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Human Molecular Genetics Advance Access originally published online on November 13, 2008
Human Molecular Genetics 2009 18(3):517-524; doi:10.1093/hmg/ddn379
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cerebrocostomandibular-like syndrome and a mutation in the conserved oligomeric Golgi complex, subunit 1

Renate Zeevaert1, François Foulquier2,3, Boyan Dimitrov2, Ellen Reynders4,5, Rita Van Damme-Lombaerts1, Emil Simeonov6, Wim Annaert4,5, Gert Matthijs2 and Jaak Jaeken1,*

1 Department of Pediatrics, University Hospitals Leuven, Herestraat 49, BE-3000 Leuven, Belgium 2 Center for Human Genetics, University of Leuven, Leuven, Belgium, 3 Unité de Glycobiologie Structurale et Fonctionnelle UMR/CNRS 8576, IFR147, Université des Sciences et Technologies, Lille, France, 4 Laboratory for Membrane Trafficking, Center for Human Genetics, University of Leuven, Leuven, Belgium, 5 Department for Molecular and Developmental Genetics, Flanders Institute for Biotechnology (VIB), Leuven, Belgium 6 Clinical Genetics Unit, Department of Pediatrics, Medical Faculty, Sofia, Bulgaria

* To whom correspondence should be addressed. Tel: +32 16343820; Fax: +32 16343842; Email: Jaak.Jaeken{at}uz.kuleuven.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 MATERIAL AND METHODS
 FUNDING
 REFERENCES
 
We describe two patients with a cerebrocostomandibular-like syndrome and a novel mutation in conserved oligomeric Golgi (COG) subunit 1, one of the subunits of the conserved oligomeric Golgi complex. This hetero-octameric protein complex is involved in retrograde vesicular trafficking and glycosylation. We identified in both patients an intronic mutation, c.1070+5G>A, that disrupts a splice donor site and leads to skipping of exon 6, a frameshift and a premature stopcodon in exon 7. Real-time reverse transcriptase polymerase chain reaction showed in the first patient only 3% of normal transcript when compared with control. A delay in retrograde trafficking could be demonstrated by Brefeldin A treatment of this patient's fibroblasts. The costovertebral dysplasia of the two patients has been described in cerebrocostomandibular syndrome (CCMS), but also in cerebrofaciothoracic dysplasia and spondylocostal dysostosis. CCMS itself is heterogeneous because both autosomal dominant and autosomal recessive inheritance has been described. We anticipate further genetic heterogeneity because no mutations in COG1 were found in two additional patients with a CCMS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 MATERIAL AND METHODS
 FUNDING
 REFERENCES
 
The conserved oligomeric Golgi (COG) complex is a protein complex, located at the cytoplasmic side of the Golgi membrane. The COG complex consists of eight subunits organized into two lobes: lobe A (Cog1–4) and lobe B (Cog5–8). A patient with a truncating mutation in COG1, c.2659-2660insC, and a mild phenotype including mild growth retardation, psychomotor retardation, hypotonia, hepatomegaly and cerebellar atrophy, has been previously described with a sialylation defect in both N- and O-glycans (1). We identified two patients with a novel mutation in COG1 and a similar cerebrocostomandibular-like syndrome (CCMS, OMIM 117650 [OMIM] ). This syndrome is characterized by severe micrognathia, rib defects and mental retardation (2). Other features of this syndrome include growth retardation, palatal defects or Pierre–Robin sequence, vertebral anomalies, microcephaly and feeding difficulties (2–4).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
MATERIAL AND METHODS
 FUNDING
 REFERENCES
 
Description of patients
Patient 1
This boy was born after an uncomplicated pregnancy of 43 weeks from healthy consanguineous Greek-Turkish parents with a birth weight of 2550 g (p3) and a length of 46 cm (<p3 = 47 cm). Dysmorphic features included proptosis, a smooth philtrum, thin upper lip, a short neck, low implanted and posteriorly rotated ears, widely spaced nipples and a simian crease on the left hand. He had multiple congenital abnormalities including Pierre-Robin sequence (cleft palate, micrognathia and glossoptosis), costovertebral anomalies including osteopenia, ribfusions and posterior rib gaps, butterfly vertebrae, misalignment of the vertebrae and a clubfoot on the right (Fig. 1). He also presented with microcephaly, pre- and postnatal growth retardation and bilateral maculopathy. Magnetic resonance imaging (MRI) of the brain showed a megacisterna magna and a hypoplastic vermis. He was diagnosed with sepsis three times, twice with Streptococcus pneumoniae and once with Haemophilus influenzae type B. Chronic aspiration led to a bronchopneumonia with partial atelectasis of the right upper lobe. Screening for immunodeficiency was negative, despite the lack of immunity after vaccination for measles, rubella and polio. He had moderate motor and mental retardation. At the age of 3 years, he started to walk and spoke a few words. Weight (8.2 kg), length (69 cm) and head circumference (42 cm) were already far below the third percentile. At the age of 6 years, height was 82.7 cm, weight 10.75 kg and head circumference 43.5 cm. Intercurrent infections included a bronchopneumonia, sepsis of unknown aetiology, mycoplasma pneumonia and gastro-enteritis. Lymphocyte proliferation tests and immunophenotyping were normal. There was mild thrombocytopenia with some giant platelets. Serum IGF-1 was normal. Orchidopexia was performed because of cryptorchidy at the age of 8 years. At the age of 10 years, he developed an atypical, diarrhoea-negative haemolytic uremic syndrome with acute renal failure. Intravenous immunoglobulins were inefficient. Haemodialysis was performed because of anuria and hypertension during 4 months, followed by a period of partial recovery of the kidney function. No enterohaemorrhagic Escherichia coli were detected in serum, faeces or urine. Factor H, factor I and complement C3 were normal. Total complement was elevated (>160%; normal: 70–140). Von Willebrand protease ADAMTS13 activity was normal. Echocardiography showed mild pulmonary hypertension and hypertrophy of the atrial septum with an incompetent foramen ovale and a left to right shunt but this normalized after 6 months. Follow-up investigations showed minimal aortic insufficiency and parachute mitral valve. Dialysis was restarted because of irreversible renal failure 4 months later. Multiple catheter problems occurred including bleeding and thrombosis. At the age of 11 years, he received a kidney transplant. After transplantation and on immunosuppressiva, he was diagnosed with a pneumonia, a pyelonephritis, an influenza A infection and onychomycosis. Nose, ear and throat investigations showed a cholesteatoma and conductive hearing loss. A computed tomography scan identified an aberrant lateral semi-canalicular channel. A bone age delay of 3 years was present at the chronological age of 12.5 years. Mild thrombocytopenia (66 000–144 000/µl) and episodes of anaemia without haemolysis persisted.


Figure 1
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  		Figure 1. Dysmorphic features of patients 1 (A and B) and 2 (F) include short stature, microcephaly, a smooth philtrum, thin upper lip, low implanted and posteriorly rotated ears, micrognathia, short neck and widely spaced nipples; costovertebral anomalies in patients 1 (C and E) and 2 (D) include rib fusions (arrows) and butterfly vertebrae.

 
Patient 2
This boy was born after an uncomplicated pregnancy of 37 weeks as the first child of healthy and unrelated Bulgarian parents with a birth weight of 2500 g (p3) and a height of 45 cm (<p3 = 47 cm). He was referred to the clinical geneticist because of shortening of the long bones and facial dysmorphy at the age of 2 months. Clinical examination revealed down-slanting palpebral fissures, wide nasal bridge, hypertelorism, up-turned nasal tip, long filtrum, small mouth, micrognathia, fine upper lip with short frenulum, high palate, short neck with low hair line, low set, posteriorly rotated ears, right-side microtia with peculiar clefting of the helix and antihelix, a narrow ear canal, rhizomelic shortening of upper limbs, ulnar deviation of fingers, thoracic scoliosis, hypospadias-I and left-side cryptorchidism.

X-ray survey detected mild mid-facial hypoplasia, proportional shortening of all long bones of the upper limbs, multiple vertebral segmentation defects and bilateral fusions of ribs (Fig. 1). Abdominal ultrasound showed extreme left-side hydronephrosis confirmed by contrast urography. He underwent surgical correction at 6 months of age.

During a 14-year follow-up, this patient developed moderate mental retardation, convergent strabismus (since 6th month of age), microcephaly [standard deviation score (SDS) = –2.5] since 1 year of age, mixed type hearing loss since 3 years which required a prosthesis at 9 years, short stature (SDS = –3) and camptodactyly of the 4th and 5th fingers of both hands observed first at 10 years.

His development was mildly delayed with walking at 20 months and speaking since 2 years. At 14 years of age, he can only make simple sentences using a few words.

MRI of the brain at 5 years detected brain atrophy of the temporal cortex and floculonodular lobe of the cerebellum together with dilatation of the lateral ventricles, fourth ventricle and cisterna magna.

X-ray re-examination at 6.5 years revealed prominent kyphoscoliosis, small, squared iliac bones, shallow acetabular roofs, coxa valga deformation, broad femoral necks and smaller proximal femoral epiphyses with irregular medial parts. Vertebral segmentation defects and bilateral rib fusions persisted.

Glycoprotein analysis
Patient 1 showed a type 2 pattern on isoelectrofocusing (IEF) of serum transferrin (Fig. 2). IEF of apolipoprotein CIII (apoC-III), an O-glycosylated protein, showed an increased apoC-III0 and a decreased apoC-III2 band in patient 1 compared with his parents, indicating hyposialylation of apoC-III (Fig. 2). Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) of the N-glycans of serum transferrin suggested a defect in sialylation and a milder defect in galactosylation (Fig. 3).


Figure 2
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  		Figure 2. (A) Serum Tf IEF in a control (C), a positive control with Cog1 deficiency (COG1) and the patient (P) showed a type 2 pattern in the patient comparable with the other COG1 deficient patient. The number of sialic acid residues is indicated on the right; (B) IEF of serum apoC-III in mother (M), father (F) and patient (P) showed hyposialylation in the patient.

 


Figure 3
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  		Figure 3. Serum transferrin N-glycan analyses by MALDI–TOF MS in a control (A) and patient (B). The patient shows a biantennary glycan structure with two terminal Sia (*) and a relative increase in biantennary N-glycans lacking one Sia without (#1) and with (#2) Fuc. There is a smaller increase of the structure lacking one Sia and one Gal (~). Symbols: closed square, GlcNac; open circle, Man; closed circle, Gal; open diamond, Sia and open triangle, Fuc.

 
Western blot analysis of the COG subunits
Steady-state levels of the tested COG subunits on western blot in patient 1 were normal, except for Cog1 with a clearly reduced expression level. The residual amount of Cog1 was about 23% of the amount of Cog1 in a control. The amount of Cog8 was ~84% compared with control, whereas the other Cog subunit levels were not significantly decreased. Also the expression level of the mannosidase II (ManII) protein was normal on western blot (Fig. 4).


Figure 4
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  		Figure 4. Western blot showed a decrease of 77% in the Cog1 subunit and 16% decrease in the Cog8 subunit in patient (P) compared with control (C).

 
Molecular analysis
Sequencing of cDNA of patient 1 identified two transcripts: a normal transcript and a transcript lacking exon 6 with a frameshift and a premature stop codon (Fig. 5A). If expressed, the resulting truncated protein consists of 321 amino acids instead of the normal 980 amino acids. However, no truncated Cog1 was identified on western blot. This can probably be explained by instability of the transcript. The intronic mutation responsible for exon 6 skipping was c.1070+5G>A, which disrupts the splice donor site (Fig. 5B). No cryptic splice site is activated, but the donor splice site of exon 5 will be used to link exon 5 directly to exon 7. In two additional patients with cerebrocostomandibular syndrome (CCMS), no mutations were found in COG1, but in patient 2 the same mutation as in patient 1, c.1070+5G>A, was identified.


Figure 5
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  		Figure 5. (A) Schematic presentation of normal splicing (full line), the mutation c.1070+5 G>A * in intron 6 and disrupting splicing leading to skipping of exon 6 (dotted line) and a stop codon (double line) in exon 7 of COG1; (B) COG1 sequencing results for control and patient on genomic DNA showing the homozygous c.1070+5 G>A mutation in 5' region of intron 6.

 
Because we detected the presence of two transcripts in patient 1, the homozygous intronic mutation c.1070+5G>A is a leaky mutation. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantify the amount of normal and aberrant transcript (Fig. 6). The amount of normal transcript with exon 6 and the amount of total transcript (with and without exon 6) was reduced to, respectively, 3 and 13% in the patient compared with a control. Puromycin treatment of the fibroblasts led to higher expression levels of normal transcript (8% of control) and of the total transcript (37% of control) in patient 1.


Figure 6
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  		Figure 6. Real-time RT-PCR for checking expression of exon 6 and total COG1 transcript on cDNA from fibroblasts of patient and control without and with treatment with puromycin (+puro).

 
Brefeldin A (BFA) treatment of fibroblasts
In normal fibroblasts, incubation with BFA leads to Golgi collapse and redistribution of the Golgi to the endoplasmatic reticulum, leaving almost no Golgi remnants (5). After BFA treatment of the fibroblasts of patient 1, 58% of the cells showed Golgi remnants. This suggested a defect in retrograde trafficking, which was not present in control fibroblasts (Fig. 7).


Figure 7
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  		Figure 7. BFA treatment (5 µg/ml) for 6 min of control (A) and patient's (B) fibroblasts showed a delay in retrograde trafficking in 58% of the patient's cells, indicated by the presence of Golgi remnants (*).

 
Discussion and review of literature
We describe a new patient with a hyposialylation defect and an intronic c.1070+5G>A mutation in COG1 leading to aberrant splicing, skipping of exon 6, a frameshift and premature stopcodon in exon 7. Only 3% of normal transcript was present in the patient compared with control, measured by RT-PCR. The same mutation was identified in a patient with a similar phenotype including short stature, costovertebral dysplasia and structural brain anomalies. Their phenotype is similar to the features of CCMS.

Since the identification of the first patients with CCMS (6,7), about 65 patients worldwide have been described. The syndrome is characterized mainly by severe micrognathia, rib defects and mental retardation (2). A spectrum of posterior rib gap defects has been described in CCMS, ranging from a few affected dorsal rib segments to complete absence of ossification (8). Histological examination showed partial absence of cartilage and bone replaced by undifferentiated fibrous and muscular tissue (9–11). This suggests a problem with the transition from undifferentiated mesenchymal cells to cartilage (10). The posterior rib gaps can create a life threatening functional flail chest (12,13). Diminution of the rib gaps, bony bridges of the defects in some ribs and/or pseudoarthrosis formation are described by different authors (6,14). In patient 1, the X-ray of the thorax at the age of 7 months showed rib gaps and multiple rib fusions. Later, X-rays of patients 1 and 2 showed only rib fusions.

In 50% of the cases of CCMS, there is cerebral involvement (4,11) including mental retardation, microcephaly and histological anomalies. Brain anomalies like delayed myelinization (6), a form of hydranencephaly (15), complete absence of the cerebellum (14), focal glial heterotopia (11), dilated lateral ventricles, gliosis and a 5th ventricle (16) and agenesis of corpus callosum (17) have been described. Mental retardation is suggested to be secondary to perinatal respiratory distress and hypoxia caused by glossoptosis and flail chest rather than to brain abnormalities (8,12,18,19). Our patients presented both with mental retardation and cerebellar atrophy, respectively, with a hypoplastic vermis or atrophy of the floculonodular lobe.

Other less frequent presentations of CCMS include renal (10,20) and cardiac anomalies (9,19), arthrogryposis (21) and conductive hearing loss (11,20). Apart from costovertebral dysplasia, other skeletal abnormalities have been described in CCMS, including hypoplastic humerus, sacral fusion and flask-shaped configuration of the pelvis, hemivertebrae, clubfoot, hip dislocation, elbow dysplasia, pectus carinatum, hypoplasia of the sternum, clavicles and pubic rami (6,11,13,15,18).

Nearly half of the cases of CCMS are familial (22). Both autosomal dominant (10,18,23–28) and autosomal recessive inheritance (3,6,15,21,29–31) have been described. Consanguinity was reported in at least three of these cases (15,30,31).

Patients with autosomal recessive and dominant inheritance are clinically not distinguishable (22) and expression within one family can also be variable (3,4). Moreover, non-penetrance or a possible germline mosaicism cannot be excluded (12,18). We conclude that the syndrome is genetically heterogeneous.

Costovertebral dysplasia including rib fusions and hemivertebrae has also been described in spondylocostal dysostosis (SCD) (32) and cerebrofaciothoracic dysplasia (33). Mental retardation is not a feature of typical SCD (34) and patients with costocerebrofacial dysplasia mostly presented with macrocrania and structural brain defects in septum pellucidum or corpus callosum (35). Micrognathia and a cleft palate are key findings in CCMS. However, other facial dysmorphic features of our patients, such as hypertelorism, a broad short nose, low set, posteriorly rotated ears and a short neck, were also found in patients with cerebrofaciothoracic dysplasia. Other characteristics of this syndrome such as a high birth weight, a low hairline, synophris and normal postnatal growth were not present in our patients (36).

We conclude that the two patients we describe here shared most characteristics with patients described with CCMS. Features that have not been described in CCMS, or in other syndromes with costovertebral dysplasia were cryptorchidism, recurrent infections and haemolytic uremic syndrome, but these features could be accidental.

We have obtained material from two patients with CCMS, one with evidence for autosomal recessive inheritance and one with evidence for autosomal dominant inheritance, but no mutations in the COG1 gene were found. So, mutations in other genes can be responsible for a similar phenotype. Moreover, because of a large overlap of symptoms between the syndromes we hypothesize to find new patients with COG1 mutations in SCD or cerebrofaciothoracic syndrome.

Compared with a mildly affected COG1 deficient patient with an 80 amino acid truncation of the Cog1 protein and reductions of all lobe A Cog proteins, Cog8 and ManII on western blot (1), the phenotype of the present patients is more severe. In contrast, only the amount of Cog1 protein is severely reduced on western blot in patient 1 without affecting the levels of the other Cog subunits. Whereas the Cog1 protein is severely truncated, the amount of Cog8 protein is only mildly decreased and the level of ManII is normal. This might be explained by the presence of a small quantity of normal transcript.

The combination of growth retardation, microcephaly, failure to thrive, hypotonia and dysmorphic features were present in nearly all patients with a COG deficiency described so far. However, in contrast to the patients with COG7 deficiencies, the largest group so far, COG1 deficiency was non-lethal (37).

So far, the patients with COG1 and COG7 deficiency have predominantly growth retardation, whereas the patients with COG8 deficiency had predominantly neurological signs and symptoms including seizures, polyneuropathy, ataxia and oculomotor apraxia.

Apart from hypotonia and seizures, no other neurological features were described in COG7 deficient patients, possibly because of their early death. Brain atrophy was found in all described patients with COG defects but the severity and location varied.

Another common feature of COG defects is dysmorphy of face and hands, and sometimes also of the thorax, but this can vary from mild to severe.

Cardiac anomalies including both cardiopathies (atrial septal defect and ventricular septal defect) in patients with different mutations in COG7 and cardiomyopathy in both COG1 patients (left ventricular hypertrophy in the first patient and transient septal hypertrophy, mild aortic insufficiency and a parachute mitral valve in the second patient) have been described. Except for the first two siblings with COG7 deficiency (38,39), only mild liver involvement has been described with mild hepatomegaly and normal or slightly elevated transaminases.

The clinical differences between the patients with different mutations in COG1 can be explained by the more severe truncation of the protein. However, contribution of another genetic defect in the context of consanguineous parents cannot be excluded. With regard to the milder defects in interactions between COG subunits, we wonder whether this might be due to the presence of a small amount of normal transcript. Finally, we want to point out that it will be important to investigate further the function of individual COG subunits.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIAL AND METHODS
FUNDING
 REFERENCES
 
Glycoprotein analysis
Serum transferrin IEF was performed in our laboratory using the method described by Carchon et al. (40). IEF of serum apoC-III was performed based on the method described by Wopereis et al. (41). Dry IEF gels for the PhastSystem (cat. No 17-0677-01; Amersham Pharmacia Biotech) were rehydrated in a solution of 9.4 ml of 8.51 M urea, 400 µl of 60 ml/l Pharmalyte, pH 4.2–4.9, and 200 µl of ampholine 3.5–5.0 (Amersham Pharmacia Biotech). The gel was prefocused at 2000 V. After this, the 5- or 10-fold in saline diluted plasma was applied to the gel. After IEF, the isoforms of apoC-III were detected by western blot using rabbit anti-human apoC-III antibody (ANAWA Biomedical Services and Products, Wangen, Switzerland) and goat anti-rabbit horseradish peroxidase-coupled antibody as the secondary antibody and visualized by chemiluminescence (Renaissance, Perkin-Elmer, Zaventem, Belgium).

MALDI–TOF MS on serum transferrin was performed as follows. PNGase treatment released the N-glycans of serum transferrin. These were purified by solid-phase extraction and permethylated in the presence of sodium hydroxide. The permethylated glycans were then analysed by MALDI–TOF MS in negative and positive ion mode (42,43). This analysis was performed by the laboratory of Dr L. Sturiale, Istituto di Chimica e Tecnologia dei Polimeri, CNR, Catania, Italy.

Cell culture
Fibroblasts from patients and controls were cultured in Dulbecco's modified Eagle's medium (DMEM-F12) (Invitrogen, Merelbeke, Belgium) containing 10% Fetal Clone III (HyClone, Cramlington, UK) at 37°C under 5% CO2. Medium was changed every 2–3 days. Cells were splitted or harvested if they reached >95% confluence. Cells were harvested by trypsinization (Trypsine, Invitrogen) after washing the cells with Versene (Invitrogen). Medium was added and the solution was put either into a 15 ml falcon to make a pellet by centrifugation at 1700 rpm for 10 min or distributed over two T75 or T150 falcons for a next passage.

Puromycin treatment
Parallel cultures of fibroblasts were treated with puromycin (Sigma, Bornem, Belgium) 200 µg/ml for 4 h before harvest to rule out nonsense-mediated decay.

RNA extraction and cDNA synthesis
RNA purification and cDNA synthesis from fibroblasts or leukocytes was carried out with the RNeasy Kit (Qiagen, Venlo, The Netherlands). The cDNA was then prepared with oligo-dT priming (Amersham, Diegem, Belgium) and Superscript III RNase (Invitrogen) according to manufacturer's protocol.

Molecular analysis
PCR amplification of genes
Primers were designed by primer3 (http://frodo.wi.mit.edu) to amplify the entire cDNA of COG1 (Primer sequences are available on request). A 1 µl cDNA sample was used in a total volume of 50 µl with 0.75 µl DNA polymerase mix and 5 µl buffer 1 of the Expand Long Template system (Roche, Indianapolis, IN, USA). This was supplemented with 7.5 µl dNTP's 2 µmol/µl, 5 µl of the respective forward and reverse primer (Eurogentec, Seraing, Belgium) and sterile water (Baxter, Nivelles, Belgium). Amplification conditions were 2 min at 95°C, 10 cycles of 10 s at 95°C, 30 s at 65°C (–1°C each cycle) and 2 min at 68°C followed by 30 cycles of 10 s at 95°C, 30 s at 55°C and 2 min at 68°C.

For amplification of exon 6 on genomic DNA, including the intron–exon boundaries, the primers designed by primer3 were used with 0.2 µl Taq polymerase and 5 µl buffer from Roche, supplemented with 5 µl dNTP's, sterile water and 0.5–1 µl of DNA 300 ng/µl. Amplification conditions were 20 cycles 30 s at 95°C, 30 s at 65°C (–0.5°C each cycle) and 30 s at 72°C, followed by 20 cycles with annealing temperature of 55°C.

Direct sequencing
After purification of the PCR products with EXO-SAP-IT (Roche), sequencing was performed with Big Dye Terminator v3.1 cycle sequencing kit on ABI 3100 Avant (Applied Biosystems, Foster City, CA, USA). For cDNA overlapping primer pairs were designed with primer3. For sequencing on genomic DNA, the same primers were used as for amplification. Gene sequence was analysed with Seqman (DNA Star, Konstanz, Germany).

RT-PCR was performed with SYBR green for detection and quantification of PCR products. Primers were designed with Primer Express v 2.0 (Applied Biosystems) amplifying the boundaries of two exons to prevent DNA amplification (Primer sequences are available upon request). The HPRT gene was used as an internal standard in experiments with cDNA from human fibroblasts. Reactions were carried out in a 96-well microtiter plate in a final volume of 25 µl containing 12.5 µl qPCR MasterMix Plus for SYBR Green I Low ROX (Eurogentec), mixed with 2.5 µl of both forward and reverse primer (5 pmol/µl) and 2.5 µl of sterile water. Control or patient cDNA (5 µl of a 1/16 diluted sample) was added. Thermo-cycling included 40 cycles of 60°C for 1 min followed by 15 s at 95°C on a 7500 real-time PCR system (Applied Biosystems). A melting curve was implemented during the reactions to check for the possibility of primer dimer formation. The 2–{Delta}{Delta}Ct method was used to determine relative transcript abundance (44). Each assay included each sample in triplicate. Each patient sample was compared with the control sample (cDNA from control cells treated with puromycin) in three independent assays.

Protein analysis
Western blot analysis was performed as described by Morava et al. (45).

The antibodies for the Cog subunits were a gift from Professor M. Krieger (MIT, Cambridge, USA), Dr D. Ungar (University of California, La Jolla, San Diego, CA, USA) and Dr V. Lupashin (University of Arkansas, Little Rock, AR, USA). The monoclonal COG1 antibody was purchased from BD bioscience (Erembodegem, Belgium).

Cell biological techniques
BFA treatment of fibroblasts
Fibroblasts (2 x 105 cells) of control and patients were grown overnight on cover slips in a 6-well plate in 2 ml DMEM-F12 medium supplemented with 10% Fetal Clone III. BFA was diluted 1/1000 in pre-heated DMEM-F12 medium. After removal of the old medium, the BFA was added to the wells and the plate was put at 37°C for 6 min. The reaction was stopped by putting the cells on ice, removing the medium and adding 1 ml paraformaldehyde 4% to the wells.

Immunofluorescence staining
Cells on the cover slips were stained for GM130 and Man II according to the following method. Cover slips were put on sealing film (Nescofilm from Alfrera Pharma cooperation, Osaka, Japan) in a humid chamber. Cells were permeabilized with phosphate buffered saline (PBS) containing 0.5% Triton 100 for 5 min and washed three times with PBS. A blocking solution [0.1% Triton (Sigma)/1% BSA (Sigma)] in PBS containing 5% normal goat serum (Invitrogen) was put on the cover slips for at least 60 min. After removal of this blocking solution, the cover slips were covered with blocking solution containing the primary antibodies in a dilution 1/1000 and were kept overnight at 4°C. The next morning, the cover slips were washed again three times with PBS, followed by incubation with the Alexa 488- or Alexa 568-conjugated secondary antibodies (Invitrogen) (1/500) for 1 h at room temperature in the dark. Immunostaining was detected through an inverted Diaphot 300 (Nikon) microscope connected to a confocal microscope (MRC 1024; Bio-Rad) and data were collected using LASERSHARP 3.0 and counting the percentage of cells with Golgi remnants after final washes with PBS.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIAL AND METHODS
 FUNDING
REFERENCES
 
This work was supported by grants from the European Commission (Fifth Framework programme, contract LSHM-CT.2005-512131 to EUROGLYCANET; http://www.euroglycanet.org), by a Marie Curie European Reintegration Grant to F.F., and by research grants from the Research Foundation (FWO) Flanders (contracts G.0553.08 to G.M. and G.0504.06 to W.A.). R.Z. is a research assistant of the FWO. This work was further supported by an ‘Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie Vlaanderen’ (IWT) scholarship (SB53523) to E.R.; W.A. also acknowledges the financial support of the ‘Vlaams Instituut voor Biotechonologie’ (VIB).


   ACKNOWLEDGEMENTS
 
We thank Professor Dr Hubert Carchon and Sandra Van Aerschot for the isoelectrofocusing tests and Professor Dr B.C. Hamel and Dr E.P. Kirk for sending us samples of patients with CCMS.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIAL AND METHODS
 FUNDING
 REFERENCES
 

  1. Foulquier F., Vasile E., Schollen E., Callewaert N., Raemaekers T., Quelhas D., Jaeken J., Mills P., Winchester B., Krieger M., Annaert W., Matthijs G. Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II. Proc Natl Acad Sci USA (2006) 103:3764–3769.[Abstract/Free Full Text]

  2. Tachibana K., Yamamoto Y., Osaki E., Kuroki Y. Cerebro-costo-mandibular syndrome. Hum. Genet. (1980) 54:283–286.[CrossRef][Web of Science][Medline]

  3. Hennekam R., Beemer F., Huijbers W., Hustinx P., Sprang F. The cerebro-costo-mandibular syndrome: third report of familial occurrence. Clin. Genet. (1985) 28:118–121.[Medline]

  4. Megier P., Ayeva-Derman M., Esperandieu O., Aubry M.-C., Couly G., Desroches A. Prenatal ultrasonographic diagnosis of the cerebro-costo-mandibular syndrome: case report and review of the literature. Prenat. Diagn. (1998) 18:1294–1299.[Medline]

  5. Steet R., Kornfeld R. COG-7-deficient human fibroblasts exhibit altered recycling of Golgi proteins. Mol. Biol. Cell (2006) 17:2312–2321.[Abstract/Free Full Text]

  6. McNicholl B., Egan-Mitchell B., Murray J., Doyle J., Kennedy J., Crome L. Cerebro-costo-mandibular syndrome. A new familial developmental disorder. Arch. Dis. Child. (1970) 45:421–424.[Abstract/Free Full Text]

  7. Smith D., Theiler K., Schachenmann G. Rib-gap defect with micrognathia, malformed tracheal cartilages, and redundant skin: a new pattern of defective development. J. Pediatr. (1966) 69:799–803.[CrossRef][Web of Science][Medline]

  8. Hennekam R., Goldschmeding R. Complete absence of rib ossification, micrognathia and ear anomalies: extreme expression of cerebro-costo-mandibular syndrome. Eur. J. Hum. Genet. (1998) 6:71–74.[Medline]

  9. Kuhn J.P., Lee S.B., Jockin H., Wieder W. Cerebro-costo-mandibular syndrome: a case with cardiac anomaly. J. Pediatr. (1975) 86:243–244.[CrossRef][Web of Science][Medline]

  10. Miller K.E., Allen R.P., Davis W.S. Rib gap defects with micrognathia. The cerebro-costo-mandibular syndrome—a Pierre-Robin like syndrome with rib dysplasia. Am. J. Roentgenol. Radium Ther. Nucl. Med. (1972) 114:253–256.[Medline]

  11. Silverman F.N., Strefling A.M., Stevenson D.K., Lazarus J. Cerebro-costo-mandibular syndrome. J. Pediatr. (1980) 97:406–416.[CrossRef][Web of Science][Medline]

  12. Plötz F.B., van Essen A.J., Bosschaart A.N., Bos A.P. Cerebro-costo-mandibular syndrome. Am. J. Med. Genet. (1996) 62:286–292.[Medline]

  13. Williams H., Shashikant M. Cerebro-costo-mandibular syndrome: long term follow-up of a patient and review of the literature. Am. J. Roentgenol. (1976) 126:1223–1228.[Abstract]

  14. Doyle J., McNicholl B. The skeletal defects of the cerebro-costo-mandibular syndrome. J. Anat. (1969) 105:214.[Medline]

  15. Clarke E., Nguyen V. Cerebro-costo-mandibular syndrome with consanguinity. Pediatr. Radiol. (1985) 15:264–266.[Medline]

  16. Gürgey A., Cagler M., Topcu M., Kutluk T. A case of the crebro-costo-mandibular syndrome. Turk. J. Pediatr. (1985) 27:109–112.[Medline]

  17. Burton E., Oestreich A. Cerebro-costo-mandibular syndrome with stippled epiphysis and cystic fibrosis. Pediatr. Radiol. (1988) 18:365–367.[Medline]

  18. Leroy J.G., Devos E.A., Vanden Bulcke L.J., Robbe N.S. Cerebro-costo-mandibular syndrome with autosomal dominant inheritance. J. Pediatr. (1981) 99:441–443.[CrossRef][Web of Science][Medline]

  19. Wilckox B.K., Tatum S.A. Cerebro-costo-mandibular syndrome presenting as Pierre Robin sequence. Am. J. Otolaryngol. (2004) 25:258–262.[Medline]

  20. Fauré C., Valleur D., Vital L. Le syndrome cérébro-costo-mandibulaire. Trois nouvelles observations. Nouv. Presse Med. (1978) 7:445–448.[Web of Science][Medline]

  21. Kirk E.P., Arbuckle S., Ramm P.L., Adès L.C. Severe micrognathia, cleft palate, absent olfactory tract, and abnormal rib development: cerebro-costo-mandibular syndrome or a new syndrome? Am. J. Med. Genet. (1999) 84:120–124.[Medline]

  22. James P., Aftimos S. Familial cerebro-costo-mandibular syndrome: a case with unusual prenatal findings and review. Clin. Dysmorphol. (2003) 12:63–68.[Medline]

  23. Flodmark P., Wattsgard C. Cerebro-costo-mandibular syndrome. Pediatr. Radiol. (2000) 31:36–37.

  24. Harris S., Waters M.G. Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport. J. Cell Biol. (1996) 132:985–998.[Abstract/Free Full Text]

  25. Merlob P., Schonfeld A., Grunebaum M., Mor N., Reisner S.H. Autosomal dominant cerebro-costo-mandibular syndrome: ultrasonographic and clinical findings. Am. J. Med. Genet. (1987) 26:195–202.[Medline]

  26. Morin G., Gekas J., Naepels P., Goudry J., Devauchelle B., Testelin S., Sevestre H., Thépôt F., Mathieu M. Cerebro-costo-mandibular syndrome in a father and a female fetus: early prenatal ultrasonographic diagnosis and autosomal dominant transmission. Prenat. Diagn. (2001) 21:890–893.[Medline]

  27. Schrander-Stumpel C., Van Der Meer S., De Die-Smulders C., Meinecke P., Rupperecht E., Maroteaux P., Schrander J., Fryns J. Cerebro-costo-mandibular syndrome: a follow-up study with 6 patients. Genet. Couns. (1996) 7:71–72.[Medline]

  28. Van den Ende J., Schrander-Stumpel C., Rupprechts E. The cerebro-costo-mandibular syndrome: seven patients and review of the literature. Clin. Dysmorphol. (1998) 7:87–95.[Medline]

  29. Drossou-Agakidou V., Andreaou A., Soubassi-Griva V., Pandouraki M. Cerebrocostomandibular syndrome in four sibs, two pairs of twins. J. Med. Genet. (1991) 28:704–707.[Abstract/Free Full Text]

  30. Ozkinay F., Ozgür C., Gündüz C., Cankaya T., Ozkinay C. Cerebro-costo-mandibular syndrome: a case report. Pediatr. Int. (2001) 43:522–524.[Medline]

  31. Trautman M.S., Schelley S.L., Stevenson D.K. Cerebro-costo-mandibular syndrome: a familial case consistent with autosmal recessive inheritance. J. Pediatr. (1985) 107:990–991.[Medline]

  32. Rimoin D.L., Fletcher B.D., McKusick V.A. Spondylocostal dysplasia. Am. J. Med. (1968) 45:948–953.[CrossRef][Web of Science][Medline]

  33. Pascual-Castroviejo I., Santoyala J.M., Lopez-Martin V., Rodriguez-Costa T., Tendero A., Mulas F. Cerebro-facio-thoracic dysplasia: report of three cases. Dev. Med. Child. Neurol. (1975) 17:343–351.[Medline]

  34. Philip N., Guala A., Moncla A., Monlouis M., Aymé S., Giraud F. Cerebrofaciothoracic dysplasia: a new family. J. Med. Genet. (1992) 29:497–499.[Medline]

  35. Guion-Almeida M.L., Richieri-Costa A., Saavedra D., Cohen M.M. Cerebrofaciothoracic syndrome. Am. J. Med. Genet. (1996) 61:152–153.[Medline]

  36. Cilliers D., Alanay Y., Boduroglu K., Utine E., Tunçbilik E., Clayton-Smith J. Cerebro-facio-thoracic dysplasia: expanding the phenotype. Clin. Dysmorphol. (2007) 16:121–125.[Medline]

  37. Zeevaert R., Foulquier F., Jaeken J., Matthijs G. Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a novel group of Congenital Disorders of Glycosylation. Mol. Genet. Metab. (2008) 93:261–278.

  38. Spaapen L.J.M., Bakker J.A., Van Der Meer S.B., Sijstermans H.J., Steet R.A., Wevers R.A., Jaeken J. Clinical and biochemical presentation of siblings with COG7 deficiency, a lethal multiple O- and N-glycosylation disorder. J. Inherit. Metab. Dis. (2005) 28:707–714.[CrossRef][Web of Science][Medline]

  39. Wu X., Steet R.A., Bohorov O., Bakker J., Newell J., Krieger M., Spaapen L., Kornfeld S., Freeze H.H. Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder. Nat. Med. (2004) 10:518–523.[CrossRef][Web of Science][Medline]

  40. Carchon H.A., Chevigné R., Falmagne J.B., Jaeken J. Diagnosis of congenital disorders of glycosylation by capillary zone electrophoresis of serum transferrin. Clin. Chem. (2004) 50:101–111.[Abstract/Free Full Text]

  41. Wopereis S., Grünewald S., Morava E., Penzien J.M., Briones P., García-Silva M.T., Demacker P.N., Huijben K.M., Wevers R.A. Apolipoprotein C-III isofocusing in the diagnosis of genetic defects in O-glycan biosynthesis. Clin. Chem. (2003) 49:1839–1845.[Abstract/Free Full Text]

  42. Ciucanu I., Kerck F. A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. (1984) 131:209–217.[CrossRef][Web of Science]

  43. Sturiale L., Barone R., Fiumura A. Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosaemia. Glycobiology (2005) 15:1268–1276.[Abstract/Free Full Text]

  44. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods (2001) 25:402–408.[CrossRef][Web of Science][Medline]

  45. Morava E., Zeevaert R., Korsch E., Huijben K.M., Wopereis S., Matthijs G., Keymolen K., Lefeber D., De Meirleir L., Wevers R.A. A common mutation in the COG7 gene with a consistent phenotype including microcephaly, adducted thumbs, growth retardation, VSD and episodes of hyperthermia. Eur. J. Hum. Genet. (2007) 15:638–645.[Medline]


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P. Paesold-Burda, C. Maag, H. Troxler, F. Foulquier, P. Kleinert, S. Schnabel, M. Baumgartner, and T. Hennet
Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation
Hum. Mol. Genet., November 15, 2009; 18(22): 4350 - 4356.
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