NEW SYNDROME

Overgrowth With Increased Proliferation of Fibroblastand Matrix Metalloproteinase Activity Related toReduced TIMP1: A Newly Recognized Syndrome?Brian Chung,1,2 Aleksander Hinek,3 Sarah Keating,4 Rosanna Weksberg,2 Vibhuti Shah,5 Susan Blaser,6

Cynthia Hawkins,7 and David Chitayat1,2*1The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto,

Toronto, Ontario, Canada2Division of Clinical and Metabolic Genetics, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto,

Ontario, Canada3Division of Cardiovascular Research, The Hospital for Sick Children, University of Toronto, Toronto, Canada4Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada5Department of Pediatrics, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada6Division of Paediatric Neuroradiology, Department of Diagnostic Radiology, The Hospital for Sick Children, University of Toronto, Toronto,Ontario, Canada7Division of Pathology and Laboratory Medicine, Department of Pediatrics, The Hospital for Sick Children; University of Toronto, Toronto,

Ontario, Canada

Manuscript Received: 22 June 2011; Manuscript Accepted: 17 June 2012

We report on a child with prenatal onset of overgrowth associ-

ated with thick, excessive wrinkled skin and other abnormalities

including cleft palate, Chiari malformation and polymicrogyria.

His clinical features do not resemble any of the known reported

overgrowth syndromes. Genetic evaluations, including karyotype,

oligoarray, methylation-sensitive multiplex ligation-dependent

probe amplification (MLPA) for 11p11.2 region, CDKN1C se-

quencing, GPC3 sequencing and dosage analysis, and HRAS

sequencing, have been un-revealing. Immunohistochemistry

done on the patient’s cultured skin fibroblasts showed normally

assembled elastic fibers and normal pattern of chondroitin

sulfate deposition with defective deposition of Collagen I fibers.

In addition, there were high levels of immuno-detectable metal-

loproteinase 3 (MMP3) and undetectable tissue inhibitor of

metalloproteinase 1 (TIMP1). The defective collagen deposition

in the fibroblast culture could be reversed by the broad spectrum

MMP inhibitor, doxycycline. We also present evidence that the

fibroblasts of this patient have an increased rate of cellular

proliferation. We propose that this is a previously unrecognized

overgrowth syndrome associated with increased cellular prolif-

eration and defective collagen I deposition due to an imbalance

between MMP and TIMP in fibroblasts. � 2012Wiley Periodicals, Inc.

Key words: overgrowth;metalloproteinase (MMP); tissue inhib-

itors of metalloproteinase (TIMP)

INTRODUCTION

Overgrowth syndromes are a heterogeneous group of conditions

characterized by either localized or generalized excessive growth for

age and sex. Individuals with overgrowth syndromes may be large

at birth or have excessive postnatal growth with some combination

of increased weight, length and head circumference [Cytrynbaum

et al., 2005]. Many overgrowth syndromes are associated with

various malformations and some with cognitive impairment and

increased susceptibility to neoplasia [Cohen, 1999]. The molecular

causes of a handful of overgrowth syndromes have been elucidated,

although most continue to be diagnosed on the basis of the clinical

criteria. Diagnostic categorization of these conditions have been

*Correspondence to:

David Chitayat, Department of Obstetrics and Gynaecology, The Prenatal

Diagnosis and Medical Genetics Program, The Ontario Power Generation

Building, 700 University Avenue, Room 3292, Toronto, Ontario, Canada

M5G 1Z5. E-mail: dchitayat@mtsinai.on.ca

Article first published online in Wiley Online Library

(wileyonlinelibrary.com):

DOI 10.1002/ajmg.a.35570

How to Cite this Article:Chung B, Hinek A, Keating S, Weksberg R,

Shah V, Blaser S, Hawkins C, Chitayat D.

2012. Overgrowth with increased

proliferation of fibroblast and matrix

metalloproteinase activity related to reduced

TIMP1: A newly recognized syndrome?

Am J Med Genet Part A.

� 2012 Wiley Periodicals, Inc. 1

challenging because many have overlapping clinical and molecular

features [Baujat et al., 2005] and yet, there are still a significant

proportion of patients with overgrowth of an unknown etiology.

Overgrowth disorders of prenatal onset include Beckwith–Wiedemann syndrome (BWS), Simpson–Golabi–Behmel (SGB),

Perlman syndrome, Sotos syndrome,Weaver syndrome, and other

rare conditions [Cohen et al., 2002]. Prenatal diagnosis of over-

growth syndromes is challenging and the suggested algorithms

often rely on the specificultrasonographicfindingsof abnormalities

and molecular diagnosis [Vora and Bianchi, 2009]. We report on a

patient presenting with fetal macrosomia and multiple congenital

anomalies. In particular, he had coarse facial features, thick, loose,

and wrinkled skin and brain dysgenesis. Detailed clinical and

molecular assessment has made various common recognizable

overgrowth syndromes unlikely. Using immunohistochemical

techniques, we assessed the cellular proliferation and the content

of the extracellular matrix components of the cultured skin fibro-

blasts fromourpatient and foundan increased cellular proliferation

and a peculiar lack of collagen type I fibers that most likely resulted

from a primary imbalance between metalloproteinase (MMP) and

tissue inhibitor of metalloproteinase (TIMP) in fibroblasts.

MATERIALS AND METHODS

Clinical ReportOur patient was born to a 27-year-old primigravida mother of

Dutch/Welsh/Scottish/Irish descent and a 29-year-old father of

Maltese/Finnish descent. The couple was healthy and nonconsan-

guineous. Our patient was the product of the couple’s first

pregnancy. On review of family history, there was no history of

congenital malformations, intellectual disability, recurrent mis-

carriages, or macrosomia. There was no history of exposure to

teratogens.

The pregnancy was conceived naturally and was initially un-

complicated. Fetal ultrasound showed the presence of bilateral

pleural effusion at 18 weeks gestation. Fetal measurements at

19 weeks gestation were consistent with 23–24 weeks gestation

indicating an early fetal overgrowth. Fetal echocardiography was

normal and repeated ultrasound at 32 weeks detected severe poly-

hydramnios. Amnio-reduction, and subsequently thoraco-amni-

otic shunt insertion,wasperformed.Chromosomeanalysis doneon

amniotic fluid showed a normal male karyotype (46, XY). TORCH

analysis showed no evidence of an acute infection.

Delivery was by elective cesarean at 36 weeks gestation for large

fetal size and thickmeconium stained liquor. He was born cyanotic

and required ventilatory support. Nitric oxide was required due

to pulmonary hypertension. His birth weight was 4,640 g, length

56.5 cm, and head circumference 38.5 cm (all above 97th centile).

There was a wide-open anterior fontanelle, a third fontanelle and

a large posterior fontanelle. The facial features were coarse and

dysmorphic with a prominent and high wrinkled forehead, hyper-

telorism, broad nasal bridge and sagging cheeks (Fig. 1a and b).

There were full lips and a U-shaped cleft of the secondary and soft

palates. There was a short neck with redundant nuchal skin but no

FIG. 1. a and b: Clinical photograph of the patient showing coarse facial features, high and wrinkled forehead, frontal bossing, depressed nasal bridge,

long philtrum with prominent pillars, and full lips. c: 3D CT surface reconstruction demonstrates marked redundancy of the scalp.

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

webbing (Fig. 1c). The toenails were hypoplastic and there was

clinodactyly of the fifth toe bilaterally. His skin was loose and thick

and neurological examination revealed generalized hypotonia with

poor suck and neonatal reflexes could not be elicited. His external

genitalia were normal.

Feeding difficulties were noted with recurrent aspirations and a

G-tube was inserted later. His chest X-ray showed hypoinflation

with multifocal zones of segmental opacities consistent with atel-

ectasis and bilateral pleural effusion. Assessment by an otolaryng-

ologists showed laryngomalacia. BrainMRI (Fig. 2) at 13 days of life

showed extensive cortical dysplasia, with polygyria involving both

Sylvian fissures. CT venogram showed thrombosis of the left

transverse sinus and the inferior aspect of the superior sagittal

sinus. Therewas lowproteinCandanti-thrombin III initiallywhich

later became normal. He was commenced on subcutaneous enox-

aparin and subsequentCT scan showed resolution of the thrombus.

The ABR and VEP were abnormal and the electroencephalogram

(EEG) was normal. Echocardiogram showed dysplastic mitral

and tricuspid valves. Abdominal ultrasound showed slightly echo-

genic kidneys which measured 5.7 cm (right) [þ1 SD] and 6.1 cm

(left) [þ2 SD] in length at 4 months of age. Liver, gallbladder,

pancreas, and spleen were unremarkable.

At 3–4months of life, he was admitted for respiratory arrest and

emergency CT scan of the brain showed enlarged ventricles with

Chiari 1 malformation. He had posterior fossa craniectomy with

duraplasty and decompression of the right cerebellar tonsil and

bilateral tonsillopexies. At 9 months of life, he had sudden deteriora-

tion presenting with septic shock, acute renal failure, fluid overload,

and disseminated intravascular coagulation. He succumbed 4 days

after admission. The autopsy revealed multiple congenital anomalies

including macrosomia, facial dysmorphism, cleft palate, plagioce-

phaly, dysplastic atrioventricular valves, Chiari 1 malformation,

polymicrogyria, and bilateral pulmonary hypoplasia. There was

no evidence of nephroblastomatosis, or islet cell hyperplasia.

FIG. 2. T1 weighted sagittal MRI in the neonatal period (a) demonstrates macrocrania and crowding of the cerebellar tonsils at the foramen magnum

(arrow). Thrombosis of the torcular heropholi is also shown. Follow-up T2weighted sagittal MRI at 3months of age (b) reveals ventriculomegaly and

Chiari 1 malformation with protrusion of the cerebellar tonsils through the foramen magnum (arrow). T2 weighted axial image at 12 days of age (c)

shows numerous small gyri, polygyria, along the anterior Sylvian fissures (curved arrow). Parasagittal T2 weighted MRI at 3 months of age (d)

confirms frontal lobe polygyria (curved arrow).

CHUNG ET AL. 3

His postnatal chromosomal analysis, including interphase

FISH for trisomy 8 mosaicism, oligoarray analysis (Signature

Genomics�) using DNA extracted from blood and skin fibroblasts

were normal and male. Subsequent DNA analysis for the HRAS,

PTPN11, KRAS, and SOS1 genes associated with Noonan syn-

drome spectrum disorder showed no detectable mutation. Gene

dosage analysis and direct sequencing of the GPC 3 gene for SGB

syndrome was normal. Methylation-sensitive MLPA for BWS and

CDKN1C sequencing showed no detectable abnormalities.

Cell cultures. The fibroblasts were originally grown from the

explants of skin biopsies of our patient and fromanormal boyof the

same age. All primary cultures were routinely trypsinized and

fibroblasts were then plated into multiple secondary cultures

(at 100,000 cells/dish) and maintained for 7 days in Dulbecco’s

modified eagle’s medium supplemented with 10% fetal bovine

serum (FBS). The parallel cultures were also maintained in the

presence and absence of 10mg/ml doxycycline (Sigma, St

Louis, MO).

Assays of fibroblast proliferation. Cells present in 7-day-old

cultures from both groups were routinely trypsinized and counted

in a hemocytometer. Three aliquots from each culture were also

used for an assay of totalDNA, utilizing theDNeasyTissue Kit from

QIAGEN according to the manufacturer’s instructions. Results

from four parallel cultures derived from our patient and from

normal control skin were averaged.

The parallel cultures were maintained in the presence of 1mCi of[3H]-thymidine/ml for the last 72 hr. The level of radioactive

thymidine ultimately incorporated to DNA was then assessed in

each 7-day-old cultures as previously described [Hinek et al., 2008].

Results from four cultures derived from our patient and from

normal control skin were averaged.

Immunostaining. On day 7, all cultures were either fixed for

30min in cold 100% methanol at �20�C (for elastin, chondroitin

sulfate, TIMP1, andMMP3 detection) or in 4% paraformaldehyde

at room temperature (for collagen detection). Parallel cultures,

were then incubated for 1 hr, with either 10 mg/ml polyclonal

antibody to tropoelastin (Elastin Products, Owensville, MO), or

5mg/ml or monoclonal antibody to chondroitin sulfate (Sigma),

or 10mg/ml or polyclonal antibody to collagen type I (Chemicon,

Billerica, MA), or 10mg/ml monoclonal antibody to MMP3

(Oncogene Research Products, Boston, MA), or monoclonal anti-

body to TIMP1 (Sigma). The appropriate fluorescein-conjugated

secondary antibodies (Sigma)wereused todetect immunoreaction.

Nuclei were counter stained with propidium iodide (Sigma).

Secondary antibodies alone were also used as an additional control.

All cultures were examinedwith aNikon Eclipse E1000microscope

attached to a cooled charge-coupled device camera (Retiga EX;

QImaging, Surrey, BC, Canada) and a computer-generated video

analysis system (Image-Pro Plus software; Media Cybernetics,

Silver Spring, MD).

RESULTS

The initial comparison of cell number, total DNA content, and the

radioactive thymidine incorporation rates indicated that cultured

fibroblasts derived from the skin biopsy of our patient proliferate

significantly faster than control counterparts (Fig. 3). Further

analysis of immuno-histochemical profiles of secondary cultures

of dermal fibroblasts derived from normal skin and from our

patient’s skin biopsies revealed no abnormalities in production

of elastic fibers or abnormal accumulation of chondroitin sulfate-

bearing moieties (Fig. 3). Interestingly, the arrangement of the

immuno-detected chondroitin sulfate-bearing moieties would

suggest a slightly lower content of this component in the newly

produced extracellular matrix than in cultures of normal fibro-

blasts. Because results of the additional morphometric analysis of

three different cultures (50 fields per culture) did not revealed

significant differences between the total content of chondroitin

sulfate/culture, we conclude that this is only due to the fact that our

patient fibroblasts proliferated faster than normal control counter-

parts and often formed clusters containing less chondroitin sulfate

deposited between individual cells.

However, in contrast to cultures of normal fibroblasts, cultures

of our patient fibroblasts did not display any presence of immuno-

detectable inhibitor of matrix metalloproteinases (TIMP1). More-

over, our patient-derived cultures demonstrated increased levels of

FIG. 3. Results of three parallel assays (cell counting, total DNA

content, and incorporation of [3H]-thymidine) indicate that the

patient-derived fibroblasts proliferated faster than their normal

counterparts, when cultured for 7 days in medium containing 10%

FBS.

4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

immuno-detectable matrix metalloproteinase 3 (MMP 3) These

findings were associated with the peculiar lack of collagen type I

fibers that were detected in cultures of normal skin fibroblasts.

Importantly, such a lack of effective collagen deposition was

reversed after in vitro treatment with broad spectrum MMP’s

inhibitor, doxycycline (Fig. 4).

DISCUSSION

Our proband presented prenatally with fetal macrosomia, poly-

hydramnios and pleural effusions without the major malforma-

tions specific for Perlman syndrome [Alessandri et al., 2008], BWS

[Le Caignec et al., 2004;Williams et al., 2005], SGB [Li et al., 2001],

Sotos [Chen et al., 2002; Thomas and Lemire, 2008], and other

overgrowth syndromes of prenatal onset (Table I). The absence

of nephromegaly, nephroblastomatosis, and islet hyperplasia in

postmortem made the diagnosis of Perlman syndrome unlikely,

whereas subsequent molecular testings have also made the diagno-

sis of BWS and SGB unlikely. Our patient has very characteristic

thick, wrinkled and excessive skin, and deep palmer creases.

Together with the fetal macrosomia, polyhydramnios, coarse

face, thick lips and the dysplastic atrioventricular valves, another

important differential diagnosis we considered was Costello

syndrome. The natural history of Costello syndrome is character-

istically biphasic, with the first phase often marked with polyhy-

dramnios and fetal macrosomia [Lin et al., 2009; Smith et al., 2009]

and followed by a second phase of failure to thrive, short stature,

mental retardation, distinctive craniofacial appearance, and

dermatologic findings. However, no HRAS mutations could be

identified in our patient [Johnson et al., 1998]. We extended our

molecular testing to PTPN11, KRAS, and SOS1 genes because of

the phenotypic overlap of Costello with Noonan and Cardio-

facio-cutaneous syndrome and these were negative as well.

Karyotype and microarray analysis was normal and ruled out

overgrowth syndrome associated with chromosomal rearrange-

ments, for example, trisomy 8 mosaicism, 15q trisomy/tetrasomy

[Tatton-Brown et al., 2009], 22qter deletion [Baujat et al., 2005],

and tetrasomy 12p mosaicism (Pallister–Killian syndrome)

[Theisen et al., 2009].

To further characterize the phenotype of our patient, we per-

formed additional investigations on his cultured skin fibroblasts.

First, we looked at the proliferation of his fibroblasts and this

was found to be increased compared with normal control. Over-

growth syndromes can result from an increased number of cells

(hyperplasia), cellular hypertrophy, or a combination of both.

Fibroblast proliferation seems to be useful in classification of

overgrowth syndromes according to the underlying mechanism

of overgrowth. In BWS or Weaver syndromes, excessive cellular

proliferation can be demonstrated or inferred to have occurred

[Cohen et al., 2002]. Fibroblast growth is also found to be increased

in Elejalde syndrome [Elejalde et al., 1977] and Costello syndrome

[Hinek et al., 2000] but not in Perlman syndrome [Neri et al.,

1985].

To understand the mechanism behind the findings on skin

fibroblast culture in our patient, his fibroblasts were examined

for their ability to produce and maintain the extracellular matrix.

Immuno-staining techniques have been used in the delineation of

cardiovascular and dermatologic phenotypes in various genetic

conditions including Costello syndrome [Hinek et al., 2000], Cutis

Marmorata teleangiectasia congenital [Hinek et al., 2008], and

Loeys–Dietz syndrome [Barnett et al., 2008]. Meaningfully, in

contrast to Costello syndrome-derived cells, the heightened pro-

liferation of our patient fibroblasts was not associated with a

decrease in the net deposition of elastic fibers or in abnormal

accumulation of chondroitin sulfate-bearing moieties which we

previously described and mechanistically linked with the height-

ened proliferation of fibroblasts derived from Costello syndrome

patients with H-Ras mutation which was characterized by the

overactive Ras-Raf-Erk signaling pathway [Hinek et al., 2000].

The increased proliferation rates detected in our patient’s fibro-

blasts were based on three different methods. In addition to the

assay of total DNA levels, we also measured incorporation of

radioactive thymidine and a cell counting in all analyzed cultures.

By obtaining the same results by using all these methods, we could

eliminated the possibility of heightenedpolyploidyor persisted lack

of apoptosis which would have given us a false positive results,

indicating faster proliferation rate, if only one assay has been used.

Culturesof these rapidly proliferatingfibroblasts demonstrated a

peculiar deficiency in the net content of collagen type I. Since this

phenomenon was associated with a higher than normal accumula-

tion of immuno-detectable MMP3 and a parallel lack of the cell-

associatedTIMP1,we speculate that the detected impairment in the

ultimate content of collagen I fibers may be due to its accelerated

degradation of collagen I precursors, likely caused by the imbalance

between levels of MMPs (increased) and their natural inhibitor

TIMP1 (decreased) in our proband. Type I collagen is by far the

most abundant protein in human skin,making up greater than 90%

of its dry weight.We therefore believe that the reduction of collagen

I may explain the dermatologic features of our proband. Further-

more, comparison of our present findings with the previously

published immunochemical characteristics of cultured dermal

fibroblasts from cases with Costello syndrome [Hinek et al.,

2000] allowed exclusion of Costello syndrome from the differential

diagnosis of our patient. Importantly, our report clearly demon-

strates that similar phenotypic findings (coarse facial features,

loose wrinkled skin, and deep palmer creases) can be produced

by different primary mechanisms.

MMPs are a group of structurally related proteinases which are

important mediators for the turnover of extracellular matrix and

cell signaling. At least 23 MMPs have been identified in human.

With thedifference in their substrate specificity, theyplay their roles

in normal development, for example, angiogenesis, skeletogenesis,

and wound healing [Page-McCaw et al., 2007] and in pathological

process such as joint arthritis [Brinckerhoff, 1991] and tumor

invasion [Egeblad and Werb, 2002]. To ensure homeostasis in

the extracellular matrix, MMP activities are tightly regulated at

multiple levels. The existence of specific endogenous TIMP is an

important measure to ensure coordination of MMP-mediated

processes. TIMPs cannot only inhibit MMPs but is also capable

of inhibiting members of the adamalysin family with the TIMP3

inhibiting the ADAM10, ADAM 12S, and ADAM 17 [Woessner,

2001]. In our experiment, defective collagen deposition was re-

versed after in vitro treatment with a broad spectrum MMP’s

inhibitor, doxycycline, showing that the MMP is at least one of

CHUNG ET AL. 5

FIG. 4. Representativemicrographs illustrating net deposition of indicated components of extracellularmatrix andmatrix-related compounds in7-day-

old cultures of normal and patients-derived skin fibroblasts. Both cultures of normal fibroblasts and patient-derived fibroblasts demonstrate

normally assembled elastic fibers and normal pattern of chondroitin sulfate deposition. In contrast to normal fibroblasts, our patient fibroblasts do

not reveal immuno-detectable TIMP1, but demonstrate heightened levels of immuno-detectable MMP 3. These features associate with the peculiar

lack of collagen type I fibers that could be detected only in cultures of normal skin fibroblasts. The parallel cultures of patient-derived fibroblasts that

were maintained in the presence of doxycycline demonstrate normal deposition of collagen fibers (magnification �6,000).

6 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

TABLE

I.Comparisonof

Our

ProbandWithCostello,Perlman,Beckw

ith–Wiedemann,Simpson–Golabi–BehmelandSotosSyndrom

es

Syndrom

esOur

case

Costello

Perlman

Beckw

ith–Wiedemann

Simpson–Golabi–Behmel

Sotossyndrom

ePolyhydram

nios

þCommon

Common

(63%)

Common

Common

Occasional

Fetalmacrosomia

þCommon

Common

(50%)

Common

Common

Common

Fetalnephrom

egaly

––

Common

(80%)

Common

––

Fetalascites

�(Pleuraleffusion)

–Common

(50%)

––

–Neonatalovergrow

thþ

Common

Common

Common

(88%)

Common

Common

Macrocephaly

þCommon

(80%)

Common

(58%)

–Common

Common

Macroglossia

–Common

–Common

(97%)

Common

–Earlobe

grooves

––

–Common

(76%)

Common

–Deep-seteyes

––

Common

––

–Flat/broad

nasalbridge

þCommon

Common

–Common

–Lips

Thicklips

Thicklips

Evertedupperlip

––

–Cleftlip/palate

þ(Cleftpalate)

––

Occasional

Occasional

Occasional

Midlinefacialgroove

––

––

Common

–Macroglossia

–Common

–Common

(97%)

Common

–Earlobe

grooves

––

–Common

(76%)

Common

–Deep-seteyes

––

Common

––

–Flat/broad

nasalbridge

þCommon

Common

–Common

–Diaphragm

atichernia

––

–Occasional

Occasional

–Supranum

erarynipples

–Occasional

––

Common

–CVSabnormality

Dysplastic

AVvalves

Common

(60–80%)

Uncommon

Uncommon

(6.5–20%)

Common

(35–50%)

Uncommon

(6.5%)

Abdominalwalldefect

––

Abdominaldistension

(46%)

Omphalocele/

umbilicalhernia(80%)

Umbilicalhernia

Occasional

umbilicalhernia

Hepatom

egaly

þ–

Common

(76%)

Common

Common

–Isletcellhyperplasia

––

Common

(71%)

Common

Occasional

–Nephroblastom

atosis

––

Common

(77%)

Occasional

Uncommon

–Cryptochidism

–Occasional

Common

(75%)

–Common

–Polydactyly

––

–Uncommon

Common

–Loosewrinkleskin

þCommon

(>90%)

––

––

Deeppalmer

creases

þCommon

(100%)

––

––

Neonatalandinfantmortality

9Months

?Common

(57%)

Uncommon

(�20%)

Common

(50%)

?

Inform

ationandfrequency

(indicatedinbracket)from

Alessandriet

al.[2008],Baujatet

al.[2005],Cohenet

al.[2002],Cytrynbaum

etal.[2005],Hennekam

[2003],Liet

al.[2001],Linet

al.[1999],andTerespolskyet

al.[1995].

Abbreviations:AV,atrioventricular;CVS,cardiovascular;‘‘þ

’’,present;‘‘–’’,absent;‘‘?’’,unknow

nor

noinform

ation.

CHUNG ET AL. 7

the important mechanisms resulting in the defective collagen

content.

One of the major advances in MMP biology has been the

realization that extracellular proteolysis is not simply a mechanism

of destroying structure or information. Instead, MMP can release

growth factors from extracellular matrix and cell surface, activate

latent proteins and generate new bioactive molecules through its

proteolytic action [Page-McCaw et al., 2007]. This may explain the

increase in fibroblast proliferation and the overgrowth phenotype

in our patient. Further research is required to elucidate the under-

lyingmechanism causing the abnormalities detected in our patient.

Lastly, it is worthnoting that our patient had quite significant

pulmonary abnormalities. He developed pulmonary hypertension

at birth requiring nitric oxide therapy and had multiple hospital-

izations due to respiratory problems. Our patient had pulmonary

hypoplasia, some of it most probably related to the prenatal pleural

effusion. However, TIMP3 has been recently found to be important

in lung development and TIMP3 null mice have decreased bron-

chiole branching [Gill et al., 2006] and disrupted alveologenesis

[Gill et al., 2009]. Both of these phenotypes are associated with

increase inMMPactivities and can be rescued bymetalloproteinase

inhibitors.Unfortunately, thepulmonary edema and inflammation

due to sepsis had caused extensive damage to the autopsy lung

specimen and preclude us to perform further investigations to shed

light into the role of TIMP3 in our patient’s lung development.

Confirmation of the causative link between TIMP deficiency and

the described phenotypes is not possible due to the lack of more

exhaustive investigations in the post-mortem examination of this

patient. Further investigations including immunostaining are

needed in patients with various forms of overgrowth syndromes

todelineate thepossible role ofMMPbiology in tissueproliferation.

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