency we
observed in other tissues is also consistent with the
hypothesis that several CLOVES malformations are the
result of paracrine signaling.
Our findings add CLOVES to a growing list of over-
growth syndromes that result from somatic activation of
the PI3K-AKT pathway. Individuals with PTEN loss-of-
function mutations (somatic and germline) have asym-
metric soft-tissue overgrowth in association with vascular
Missense mutations in each of the AKT
Table 2. Summary of PIK3CA Mutations in Participants with CLOVES Syndrome
Mutation Lesional Tissue
Pathologic Components
Mutant Allele Frequency
DNA Sequence
Genomic DNA
PCR Subclones
CL1 c.3140A>G
resected truncal
yes FFPE 5/24 (21%) 8/49 (16%) NA NA
CL2 c.3140A>G
debulked tissue
from both feet
yes FFPE 13/64 (20%) 16/70 (23%) NA NA
saliva 0/19 (0%) 0/69 (0%)
CL3 c.1624G>A
resected truncal
yes LM frozen 1/13 (8%) 4/48 (8%) 0/80 (0%)
CL4 c.1624G>A
resected buttock
and thigh mass
yes combined LM
and VM
frozen 2/16 (13%) 3/48 (6%) 5/48 (10%)
blood 0/18 (0%) 0/40 (0%)
CL5 c.1258T>C
resected truncal
yes combined AVM fresh 8/44 (18%) 2/70 (3%) 16/62 (26%)
blood NA NA 0/68 (0%)
CL6 c.1258T>C
left-leg amputation
below the knee
yes combined LM
and VM
fresh 12/40 (30%) 8/70 (11%) 23/65 (35%)
saliva 0/55 (0%) 0/70 (0%)
The following abbreviations are used: FFPE, formalin-fixed paraffin-embedded; NA, not available; LM, lymphatic malformation; VM, venous malformation; and
AVM, arteriovenous malformation.
cDNA prepared from the same RNA stock that was used for massively parallel RNA sequencing.
The American Journal of Human Genetics 90, 1108–1115, June 8, 2012 1111
family members have been reported in individuals with
Proteus syndrome (AKT1), individuals with asymmetric
overgrowth and hypoglycemia (AKT2), and an individual
with hemimegalencephaly (AKT3).
The nonoverlap-
ping phenotypes associated with the different mutations
might reflect the stage of development at which the
genetic alterations arose and the restricted ability of
a cell type or tissue to be affected by the consequences.
In a PIK3CA activating mutation, it is reasonable to spec-
ulate that differing phenotypes in the CLOVES spectrum
might occur depending upon when the mutation arises
during development; such is the case for the broad range
of phenotypes that occur in individuals with somatic
mutations in GNAS and PTEN.
Similar to CLOVES
syndrome, Klippel-Trenaunay syndrome (KTS [MIM
149000]) exhibits features such as capillary, lymphatic,
and venous anomalies with overgrowth.
KTS might also result from mutations in components of
the PI3K-AKT pathway. We have begun testing this
hypothesis by screening DNA extracted from lesional
tissue in individuals with KTS for the c.1258T>C and
the c.3140A>GPIK3CA missense mutations, and we
have found mosaicism for c.3140A>G in 3 of 15 individ-
uals examined (Figure S2).
Massively parallel sequencing of DNA or RNA recov-
ered from anomalous tissue can facilitate the identifica-
tion of a somatically arising mutation that is responsible
for the disorder. We employed this strategy to identify
activating mutations in PIK3CA as the cause of CLOVES
syndrome. These same activating mutations have been
detected in several types of cancer, and pharmacologic
inhibitors of PIK3CA are being developed for the
Figure 2. Somatic Activating PIK3CA Mutations in CLOVES Syndrome
(A) Demonstration of the PIK3CA c.1624G>A somatic mosaic mutation in participant CL4. Massively parallel sequencing of this indi-
vidual’s lesional tissue identified a c.1624G>A mutation in 2 of 16 reads (Table 2). With blood DNA and lesional-tissue DNA as templates,
PCR amplimers encompassing the candidate mutation were generated and Sanger sequenced. On the top left is an electropherogram of
blood-DNA amplimers showing only a wild-type sequence. On the top right is an electropherogram of lesional-tissue amplimers
showing wild-type and mutant sequences. Blood and lesional-tissue amplimers were subcloned, and 48 individual colonies were
sequenced. In the middle on the left, all subclones from blood-DNA amplimers contain a wild-type sequence; a representative electro-
pherogram of a wild-type sequence from a single clone is shown. In the middle on the right, 3 of 48 subclones from lesional-DNA
amplimers contain a mutant sequence; a representative electropherogram of a mutant sequence from a single clone is shown. With
mRNA recovered from lesional tissue as a template, RT-PCR was performed with primers in exons flanking the exon containing the
candidate mutation. On the bottom left, a Sanger-sequence electropherogram of the RT-PCR amplimers shows wild-type and mutant
sequences. Five of the 48 subclones from the RT-PCR amplimers contain a mutant sequence, one of which is shown at the bottom right.
(B) A schematic of the PI3K-AKT signaling pathway indicates the overgrowth syndromes currently associated with mutations in this
pathway. Binding of a growth factor to a receptor-tyrosine kinase activates a PI3K family member, including PIK3CA, which converts
phosphatidylinositol-3,4-bisphosphate (PIP2) to the 3,4,5-triphosphate (PIP3) in a reaction that is antagonized by PTEN.
Membrane-associated PIP3 facilitates the localization and phosphorylation of PDK1, which then activates AKT by phosphorylation
at Thr308. AKT is further activated by Ser742 phosphorylation by the PDK2 complex including mTOR (FRAP1 [MIM 601231]). The
following abbreviation is used: PTEN-HTS, PTEN Hamartoma Tumor Syndrome.
(C) PIK3CA mutations constitutively activate the PI3K-AKT pathway. In the first column are immunoblot luminescence images of
protein lysates prepared from normal subcutaneous adipose tissue. In the second column is hamartomatous tissue from an individual
with a known PTEN mutation. In the right columns is lipomatous tissue from CLOVES-affected participants CL5 and CL6 (the blank lane
between samples was removed). Lysates were separated by 4%–8% SDS-PAGE, transferred to immobilon P, and immunodetected with
antibodies (Cell Signaling, Cambridge, MA, USA) that recognize total AKT, phosphorylated forms of PDK1 and AKT1, and beta-actin
(as a loading control). Compared to lysates from adipose tissue from an unaffected individual or lysates from lesional tissue from an indi-
vidual with a heterozygous PTEN mutation, lysates from the CLOVES lipomatous tissue show marked increases in the activated forms of
PDK1 and AKT1.
1112 The American Journal of Human Genetics 90, 1108–1115, June 8, 2012
treatment of these tumors.
These same inhibitors
might have therapeutic applications for individuals
with CLOVES syndrome or other overgrowth anomalies
that are also the result of somatic activating mutations
in PIK3CA.
Supplemental Data
Supplemental Data include two figures and three tables and can be
found with this article online at
We are grateful for the active support of the CLOVES
syndrome community ( and www. and to the individuals and families that
participated in this study. We also acknowledge Cameron Trenor
III, Arin Greene, Joseph Upton III, Rebecca Fevurly, Denise Adams,
and all of the members of the Vascular Anomalies Center, Boston
Children’s Hospital for their dedication to the care of individuals
with CLOVES and other vascular problems. We thank Mr. Ryan
Neff for providing computation assistance, and we thank Joseph
Figure 3. Detection of PIK3CA Somatic Mosaicism in Multiple Tissue Types
(A) At 18 years of age, participant CL6 shows overgrowth of the lower limbs, right-foot polydactyly, and lymphatic and venous anom-
alies of the lower-left extremity.
(B) The participant’s resected lower limb. The scale bar represents 10 cm.
(C) The participant’s radiograph (after venous contrast injection) demonstrates a dilated and aberrant venous system.
(D–M) Resection specimen and photomicrographs of hematoxylin- and eosin-stained sections from sampled areas used for DNA isola-
tion. The locations of tissue sampling are indicated by boxes. The scale bar indicates 200 mm.
(D) A transverse section at the base of the calf shows massive lipomatous overgrowth involving subcutaneous tissue and skeletal muscle
(I) and obliteration of tissue planes. The stranded appearance of subcutaneous tissue is due to extensive lymphatic malformation (H).
The asterisk shows an abnormally dilated vein that was dissected (E) and sampled (J).
(F) A metatarsal-phalangeal joint with degenerative articular changes (top) and synovial expansion by fat and venous malformation.
Also shown are sampled sections of the total joint (K), synovium with fat and venous malformation (L), and articular cartilage and
bone (M).
(G) A transmetatarsal section shows extensive involvement of lipomatous overgrowth and lymphatic malformation.
(N) A tube containing adipocytes (yellow layer) freshly isolated after collagenase treatment by centrifugation.
DNA was amplified from each of the aforementioned tissues. Amplimers were subcloned, and the frequency of mutant alleles was deter-
mined. The numbers and percentages of mutant alleles are listed below the corresponding tissue samples.
The American Journal of Human Genetics 90, 1108–1115, June 8, 2012 1113
Gleeson and colleagues, who shared information about similar
findings in individuals with hemimegalencephaly. This work was
funded in part by the Manton Center for Orphan Disease Research
(pilot grant 94824-01 to K.C.K.) and the Stuart and Jane Weitzman
Family Vascular Anomalies Fund at Boston Children’s Hospital,
the National Institutes of Health-National Institute of Arthritis
and Musculoskeletal and Skin Diseases grant AR053237, and the
Howard Hughes Medical Institute.
Received: April 30, 2012
Revised: May 11, 2012
Accepted: May 15, 2012
Published online: May 31, 2012
Web Resources
The URLs for data presented herein are as follows:
1000 Genomes,
dBSNP Build 132,
Integrative Genomics Viewer,
NHLBI Exome Variant Server,
Online Mendelian Inheritance in Man (OMIM), http://www.
RNA-Seq Unified Mapper (RUM),
University of California-Santa Cruz Genome Bioinformatics,
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    • "The most common mutation is a postzygotic change affecting amino acid 1047, with p. His1047Arg in 19, and p.His1047Leu in eight of 35 individuals reviewed by Keppler-Noreuil et al. [2014] (Table II). While the CLOVES syndrome or fibroadipose hyperplasia phenotype appears to be common in individuals with a missense mutation at codon 1047, individuals with a clinical diagnosis of Klippel–Trenaunay syndrome, or isolated lymphatic malforma- tion [Kurek et al., 2012; Luks et al., 2015] also had a mutation affecting this amino acid. Keppler-Noreuil et al. [2014] differentiated between mutations at p.His1047, which affect the catalytic domain of the protein product, and multiple other changes in the coiled domain, and concluded that the majority of patients with CLOVES syndrome had mutations in the latter. "
    [Show abstract] [Hide abstract] ABSTRACT: Wilms tumor and nephroblastomatosis are associated with syndromic conditions including hemihyperplasia. Hemihyperplasia is genetically heterogeneous and may be the result of genomic abnormalities seen in Beckwith-Wiedemann syndrome, mosaic chromosome or genomic abnormalities, or somatic point mutations. Somatic missense mutations affecting the PI3K-AKT-MTOR pathway result in segmental overgrowth and are present in numerous benign and malignant tumors. Here, we report a fourth patient with asymmetric overgrowth due to a somatic PIK3CA mutation who had nephroblastomatosis or Wilms tumor. Similar to two of three reported patients with a somatic PIK3CA mutation and renal tumors, he shared a PIK3CA mutation affecting codon 1047, presented at birth with asymmetric overgrowth, and had fibroadipose overgrowth. Codon 1047 is most commonly affected by somatic mutations in PIK3CA-related overgrowth spectrum (PROS). While the fibroadipose overgrowth phenotype appears to be common in individuals with PIK3CA mutations at codon 1047, individuals with a clinical diagnosis of Klippel-Trenaunay syndrome or isolated lymphatic malformation also had mutations affecting this amino acid. Screening for Wilms tumor in individuals with PROS-related hemihyperplasia may be considered and, until the natural history is fully elucidated in larger cohort studies, may follow guidelines for Beckwith-Wiedemann syndrome, or isolated hemihyperplasia. It is not known if the specific PIK3CA mutation, the mosaic distribution, or the clinical presentation affect the Wilms tumor or nephroblastomatosis risk in individuals with PROS. © 2016 Wiley Periodicals, Inc.
    Article · May 2016
    • "In addition to macrodactyly, both patients developed lipomatous overgrowth of the trunk and the limbs and vascular anomalies including lymphatic, capillary and venous malformations . The affected female patient also had a hypoplastic right kidney (Kurek et al., 2012 ). A subsequent study reported the prenatal diagnosis of CLOVES syndrome in a 27-week-old fetus carrying the same somatic mutation of the PIK3CA gene. "
    [Show abstract] [Hide abstract] ABSTRACT: Isolated macrodactyly (OMIM 155500) belongs to a heterogeneous group of overgrowth syndromes. It is a congenital anomaly resulting in enlargement of all tissues localized to the terminal portions of a limb and caused by somatic mutations in the phosphatidylinositol 3-kinase catalytic alpha (PIK3CA, OMIM 171834) gene. Here we report a Hungarian girl with macrodactyly and syndactyly. Genetic screening at hotspots in the PIK3CA gene identified a mosaic mutation (c.1624G>A, p.Glu542Lys) in the affected tissue, but not in the peripheral blood. To date, this somatic mutation has been reported in eight patients affected by different forms of segmental overgrowth syndromes. Detailed analysis of the Hungarian child and previously reported cases suggests high phenotypic diversity associated with the p.Glu542Lys somatic mutation. The identification of the mutation provides a novel therapeutic modality for the affected patients: those who carry somatic mutations in the PIK3CA gene are potential recipients of a novel “repurposing” approach of rapamycin treatment.
    Article · Feb 2016
    • "'Oncogenic' PIK3CA mutations are found across the PIK3CA gene but mainly occur in hotspots and lead to increased lipid binding and/or basal activity of p110α through a multitude of mechanisms that mimic and enhance the dynamic allosteric/ intramolecular events that normally activate wild-type PI3Kα [5]. PIK3CA and PI3K pathway components have recently been found to be somatically mutated in a spectrum of congenital or early-childhood-onset human overgrowth disorders678910. PI3K pathway mutations in these disorders are almost exclusively found in the tissue of mesodermal origin, with overgrowth frequently observed in adipose, muscle and skeletal tissue. "
    [Show abstract] [Hide abstract] ABSTRACT: The PI3K lipid kinases are involved in signal transduction and intracellular vesicular traffic, endowing these enzymes with multiple cellular functions and important roles in normal physiology and disease. In this mini-review, we aim to distill from the vast PI3K literature the key relevant concepts for successful targeting of this pathway in disease. Of the eight isoforms of PI3K, the class I PI3Ks have been implicated in the aetiology and maintenance of various diseases, most prominently cancer, overgrowth syndromes, inflammation and autoimmunity, with emerging potential roles in metabolic and cardiovascular disorders. The development of class I PI3K inhibitors, mainly for use in cancer and inflammation, is a very active area of drug development. In 2014, an inhibitor of the p110δ isoform of PI3K was approved for the treatment of specific human B cell malignancies. The key therapeutic indications of targeting each class I PI3K isoform are summarized and discussed.
    Article · Dec 2015
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