Simpson-Golabi-Behmel Syndrome Research Articles

LipiD-QuanT: a novel method to quantify lipid accumulation in live cells

Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here.

Modulation of triglyceride accumulation in adipocytes by psychopharmacological agents in vitro

Weight gain is a major problem during psychopharmacological treatment. Research has concentrated on the appetite inducing properties and mechanisms of these drugs in the central nervous system. The potential contribution of direct effects of drugs on metabolically relevant peripheral cells such as adipocytes is less well understood.We examined the influence of the antidepressant imipramine, the antipsychotic clozapine, and the mood stabilizer lithium on preadipocytes and adipocytes in vitro, using Simpson-Golabi-Behmel syndrome (SGBS) cells, an established human preadipocyte model. Parameters of cell differentiation and signaling, and cell metabolism were measured.We found significantly increased triglyceride accumulation in adipocytes after supplementation with imipramine and lithium at therapeutic concentrations, compared to non-supplemented control samples. However, gene expression levels of an early marker of adipogenesis, the peroxisome proliferator-activated receptor gamma (PPAR-γ) and a late marker of adipogenesis, the fatty acid binding protein 4 (FABP4), as well as expression of adiponectin (ADIPOQ) did not change significantly in the presence of these psychopharmacological agents.The results suggest a direct influence of imipramine and lithium but not clozapine on fat storage of adipocytes. The underlying mechanisms of fatty acid storage and adipocyte differentiation however remain to be elucidated.

Whole exome sequencing identifies a novel frameshift mutation in GPC3 gene in a patient with overgrowth syndrome

Overgrowth syndromes are a heterogeneous group of diseases characterized by focal or generalized overgrowth. Many of the syndromes have overlapping clinical features and it is difficult to diagnose the condition based on clinical features alone. In the present study we report on a patient with overgrowth syndrome where extensive investigation did not reveal the cause of disease. Finally exome sequencing revealed a novel hemizygous single base pair deletion in exon 8 of GPC3 gene (chrX:132670203delA) resulting in a frameshift and creating a new stop codon at 62 amino acids downstream to codon 564 (c.1692delT; p.Leu565SerfsTer63) of the protein. The mutation was confirmed by Sanger sequencing. The mother was found to be heterozygous for the mutation. This variation is not reported in the 1000 Genomes, Exome Variant Server (EVS), Exome Aggregation Consortium (ExAC) and dbSNP databases and the region is conserved across primates. Exome sequencing was helpful in establishing diagnosis of Simpson–Golabi–Behmel syndrome type 1 (SGBS1) in a patient with unknown overgrowth syndrome.

Fibroblast growth factor-1 inhibits Wnt/β-catenin pathway during adipogenesis

To determine the time course and potential mechanism of fibroblast growth factor-1 (FGF-1) in the regulation of adipogenesis. We cultured human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes with recombinant FGF-1 and harvested cells at various stages prior to and during differentiation; at cell proliferation (D-3), confluence (D0), early (D3), middle (D7) and mature (D14) stages of differentiation. We determined lipid accumulation in mature adipocytes by morphological observation and quantitative measurement of oil red O staining. We also examined the expression of adipogenic genes and related markers involved in the Wnt/β-catenin pathway using quantitative Real-time PCR and Western blot. Compared to control SGBS cells, treatment with FGF-1 increased lipid accumulation; induced a sustained increase in the mRNA for peroxisome proliferater-activated receptor γ (PPARγ), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), adiponectin and glucose transporter type 4 (GLUT4); and promoted a sustained decrease in expression of markers of the Wnt/β-catenin pathway, β-catenin and transcription factor 4 (TCF4). The adipogenic effects of FGF-1 are apparent throughout the whole priming and differentiation period in human SGBS pre-adipocytes. Furthermore, our results suggest that FGF-1 promotes adipogenesis, at least in part, via a sustained decrease in activity of the Wnt/β-catenin pathway.

Angiotensin II directly impairs adipogenic differentiation of human preadipose cells.

Angiotensin II reduces adipogenic differentiation of preadipose cells present in the stroma-vascular fraction of human adipose tissue, which also includes several cell types. Because of the ability of non-adipose lineage cells in the stroma-vascular fraction to respond to angiotensin II, it is not possible to unequivocally ascribe the anti-adipogenic response to a direct effect of this hormone on preadipose cells. Therefore, we used the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain to investigate the consequences of angiotensin II treatment on adipogenic differentiation under serum-free conditions, by assessing expression of typical adipocyte markers perilipin and fatty acid-binding protein 4 (FABP4), at the transcript and protein level. Reverse transcription-polymerase chain reaction showed that perilipin and FABP4 transcripts were, respectively, reduced to 0.33±0.07 (P<0.05) and 0.41±0.19-fold (P<0.05) in SGBS cells induced to adipogenic differentiation in the presence of angiotensin II. Western Blot analysis corroborated reduction of the corresponding proteins to 0.23±0.21 (P<0.01) and 0.46±0.30-fold (P<0.01) the respective controls without angiotensin II. Angiotensin II also impaired morphological changes associated with early adipogenesis. Hence, we demonstrated that angiotensin II is able to directly reduce adipogenic differentiation of SGBS preadipose cells.

Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One-Carbon Cycle Energy Producing Pathway.

Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS) preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate association approach was employed to examine the influence of fructose on the intermediary metabolism of glucose. Increasing concentrations of fructose robustly increased the oxidation of [1,2-13C2]-d-glucose to 13CO2 (p < 0.000001). However, glucose-derived 13CO2 negatively correlated with 13C labeled glutamate, 13C palmitate, and M+1 labeled lactate. These are strong markers of limited tricarboxylic acid (TCA) cycle, fatty acid synthesis, pentose cycle fluxes, substrate turnover and NAD+/NADP+ or ATP production from glucose via complete oxidation, indicating diminished mitochondrial energy metabolism. Contrarily, a positive correlation was observed between glucose-derived 13CO2 formed and 13C oleate and doses of fructose which indicate the elongation and desaturation of palmitate to oleate for storage. Collectively, these results suggest that fructose preferentially drives glucose through serine oxidation glycine cleavage (SOGC pathway) one-carbon cycle for NAD+/NADP+ production that is utilized in fructose-induced lipogenesis and storage in adipocytes.

MFAP5is related to obesity-associated adipose tissue and extracellular matrix remodeling and inflammation

Low-grade inflammation is involved in adipose tissue (AT) and extracellular matrix (ECM) remodeling and induces deposition of ECM proteins in AT. We have previously shown that MFAP5 (microfibrillar-associated protein 5) expression decreases in AT after weight loss. The aim of this study was to investigate MFAP5 localization in human AT and gene expression in adipocytes and the role of MFAP5 in adipocyte metabolism and inflammation. MFAP5 protein localization and gene expression were studied with immunohistochemistry and quantitative reverse transcriptase PCR (RT-qPCR) in human subcutaneous AT and cultured Simpson-Golabi-Behmel syndrome (SGBS) adipocytes, respectively. The effect of MFAP5 knock-down by siRNA on gene expression and insulin action was examined with RT-qPCR, western blot, and insulin-stimulated glucose uptake. The effect of different cytokines on MFAP5 gene and protein expression was investigated in cultured human SGBS preadipocytes. MFAP5 protein was highly expressed in AT, and gene expression decreased during adipocyte differentiation in SGBS cells. Treatment of preadipocytes with TNFα and TGFβ1 increased MFAP5 gene and protein expression. Furthermore, MFAP5 knock-down decreased the expression of genes involved in inflammation. Our results demonstrate that factors involving low-grade inflammation modulate MFAP5 expression and that the modified expression of MFAP5 may further regulate AT inflammation.

Additive regulation of adiponectin expression by the mediterranean diet olive oil components oleic Acid and hydroxytyrosol in human adipocytes.

Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (P<0.05) prevented TNF-α-induced suppression of total adiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression.

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.

Intestinal epithelial cells promote secretion of leptin and adiponectin in adipocytes

Although leptin and adiponectin are the predominant adipokines, how their circulating levels are regulated is incompletely understood. The present study tested whether intestinal epithelial cells influence the expression and secretion of these adipokines by adipocytes. Leptin gene expression and secretion by cultured human primary adipocytes and Simpson–Golabi–Behmel Syndrome adipocytes increased upon coculture with human enterocytic Caco-2 cells or incubation in conditioned medium of Caco-2 cells. Although adiponectin secretion increased, its mRNA levels decreased. Tissue homogenate of the ileum (but not the jejunum, colon, or liver) of nonobese C57BL/6J mice also stimulated leptin and adiponectin secretion by cultured murine 3T3-L1 adipocytes. However, ileal homogenate of obese KK-Ay mice had no effect on leptin and adiponectin secretion. We propose that as yet unidentified humoral factors released from intestinal epithelial cells are involved in regulating circulating leptin and adiponectin levels. Decreased production of such factors may contribute to hyperphagia in KK-Ay mice.

Anesthetic Management of a Child with Simpson-Golabi-Behmel Syndrome—A Case Report

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked recessive condition associated with pre-natal and postnatal overgrowth with visceral and skeletal anomalies. Abnormal airway anatomy may complicate the perioperative management of SGBS. However, there have been few reports regarding the anesthetic management of children with SGBS. We present the preoperative evalua-tion and intraoperative management of a 1.5-year-old child with SGBS undergoing plastic surgery for cleft palate. Sevoflurane-fentanyl anesthesia without a neuromuscular blocker was planned, due to the preoperative evaluation that airway management would be complicated by the macroglossia and short neck characteristic of SGBS. Fortunately, there was no difficulty in endotracheal intubation in the present patient due to careful planning of general anesthesia and meticulous airway management. Appropriate preoperative preparation for a difficult airway by the anesthe-siologist can ameliorate the difficulties inherent to the anesthetic management of patients with SGBS.

Abstract 5303: Uptake and Efflux of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in human adipocytes

Abstract Obesity has been linked to the development of numerous chronic diseases, including breast cancer. Although the association between obesity and breast cancer risk is clear in postmenopausal women, high body mass index (BMI) appears to protect against breast cancer in pre-menopausal women. The mechanistic basis of this “obesity paradox” is unknown. One hypothesis is that abundant adipose tissue in the mammary gland functions as a storage depot for lipid-soluble carcinogens of dietary origin, initially offering protection against exposures. Heterocyclic amines (HCAs) are pro-carcinogens that are formed from high-temperature cooked meats. Exposures to these compounds are ubiquitous in Western diets and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP), the most mass-abundant HCA, is classified as a “probable human carcinogen.” To determine rates of influx of PhIP into adipocytes, differentiated Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes were treated with 5 µM plus 0.02 µCi 14C-PhIP for 0.5, 1, 2, 5, 10, 15, 30, 45, and 60 minutes, and media was removed at time points. Cells were re-suspended in scintillation fluid, and counted with a 1450 LSL &amp; Luminescence Count (Perkin Elmer). Influx was rapid and PhIP continued to accumulate in the cells between 15 and 60 minutes. To determine efflux, SGBS cells were exposed to 5 µM plus 0.02 µCi 14C-PhIP and allowed to equilibrate for 30 minutes. Media was removed and the cells were washed with PBS. Radioactivity retained in SGBS cells was counted at 0.5, 1, 5, 10, 30, and 60 minutes. SGBS cells retained 50% of the radioactivity for more than 10 minutes, compared to other studies where PhIP efflux from pancreatic acinii and hepatocytes occurs within the first 15 seconds. Our data demonstrates that human adipocyte efflux is significantly slower than those in other type of cells reported in the literature, suggesting that human adipocytes can sequester PhIP, thereby protecting the breast from dietary exposure to carcinogens. Citation Format: Aiwei Yao-Borengasser, Lora J. Rogers, Susan A. Kadlubar. Uptake and Efflux of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in human adipocytes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2014-5303

Simpson-Golabi-Behmel syndrome types I and II.

Simpson-Golabi-Behmel syndrome (SGBS) is a rare overgrowth syndrome clinically characterized by multiple congenital abnormalities, pre/postnatal overgrowth, distinctive craniofacial features, macrocephaly, and organomegaly. Abnormalities of the skeletal system, heart, central nervous system, kidney, and gastrointestinal tract may also be observed. Intellectual disability, early motor milestones and speech delay are sometimes present; however, there are a considerable number of individuals with normal intelligence.Genomic rearrangements and point mutations involving the glypican-3 gene (GPC3) at Xq26 have been shown to be associated with SGBS. Occasionally, these rearrangements also include the glypican-4 gene (GPC4). Glypicans are heparan sulfate proteoglycans which have a role in the control of cell growth and cell division.Although a lethal and infrequent form (also known as SGBS type II) has been described, only the classical form of SGBS is reviewed in this work, whereas only some specific features on SGBS type II are commented.We review all clinical and molecular aspects of this rare disorder, updating many topics and suggest a follow-up scheme for geneticists and primary care clinicians.

A patient with a unique frameshift mutation in GPC3, causing Simpson–Golabi–Behmel syndrome, presenting with craniosynostosis, penoscrotal hypospadias, and a large prostatic utricle—Am J Med Genet Part A 161A: 3121–3125

A patient with a unique frameshift mutation in <i>GPC3</i>, causing Simpson–Golabi–Behmel syndrome, presenting with craniosynostosis, penoscrotal hypospadias, and a large prostatic utricle—Am J Med Genet Part A 161A: 3121–3125

Establishment of lipofection for studying miRNA function in human adipocytes.

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.

Somatic CTNNB1 mutation in hepatoblastoma from a patient with Simpson–Golabi–Behmel syndrome and germline GPC3 mutation

Simpson-Golabi-Behmel syndrome is a rare overgrowth syndrome caused by the GPC3 mutation at Xq26 and is clinically characterized by multiple congenital abnormalities, intellectual disability, pre/postnatal overgrowth, distinctive craniofacial features, macrocephaly, and organomegaly. Although this syndrome is known to be associated with a risk for embryonal tumors, similar to other overgrowth syndromes, the pathogenetic basis of this mode of tumorigenesis remains largely unknown. Here, we report a boy with Simpson-Golabi-Behmel syndrome who had a germline loss-of function mutation in GPC3. At 9 months of age, he developed hepatoblastoma. A comparison of exome analysis results for the germline genome and for the tumor genome revealed a somatic mutation, p.Ile35Ser, within the degradation targeting box of β-catenin. The same somatic mutation in CTNNB1 has been repeatedly reported in hepatoblastoma and other cancers. This finding suggested that the CTNNB1 mutation in the tumor tissue represents a driver mutation and that both the GPC3 and the CTNNB1 mutations contributed to tumorigenesis in a clearly defined sequential manner in the propositus. The current observation of a somatic CTNNB1 mutation in a hepatoblastoma from a patient with a germline GPC3 mutation supports the notion that the mutation in GPC3 may influence one of the initial steps in tumorigenesis and the progression to hepatoblastoma.

Proinflammatory Cytokine Induction of 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) in Human Adipocytes Is Mediated by MEK, C/EBPβ, and NF-κB/RelA

Levels of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regenerates active glucocorticoids, are selectively elevated in adipose tissue in human obesity and metabolic syndrome, both conditions associated with chronic low-grade inflammation. 11β-HSD1 expression is induced by proinflammatory cytokines in a variety of cell types, including in human adipocytes differentiated in vitro. Our objective was to determine the mechanisms by which proinflammatory cytokines induce 11β-HSD1 in human adipocytes. The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11β-HSD1 mRNA levels in human primary adipocyte fractions and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes (P<.001). Inhibition of the MAPK/ERK kinase (MEK) attenuated CCAAT/enhancer binding protein (C/EBP) β phosphorylation at Thr235 and IL-1α/TNFα induction of 11β-HSD1 (P≤.007). The small interfering RNA-mediated knockdown of C/EBPβ and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11β-HSD1 (P≤.001). Moreover, induction of 11β-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPβ and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPβ and NF-κB/RelA located to the 11β-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPβ and NF-κB/RelA signaling, attenuated the IL-1α induction of 11β-HSD1 (P≤.002). Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11β-HSD1 expression at this site via MEK, C/EBPβ, and NF-κB/RelA. These molecules/signaling pathways are, therefore, potential targets for drugs, including metformin and acetylsalicylic acid, to prevent/decreased up-regulation of 11β-HSD1 in human obese/metabolic syndrome adipose tissue.

A patient with Simpson–Golabi–Behmel syndrome, biliary cirrhosis and successful liver transplantation

Simpson-Golabi-Behmel syndrome type 1 (SGBS1) -OMIM 312870- is a rare X-linked inherited overgrowth syndrome caused by a loss of function mutation in the GPC3 gene. Affected patients present a variable phenotype with pre- and post-natal macrosomia, distinctive facial dysmorphism, organomegaly, and multiple congenital anomalies. Intellectual disability is not constant. About 10% of patients have an increased risk of developing embryonic tumors in early childhood. Only one case of biliary disease has been described so far. GPC3 is localized on Xq26. It encodes for glypican 3, a heparan sulfate proteoglycan, which among its different known roles, negatively regulates liver regeneration and hepatocyte proliferation. This report concerns a male with a SGBS1, carrier of a GPC3 pathogenic mutation, and neonatal liver disease, who developed an early biliary cirrhosis. Together with the associated risk of cancer and developmental delay, liver transplantation was discussed and then successfully performed at the age of 19 months. A hypothesis on the role of GPC3 in the patient's liver disease is also proposed.

Duplication of exon 2 of the GPC3 gene in a case of Simpson‐Golabi‐Behmel syndrome

In the May issue of the Journal, Mateos et al. [2013] reported a case of Simpson-Golabi-Behmel syndrome (SGBS), with a novel mutation of theGPC3 gene consisting of a duplication of exons 2–4 which causes a frameshift leading to a protein truncated of its last 183 amino-acids including the C-terminus. This report pinpointed that duplication of the GPC3 gene had not been reported before. We would like to emphasize that duplication screening should be part of the molecular analysis in SGBS by describing another duplication of GPC3. In a recent paper [Cottereau et al., 2013] we reported a large series of patientswith SGBS including one casewith aduplication of exon 2. This duplication was detected bymultiplex ligation-dependent probe amplification (MLPA) analysis in a series of 48 patients referred for SGBS, in whom no GPC3 deletion or point mutation had been identified (Fig. 1). This result was confirmed by quantitativemultiplex fluorescent PCR (PCR of exons 2, 3, and 8 ofGPC3 compared with exon 22 of the CFTR gene as internal control) (results not shown). RT-PCR analysis performed on mRNA extracted from a lymphoblastoid cell line of the patient, with primers designed in exons 1 and 3, respectively, showed a fragment with a size increase of around 160 pb compared to a normal control (Fig. 2). Sequencing of this fragment showed that it was a tandem duplication of exon 2. Unlike the previously reported case, this duplication conserves the reading frame and leads to an insertion of 54 residues from Gly59 to Gln112, in the GPC3 protein. This insertion includes, in the fourteen cysteine motif, three additional cysteine residues (p.Cys65, p.Cys72 and p.Cys73) which probably disrupt the conserved glypican three-dimensional structure of the protein thus altering its function [Filmus and Capurro, 2008]. This duplication was observed in a sporadic case (DNA of his mother was unavailable) who had a clinical picture typical of SGBS. He had been examined at the age of 20 years for tall stature. The diagnosis of SGBS had been suggested on themedical history of the patient because of macrosomia at birth followed by advanced statural growth (height at 214 cm) but normal OFC (þ0.8 SD) and BMI (20.1), diaphragmatic hernia operated on in the neonatal period, cryptorchidism, and on examination pectus excavatum, one pair of extra nipples, inguinal hernia, large hands, and suggestive dysmorphic features (macrognathia, median groove of the

A patient with a unique frameshift mutation in GPC3, causing Simpson–Golabi–Behmel syndrome, presenting with craniosynostosis, penoscrotal hypospadias, and a large prostatic utricle

We present a Hispanic male with the clinical and molecular diagnosis of Simpson-Golabi-Behmel syndrome (SGBS). The patient was born with multiple anomalies not entirely typical of SGBS patients, including penoscrotal hypospadias, a large prostatic utricle, and left coronal craniosynostosis. In addition, he demonstrated endocrine anomalies including a low random cortisol level suspicious for adrenal insufficiency and low testosterone level. To our knowledge, this is the first report of a prostatic utricle in SGBS and the second report of craniosynostosis. The unique disease-causing mutation likely arose de novo in the mother. It is a deletion-insertion that leads to a frameshift at the p.p. S359 [corrected] residue of GPC3 and a premature stop codon after five more amino acids. p. S359 [corrected] is the same residue that is normally cleaved by the Furin convertase, although the significance of this novel mutation with respect to the patient's multiple anomalies is unknown. We present this case as the perinatal course of a patient with unique features of SGBS and a confirmed molecular diagnosis.