Lambert P Van Den Heuvel
email: b.vandenheuvel@cukz.umcn.nl

Paulien Smits

Ann Saada

Saskia B Wortmann

Angelien J Heister

Chaya Miller

Dorothea Haas

Ralph Hantschmann

Richard J T Rodenburg

Jan A M Smeitink

M Brink

R Pfundt

Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy

Keywords: MRPS22, combined OXPHOS deficiency, mitochondrial translation, Cornelia de Lange-like phenotype

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mitochondrial disorders are generally caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. The OXPHOS system, comprising five enzyme complexes located in the mitochondrial inner membrane, is responsible for the production of most of the cell's ATP.

Synthesis of this energy-generating system is controlled by both the mitochondrial and the nuclear genomes (mtDNA and nDNA). The mtDNA codes for 13 subunits of the OXPHOS complexes I, III, IV and V as well as the tRNAs and rRNAs required for the translation of these transcripts. All other mitochondrial proteins, including over 70 OXPHOS subunits and all proteins of the mitochondrial translation machinery, are encoded by nuclear genes. The majority of mutations associated with combined OXPHOS deficiencies due to impaired mitochondrial translation are located in mtDNA genes; [START_REF]MITOMAP: A Human Mitochondrial Genome Database[END_REF] however, the list of nDNA mutations is steadily growing with defects reported in mitochondrial translation factors, [START_REF] Antonicka | The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1[END_REF][START_REF] Coenen | Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency[END_REF][START_REF] Smeitink | Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs[END_REF][START_REF] Valente | Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu[END_REF] mitochondrial ribosomal proteins, [START_REF] Miller | Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation[END_REF][START_REF] Saada | Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation[END_REF] mitochondrial tRNA synthetases, [START_REF] Edvardson | Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia[END_REF][START_REF] Scheper | Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation[END_REF][START_REF] Isohanni | DARS2 mutations in mitochondrial leukoencephalopathy and multiple sclerosis[END_REF] and tRNA modifying enzymes. [START_REF] Bykhovskaya | Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA)[END_REF][START_REF] Fernandez-Vizarra | Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA)[END_REF][START_REF] Zeharia | Acute infantile liver failure due to mutations in the TRMU gene[END_REF] We investigated a group of 33 patients with combined OXPHOS deficiencies (and normal complex II activities) by mutational analysis of the entire mtDNA, polymerase gamma and nuclear genes implicated in mitochondrial translation. Here we report a mutation in mitochondrial ribosomal protein MRPS22 detected in one patient and establish its pathogenicity.

Materials and methods

Case report

The male patient was the first child of healthy, first grade consanguineous Pakistani parents.

Fetal ultrasound disclosed microcephaly with dilatation of the third ventricle and a left ventricular hypertrophic cardiomyopathy (HCM). The patient was born by spontaneous vaginal delivery at 35 weeks of gestation with a birth weight of 2610 grams (P50), length 44 cm (P10) and head circumference 29 cm (<P3). Physical examination showed Cornelia de Lange-like dysmorphic features (synophrys, low implanted posteriorly rotated ears, retrognathia, redundant skin over the neck, hypospadias, non-pitting edema of the limbs, back and eyelids, but no distal limb anomalies). Generalized muscle hypotonia and paucity of spontaneous movements were seen. Echocardiography showed biventricular and septal HCM with good function and pulmonary hypertension. MRI revealed corpus callosum hypoplasia and dilatation of the third ventricle, a subdural hematoma in the right fronto-parietal region, leukencephalopathy and delayed myelinisation. Chronic, severe metabolic acidosis with increased plasma lactate (max: 9 mmol/l, N: <2.1 mmol/l), elevation of alanine in plasma (684 µmol/l, N: <400 µmol/l) and elevated urinary excretion of Krebs cycle intermediates were noted. In the following months, dysphagia and failure to thrive necessitated gastrostomy. The biventricular HCM remained stable over the years with a shortening fraction of 39%. ECG revealed Wolf-Parkinson-White syndrome. At the age of two years, he developed tonic seizures and EEG displayed a low background activity with multifocal sharp-slow waves.

Seizures and EEG abnormalities disappeared spontaneously. Ophthalmological examination and hearing tests were repeatedly normal. Growth remained poor; growth parameters at the age of four years: weight 8.5 kg (<<<P3), length 80 cm (<<<P3), head circumference 40 cm (<<<P3). The facial features got less pronounced over the years. The boy hardly developed; now, at the age of 5.5 years, he shows a severe truncal hypotonia and tetraspasticity (left>right) with sparse but dystonic movements. He is unable to sit freely and communicate, but he reacts to light and sounds.

Cell culture

Human skin fibroblasts were cultured in M199 medium (Gibco, Breda, The Netherlands) supplemented with 10% fetal calf serum (FCS) and antibiotics.

For complementation experiments, human skin fibroblasts were grown in DMEM (Dulbecco's modified Eagle's medium, Kibbutz Beit Haemek, Israel) with 4.5 g/l glucose, in the presence of 10% FCS, 50 µg/ml uridine and 110 µg/ml pyruvate. Selection of transfected cells was done in glucose free RPMI (Roswell Park Memorial Institute) supplemented with 50 µM galactose and 10% dialyzed FCS. [START_REF] Robinson | Use of fibroblast and lymphoblast cultures for detection of respiratory chain defects[END_REF] Viability was assessed by trypan blue exclusion.

Enzyme measurements

The activities of the OXPHOS enzyme complexes were measured in cultured skin fibroblasts as described previously; citrate synthase (CS) served as a control. [START_REF] Janssen | Some practical aspects of providing a diagnostic service for respiratory chain defects[END_REF][START_REF] Smeitink | Human NADH:ubiquinone oxidoreductase[END_REF]

Pulse labeling of mitochondrial translation products

In vitro labeling of mitochondrial translation was performed as described elsewhere, [START_REF] Boulet | Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and raggedred fibers (MERRF)[END_REF] with a few minor adaptations. In short, cells were labeled for 60 min at 37 ºC in methionine-free DMEM with 10% dialyzed FCS, 200 µCi/ml [ 35 S]methionine (Tran 35 S-Label, MP Biomedicals, Eindhoven, The Netherlands) and 100 µg/ml emetine, and subsequently chased for 10 min in regular DMEM with 10% FCS. Total cellular protein (100 µg) was resuspended and incubated for 10 min in PBS containing 2% lauryl maltoside. Unsolubilized material was removed by centrifugation at 10,000g for 10 min and loading buffer was added to the supernatant to a final concentration of 100 mM Tris-HCl (pH 6.8), 20% glycerol, 1% SDS, 0.02% Coomassie Blue G-250 and 1% mercaptoethanol. Samples were run on 16% polyacrylamide gels, [START_REF] Schagger | Blue Native electrophoresis for isolation of membrane protein complexes in enzymatically active form[END_REF] which were subsequently scanned on a FLA5100 (Fujifilm Life Science, Düsseldorf, Germany). Equal protein loading was confirmed by Coomassie blue staining.

Homozygosity mapping

Patient DNA was isolated from cultured skin fibroblasts by salt extraction. [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] Whole-genome homozygosity mapping was performed using the GeneChip Human Mapping 10K Array Xba 142 version 2.0 (Affymetrix, High Wycombe, UK) in accordance with the manufacturer's guidelines. [START_REF] Matsuzaki | Parallel genotyping of over 10,000 SNPs using a oneprimer assay on a high-density oligonucleotide array[END_REF] This array contains 10,204 single neutral polymorphisms (SNPs) with a mean intermarker distance of 258 kb, equivalent to 0.36 cM, and an average SNP heterozygosity of 0.38.

Sequence analysis

Genomic DNA (gDNA) was extracted from cultured skin fibroblasts (patient) or blood (parents and controls) by standard procedures. [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] Total RNA was isolated from patient fibroblasts using RNA-Bee (Tel-Test, Texas, USA); complementary DNA (cDNA) was prepared using superscript II reverse transcriptase (Invitrogen, Breda, The Netherlands).

Blue-native and SDS-PAGE analysis and in-gel activity assay

Blue-native (BN) PAGE was used for separation of the OXPHOS complexes on 5%-15% polyacrylamide-gradient gels as described before. [START_REF] Calvaruso | Electrophoresis techniques to investigate defects in oxidative phosphorylation[END_REF] After electrophoresis of 40 µg mitochondrial protein from fibroblasts, gels were further processed for complex I in-gel activity assay and Western blotting. Assembly of the OXPHOS complexes was investigated using monoclonal antibodies against subunits of complexes I, III, V (Molecular Probes, Leiden, The Netherlands), II and IV (MitoSciences, Oregon, USA).

One-dimensional 10% SDS-PAGE analysis was performed as described previously. [START_REF] Ugalde | Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies[END_REF] For detection of MRPS22, a polyclonal antibody raised against a GST-MRPS22 (full length) fusion protein (ProteinTech Group, Illinois, USA) was used.

Transfection with wild-type MRPS22 cDNA

Wild-type MRPS22 cDNA was cloned into the pLenti6/V5-D-TOPO 6969-bp expression vector by directional TOPO cloning (primers and PCR conditions are available upon request).

The recombinant vector was propagated in One Shot Stbl3 Escherichia coli and introduced into 293FT cells after cotransfection with pLP1, pLP2 and pLP/VSVG plasmids using lipofectamine according to the manufacturer's instructions (ViraPower, Invitrogen, California, USA). Control and patient cells were infected with the lentiviral construct carrying the wildtype MRPS22, and clones of stably transfected cells were established by blasticin (4 µg/ml) selection and by selection on glucose free medium. [START_REF] Saada | Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation[END_REF][START_REF] Saada | C6ORF66 is an assembly factor of mitochondrial complex I[END_REF] Patient cells were also transiently transfected with the lentiviral vector carrying an expression control plasmid, pLenti6/V5-GW/lacZ (Invitrogen).

Determination of complex IV levels

Protein was determined by Bicinchoninic Acid (BCA) protein assay kit (Sigma-Aldrich, Rehovot, Israel). Amount of complex IV was estimated using the MitoProfile Biogenesis Dipstick assay kit (MitoSciences) according to the manufacturer's instructions. Results were obtained after digital imaging with TINA 2.0 software.

Determination of mitochondrial 12S rRNA and 16S rRNA levels

Total mRNA isolated using TRI reagent (Sigma-Aldrich) was reverse transcribed with ImProm-II reverse transcriptase (Promega, Wisconsin, USA). The cDNA was amplified in a PCR reaction to detect the expression of specific nucleic acid sequences using SYBR Green (Applied Biosystems, Roche, New Jersey, USA). β-Actin served as an endogenous control and mitochondrial 12S rRNA or 16S rRNA as the target cDNAs (primers are available upon request). The levels of all cDNAs were equalized and real-time PCR reactions were performed using each primer set separately. The relative expression of the target transcript was calculated with the comparative Ct method (Applied Biosystems User Manual) and analyzed by two-tailed Student's t-test.

Results

Enzyme activities of OXPHOS complexes I, III and IV in patient fibroblasts were reduced to 45%, 59% and 36% of the lowest control value, respectively; complex II was not affected (Table 1). BN-PAGE analysis confirmed this combined OXPHOS deficiency: complex I and IV were hardly detectable and complex III showed a slight decrease in the amount of fully assembled complex, whereas the level of complex II was normal (Figure 1). Additionally, complex I activity was again clearly lowered.

Pulse labeling of mitochondrial protein synthesis products revealed a marked and generalized mitochondrial translation defect (Figure 2): total incorporation of [ 35 S]methionine was 26% of that in controls. The decrease in mitochondrial translation varied between different subunits, with the translation rates of ND5, ND6 and COIII/COII (10-15%) being most severely and of ATP8 and ND4L/ATP6 (60-70%) being the least affected.

After excluding mutations in the mtDNA, polymerase gamma (POLG and POLG2) and all nine known mitochondrial translation factors (MTIF2, MTIF3, TSFM, TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF) by direct sequencing, we used homozygosity mapping to guide further investigations. Multiple homozygous regions were found, containing mitochondrial ribosomal proteins, among others. Based on the mitochondrial translation deficit in patient fibroblasts and previously reported mutations, we selected MRPS16 (located in the largest homozygous region, spanning 110.9 Mb on chromosome 10) and MRPS22 (located in a region of 16.1 Mb between SNPs rs977683 and rs1112189 on chromosome 3) as plausible candidate genes. Sequence analysis revealed a homozygous T to C transition at position 644 of the coding sequence of MRPS22, which is predicted to result in the substitution of the highly conserved leucine for proline at position 215 (Figure 3A-B). The mutation was heterozygous in both parents and absent in 109 ethnically matched controls.

Since MRPS22 has no ortholog in bacteria and no crystal structure exists for modeling, we used the PSIPRED server to predict the secondary structure of the MRPS22 protein. [START_REF] Jones | Protein secondary structure prediction based on position-specific scoring matrices[END_REF] The mutation appears to be located in the middle of a helix (Figure 3C) and presumably disturbs it due to the lack of stabilizing hydrogen bond formation by proline and the possible introduction of a kink. The homology-based tools PolyPhen (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph/index.html) [START_REF] Ramensky | Human non-synonymous SNPs: server and survey[END_REF] and SIFT (Sorting Intolerant From Tolerant, http://sift.jcvi.org/) [START_REF] Ng | SIFT: Predicting amino acid changes that affect protein function[END_REF] were applied to indicate the functional impact of the missense mutation. Both PolyPhen and SIFT predicted the mutation to be deleterious (with a PSIC score difference of 2.243 and a score of 0.00, respectively). Western blotting revealed the (near) absence of MRPS22 in patient fibroblasts (Figure 3D).

To investigate whether the MRPS22 deficit causes the OXPHOS system deficiency, we introduced wild-type MRPS22 cDNA into patient cells. The complex IV enzyme activity increased from 40% to 109-118% and its protein level went from 51% to 116-120% of the control values (Table 2). Quantification of mitochondrial rRNA levels showed a restoration of 12S and 16S rRNA content from 36% and 77% to 71-109% and 70-120% of control levels (Figure 4A-B), respectively, resulting in normal 12S rRNA/16S rRNA ratios (Figure 4C).

Complex IV activity and amount as well as rRNA content were decreased after transfection of patient fibroblasts with a control vector. Subsequently, genes known to be implicated in Cornelia de Lange syndrome were screened for mutations to shed light on the genetic origin of the Cornelia de Lange-like dysmorphic features. No mutations were found in the genes NIPBL, SMC1A and SMC3. In

Discussion

The present study confirms the importance of mitochondrial ribosomal protein MRPS22 for mitochondrial translation and consequently for the formation and functioning of the OXPHOS system. We described the second mutation in MRPS22 and determined its pathogenic role in the patient's mitochondrial disorder.

The combined OXPHOS complex I, III and IV deficiency, impairment of mitochondrial translation and absence of mutations in the mtDNA in our patient suggested that the genetic defect was located in a nuclear gene encoding a constituent of the mitochondrial translation machinery. Sequence analysis of all mitochondrial translation factors followed by homozygosity mapping led to the identification of a homozygous missense mutation in MRPS22, predicted to substitute a highly conserved leucine for proline (Leu215Pro). In patient fibroblasts, the MRPS22 protein was undetectable and expressing the wild-type MRPS22 cDNA rescued the disease phenotype, evinced by the restoration of OXPHOS complex IV amount and activity as well as 12S rRNA transcript level, establishing the MRPS22 defect as the cause of the disease.

Human MRPS22 is a 360-amino acid protein that has been found in a limited number of species, having no ortholog in fungi or prokaryotes. [START_REF] Smits | Reconstructing the evolution of the mitochondrial ribosomal proteome[END_REF] The exact location of MRPS22 in the three dimensional structure of the ribosome and its exact function are currently unknown.

Recently, a MRPS22 defect was shown to strongly hamper assembly of the small ribosomal subunit, while assembly of the large subunit and part of the small subunit were only mildly affected. [START_REF] Emdadul | The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria[END_REF] This is consistent with both our and previous findings that mutations in mitochondrial ribosomal small subunit proteins cause a marked decrease in the abundance of the 12S rRNA transcript and nearly no reduction in the level of 16S rRNA, [START_REF] Miller | Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation[END_REF][START_REF] Saada | Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation[END_REF] since rRNAs are readily degraded unless they are incorporated into a ribosomal subunit. [START_REF] Dennis | Regulation of ribosomal protein synthesis in Escherichia coli B/r[END_REF] Thus although MRPS22 is evolutionary not well conserved, it is indispensable for the assembly of the human mitochondrial ribosome and consequently, as we have shown, for effective mitochondrial protein synthesis. Previously, a missense mutation in MRPS22 (Arg170His) [START_REF] Saada | Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation[END_REF] and a nonsense mutation in MRPS16 (Arg111X), [START_REF] Miller | Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation[END_REF] which is highly conserved and essential for ribosome assembly, [START_REF] Smits | Reconstructing the evolution of the mitochondrial ribosomal proteome[END_REF][START_REF] Emdadul | The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria[END_REF][START_REF] Cavdar | The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present[END_REF] were reported to be lethal. The clinical phenotypes of patients with MRPS16 or MRPS22 mutations bear resemblances. All three mutations are associated with muscle hypotonia, lactic acidemia and edema of the limbs. Furthermore, both MRPS22 defects led to hypertrophic cardiomyopathy. The patient harboring the MRPS22 Arg170His mutation additionally presented with tubulopathy and no brain abnormalities were noted, in contrast to the patient reported in this study. It remains unclear whether the brain abnormalities observed in our patient are primarily due to the genetic defect or developed secondary to intrauterine ischemia; presumably both contributed. The dysmorphic features and brain abnormalities of our patient are similar to those seen in the MRPS16 patient, however, the latter did not develop hypertrophic cardiomyopathy. Based on the identification of a MRPS22 mutation and the absence of mutations or copy number variations responsible for the Cornelia de Lang-like symptoms in our patient, we advise to perform echocardiography, metabolic screening (plasma lactate, plasma amino acid analysis and urinary organic acid analysis) and cerebral MRI in patients with dysmorphic features suggestive of Cornelia de Lange syndrome.

Notably, in our patient these features were quite prominent at birth but diminished over the years.

The patient reported in this study was part of a cohort of 33 patients with combined OXPHOS deficiencies. In 7 other patients from this cohort, the causative genetic defect was found. Mutations were identified in the mitochondrial tRNA Lys , tRNA Arg and tRNA Trp genes (4 patients), [START_REF] Smits | Functional consequences of mitochondrial tRNA Trp and tRNA Arg mutations causing combined OXPHOS defects[END_REF] POLG (1 patient) and translation factors TSFM and GFM1 (2 patients), including 3 mutations that had not been published before. Our findings emphasize that nuclear gene mutations contribute substantially to mitochondrial translation deficits. Vastly exceeding 100 proteins, among which 81 mitochondrial ribosomal proteins, 29 are (in)directly involved in the mitochondrial translation process, whereas in merely nine of these proteins mutations have been found up till now. Therefore, many more such defects are expected to be discovered.

Progress in high-throughput sequencing technologies will hopefully make the difficult task of selecting the right candidate genes obsolete. In the meantime, supernumerary proteins such as MRPS22, lacking homologs in bacteria and other lower species, should not be disregarded during investigations of mitochondrial disorders since they evidently perform important roles.

Titles and legends to figures

  Sequence analysis of MRPS22 (GenBank accession number NM_020191.2) was carried out on cDNA in two overlapping fragments; the parts of the first and last exon not covered, including the intron-exon boundaries, were amplified on gDNA. The presence of the mutation in exon 4 was confirmed on gDNA and subsequently screened for in both parents and 109 Pakistani controls. Sequence analysis of the entire open reading frame including the intronexon junctions of NIPBL, SMC1A and SMC3 (GenBank accession numbers NM_015384.4, NM_005445.3 and NM_006306.2, respectively) was performed on gDNA. Primer sequences and PCR conditions used for amplification can be obtained upon request. Both strands were used for direct sequencing on a 3130xl Genetic Analyzer, using the BigDye terminator v1.1 cycle sequencing kit according to the manufacturer's protocol (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Sequence data were analyzed with Sequencher 4.8 software (Gene Codes, Michigan, USA) by comparison with the reference sequences. Affymetrix NspI SNP array hybridisation and analysis Copy number variation (CNV) screening by means of microarray analyses was carried out on the Affymetrix GeneChip 250k (NspI) SNP array platform (Affymetrix, Inc., Santa Clara, CA), which contains 25-mer oligonucleotides representing a total of 262,264 SNPs. Hybridisations were performed according to the manufacturer's protocols. Copy numbers were determined using the 2.0 version of the CNAG (Copy Number Analyzer for Affymetrix GeneChip mapping) software package, 21 by comparing SNP probe intensities from patient DNA with those of a sex-matched pooled reference DNA sample (DNA from ten healthy male individuals). The average resolution of this array platform, described by McMullan et al., 22 is 150-200 kb.

  of patient DNA on the 250K NspI SNP array revealed a normal male profile, thus excluding any potentially clinically relevant copy number variants.

Fig. 1 .

 1 Fig. 1. BN-PAGE analysis in patient fibroblasts. Antibodies directed against specific

Fig. 2 .

 2 Fig. 2. In vitro labeling of mitochondrial translation products. The patient (P) translation rates of the different subunits are expressed as percentages of the average [ 35 S]methionine

Fig. 3 .

 3 Fig. 3. MRPS22 mutation, conservation of the mutated residue, protein structure prediction

Fig. 4 .

 4 Fig. 4. Restoration of mitochondrial rRNA levels by MRPS22 transfection. Patient (P) and

Acknowledgements

This work was supported by the European Union's Sixth Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE), and in part by the Association Française contre les Myopathies (AFM) and the Israeli Ministry of Health to A.S. We thank Ilse Pronk and Benigna Geurts (Nijmegen Center for Mitochondrial Disorders) for their work on the sequence analysis of MRPS22 and Hanka Venselaar (Center for Molecular and Biomolecular Bioinformatics, Nijmegen) for the secondary structure prediction. Maleeha Azam and Dr. Raheel Qamar (COMSATS Institute of Information Technology, Islamabad) are acknowledged for providing the Pakistani control samples. We are grateful to Dr. Michael Marusich (MitoSciences, Oregon) for supplying the MitoProfile assay kit. Finally, Dr. Avraham Shaag (Hadassah-Hebrew University Medical Center, Jerusalem) is acknowledged for helpful comments regarding the rRNA quantification.

Abstract

The oxidative phosphorylation (OXPHOS) system is under control of both the mitochondrial and the nuclear genomes; 13 subunits are synthesized by the mitochondrial translation machinery. We report a patient with Cornelia de Lange-like dysmorphic features, brain abnormalities and hypertrophic cardiomyopathy, and studied the genetic defect responsible for the combined OXPHOS complex I, III and IV deficiency observed in fibroblasts. The combination of deficiencies suggested a primary defect associated with the synthesis of mitochondrially encoded OXPHOS subunits. Analysis of mitochondrial protein synthesis revealed a marked impairment in mitochondrial translation. Homozygosity mapping and sequence analysis of candidate genes revealed a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. The mutation predicts a Leu215Pro substitution at an evolutionary conserved site. Mutations in genes implicated in Cornelia de Lange syndrome or copy number variations were not found. Transfection of patient fibroblasts, in which MRPS22 was undetectable, with the wild-type MRPS22 cDNA restored the amount and activity of OXPHOS complex IV as well as the 12S rRNA transcript level to normal values. These findings demonstrate the pathogenicity of the MRPS22 mutation and stress the significance of mutations in nuclear genes, including genes that have no counterparts in lower species like bacteria and yeast, for mitochondrial translation defects.

4

Conflict of interest

The authors declare no conflict of interest. 1 Measured in mitochondrial enriched fractions from fibroblasts. [START_REF] Antonicka | The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1[END_REF] The activity of citrate synthase was 208 mU/mg protein (reference range 144 -257 mU/mg protein).

Tables

Decreased activities as compared to controls are in bold.


Emma L Baple

Rebecca L Poole

Sahar Mansour

Catherine Willoughby

Karen Temple

Louise E Docherty

Rohan Taylor

Deborah Jg Mackay

An atypical case of hypomethylation at multiple imprinted loci

Keywords: Angelman, Prader-Willi, Beckwith-Wiedemann, Imprinting, hypomethylation

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are caused by genetic and epigenetic mutations of the imprinted gene cluster on chromosome 15q13.

Although the imprinting mutations causing PWS and AS are essentially opposite in nature, remarkably, a small number of patients have been reported with clinical features of PWS but epigenetic mutations consistent with AS.

We report here a patient who presented with clinical features partially consistent with both PWS and BWS (Beckwith-Wiedemann syndrome). Epimutations were found at both the AS/PWS and BWS loci, and additionally at the H19, PEG3, NESPAS and GNAS loci. This patient is therefore the first described case with a primary epimutation consistent with AS accompanied by hypomethylation of other imprinted loci (AS-HIL).

Introduction

Imprinted genes are limited in expression potential by their parent of origin, which often coincides with parental-allele specific DNA methylation. Imprinting disorders typically have heterogeneous clinical presentations. Some features, such as prenatal overgrowth, macroglossia, or truncal obesity, are associated with more than one imprinting disorder, suggesting that imprinted genes control many and overlapping facets of human development. Moreover, in recent years, patients have been identified with imprinting disorders affecting more than one imprinted locus, and their clinical features may diverge from the 'classical' presentation of the disorder, possibly reflecting the complement of genes affected. [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF] Angelman syndrome (AS) and Prader-willi syndrome (PWS) are distinct genetic conditions caused by abnormalities at the 15q11-13 imprinted region. Patients with classical Angelman syndrome present with severe mental retardation, speech impairment, ataxia and seizures. They have a characteristic behaviour pattern of hyperactivity and a happy personality and episodes of inappropriate bursts of laughter.

Dysmorphic features include microcephaly, macrostomia and prominence of the jaw. [START_REF] Buggenhout | Angelman Syndrome[END_REF] Prader-Willi syndrome is characterised by global developmental delay, hypotonia and feeding difficulties with failure to thrive in infancy. There is rapid weight gain usually between the ages of 1 and 4 years. This is due to hyperphagia and results in truncal obesity. Patients have short stature and small hands and feet, and males have hypogonadism. The face is characterised by almond shaped eyes and an upward slant of the palpebral fissures. Temper tantrums and a tendency to an obsessive compulsive phenotype are commonly seen. [START_REF] Cassidy | Prader-Willi syndrome[END_REF] AS is most commonly (~70%) caused by interstitial deletion of the maternally derived chr15q11-13. Other causes include paternal uniparental disomy (UPD) (1-2%), imprinting defect (3-5%) and mutations in UBE3A (5-10%). Prader-Willi syndrome is caused by a paternal deletion of the same region (~70%) or maternal UPD (~29%). A primary imprinting abnormality is rare. [START_REF] Horsthemke | Mechanisms of imprinting of the Prader-Willi/Angelman region[END_REF] There are a small number of reported cases of patients with clinical features of PWS but a molecular diagnosis of AS. [START_REF] Gillessen-Kaesbach | A previously unrecognised phenotype characterised by obesity, muscular hypotonia and ability to speak in patients with Angelman syndrome caused by an imprinting defect[END_REF][START_REF] Molfetta | A further case of a Prader-Willi syndrome phenotype in a patient with Angelman syndrome molecular defect[END_REF][START_REF] Camprubí | Imprinting center analysis in Prader-Willi and Angelman syndrome patients with typical and atypical phenotypes[END_REF] In the majority of these cases partial loss of maternal methylation is observed.

We report here a three year-old girl who presented with features of both PWS and a second imprinting disorder, Beckwith-Wiedemann syndrome (BWS). It was hypothesised that a methylation anomaly at more than one locus might explain the phenotype. Therefore, imprint status was investigated at both the PWS/AS and BWS regions, and thereafter at other imprinted loci.

Case report

The patient is one of 4 children born to unrelated Sri Lankan parents. Conception was natural and the pregnancy uncomplicated. Her birth weight at term was 3640g (55 th centile). A large, protruding tongue was noted in the neonatal period, the umbilicus was normal. There were no feeding problems, normal tone and no history of hyperor hypoglycaemic episodes. Parathyroid, calcium and phosphate levels were normal.

There was no history of organomegaly. Motor development was normal and she walked independently at 14 months. She was a shy child with some developmental delay particularly with speech, having no words by 2 years. She had excessive weight gain in her second year and by 3.5 years her OFC was 53.3 cm (98 th centile), height 96.9cm (<25 th centile) and weight 19.5kg (95 th centile). At the age of three and a half years she has remained seizure free and is not ataxic. In addition to her large tongue, she has a round face and bilateral single palmar creases . She has two words and points to indicate her needs. In light of her macroglossia, Beckwith-Wiedemann syndrome (BWS) was suspected; however, because of the developmental delay and increased BMI, Prader-Willi syndrome (PWS) also seemed a possibility.

Materials and Methods

Ethical permission for this study was obtained from Southampton and South West

Hampshire Research ethics committee, REC reference number: 07/H0502/85. DNA was extracted from peripheral blood by standard procedures.

Initially methylation was determined at the PWS/AS by Methylation specific PCR (MS-PCR) according to Kubuta et al [START_REF] Kubota | Methylation-specific PCR simplifies imprinting analysis[END_REF] , and by methylation specific MLPA (MS-MLPA) at 11p15 using MRC Holland BWS kit ME030B according to the manufacturer's instructions (as described by Scott et al [START_REF] Scott | Methylation-specific multiplex ligationdependent probe amplification (MS-MLPA) robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation[END_REF] ). KCNQ1OT1 and H19 results were confirmed by Methylation-sensitive high resolution melt analysis according to Alders et al [START_REF] Alders | Determination of KCNQ1OT1 and H19 methylation levels in BWS and SRS patients using methylation-sensitive high-resolution melting analysis[END_REF]

(MS-HRM).

Subsequently, methylation specific PCR (MS-PCR) analysis of the patient and parental samples was carried out at the ARHI, PLAGL1,MEST1,GRB10, KCNQ1OT1, H19, IGF2P0, DLK1, SNRPN, PEG3, GNAS, NESP55 and NESPAS loci as described previously [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF][START_REF] Poole | Investigation of 90 patients referred for molecular cytogenetic analysis using aCGH uncovers previously unsuspected anomalies of imprinting[END_REF] . Pyrosequencing of SNRPN, GNAS, NESPAS and PEG3 was performed as previously described [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF][START_REF] White | Quantitative analysis of SNRPN gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome[END_REF] .

Microsatellite analysis around the SNRPN, KCNQ1, H19 and PEG3 regions was performed using published microsatellites by standard methods. The markers investigated were: D15S817, D15S128, D15S1506, D15S122, D11S4177, D11S922, D11S4046, D11STH2, D11S1318, D11S4088, D11S4146, D19S210, and D19S887.

ZFP57 sequencing was performed as previously described [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF] .

Results

Initial testing of the SNRPN DMR by MS-PCR and the 11p15.5 region using MS-MLPA revealed a reduced intensity of the methylated maternal band at the SNRPN DMR, partial hypomethylation at KvDMR and subtle hypomethylation at H19. The results at KvDMR and H19 were confirmed by MS-HRM (data not shown).

To extend these findings, MS-PCR was carried out at 13 imprinted loci across the genome. In addition to confirming the abnormalities at 11p15 and 15q13, partial loss of maternal methylation was seen at GNAS, NESPAS and PEG3 (Figure 1). The

SNRPN epimutation and the additional epimutations at GNAS, NESPAS and PEG3

were confirmed by pyrosequencing.

Methylation analysis of the parental samples revealed normal methylation levels at all the loci investigated.

Microsatellite analysis of the patient and her parents around the regions of methylation abnormality showed biparental inheritance, excluding multiple regions of uniparental disomy as a cause of the imprinting anomalies. Sequencing of the ZFP57 gene revealed no mutations (data not shown).

Discussion

We describe here a patient with clinical features suggestive of both PWS and BWS, who was found to exhibit hypomethylation of multiple imprinted loci (HIL).

The clinical features of this patient were remarkably mild given the extent of her methylation abnormlities. Macroglossia and single palmar creases are both diagnostic features of BWS, and consistent with the imprinting anomaly observed. However, the normal birth weight, umbilicus, glycaemia and lack of hemihypertrophy were inconsistent with BWS. The patient's high BMI and developmental delay were consistent with the initial clinical suspicion of PWS, though normal tone and normal facial appearance were inconsistent with PWS. Moreover, she had no ataxia, microcephaly or seizures to suggest AS. We suggest that this case is an example of the clinical picture that may emerge when hypomethylation affects multiple imprinted loci.

Methylation abnormalities at multiple imprinted loci have been described previously in other imprinting syndromes including Transient Neonatal diabetes (TND [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF][START_REF] Mackay | A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus[END_REF] ), BWS [START_REF] Rossignol | The epigenetic imprinting defect of patients with Beckwith-Wiedemann syndrome born after assisted reproductive technology is not restricted to the 11p15 region[END_REF][START_REF] Bliek | Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells[END_REF][START_REF] Bliek | Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith-Wiedemann syndrome[END_REF] and Russell-Silver syndrome [START_REF] Azzi | Multilocus methylation analysis in a large cohort of 11p15-related foetal growth disorders (Russell Silver and Beckwith Wiedemann syndromes) reveals simultaneous loss of methylation at paternal and maternal imprinted loci[END_REF][START_REF] Turner | Methylation analysis of 79 patients with growth restriction reveals novel patterns of methylation change at imprinted loci[END_REF] . The only gene mutation so far associated with such disorders is mutation of ZFP57, which was found in approximately 50% of patients with TND HIL [START_REF] Mackay | Hypomethylation of multiple imprinted loci in individuals with transient neonatal diabetes is associated with mutations in ZFP57[END_REF] . This patient showed no evidence of a ZFP57 mutation, and her pattern of methylation abnormalities seems distinct from that seen in either TND HIL or BWS HIL. Interestingly, the epimutations observed include mosaic hypomethylation of the maternally methylated SNRPN, KCNQ1OT1, PEG3 and NESPAS loci and the paternally methylated H19 locus. This pattern of abnormalities suggests failure of imprint maintenance, rather than its establishment.

It would be interesting to study further cases with an epimutation consistent with AS but presenting with features of PWS, as it is likely that some of these cases will also harbour additional methylation changes. This case is a significant addition to the weight of evidence suggesting that patients with HIL may go undiagnosed because their presentation may blur the boundaries of recognised imprinting disorders. We therefore suggest that epigenetic analysis at multiple loci should be carried out in patients presenting with clinical signs that could be consistent with more than one of the classical imprinting disorders. It is becoming clear that the clinical picture in patients with HIL is extremely variable and that epigenetic as well as genetic mutations should be suspected in any patient with an unusual syndromic presentation.

Titles and legends

Legend to Figure 1.

Electropherograms of methylation-specific PCR amplicons. The successive panels represent analysis of the SNRPN, KCNQ1OT1, H19, PEG3, GNAS and NESPAS DMRs, respectively.

In each panel, the top trace illustrates a normal control, and the lower trace the patient.

Bars labelled me and un indicate "methylated" and "unmethylated" peaks, respectively. Hypomethylation in the patient is indicated by a reduction in the methylated peak height relative to the unmethylated peak when compared to the control.

Kv 2 ctrl 2 nilakis

Acknowledgements

We thank the family for supplying blood samples for scientific research. A part of this work was supported by the Newlife foundation (formerly BDF).

Conflict of interest

The authors declare no conflict of interest.


Richard E Stead

Mbchb Anthony

J King

MD Anthony J King
email: anthony.king@nuh.nhs.uk

Outcome of Trabeculectomy with Mitomycin C in Patients with Advanced Glaucoma

Keywords: advanced glaucoma, trabeculectomy, intraocular pressure

Background/Aims

To determine the medium term intraocular pressure control and visual outcomes for patients with advanced glaucoma undergoing trabeculectomy with mitomycin C.

Methods

All patients with advanced glaucoma (MD -20dB or above) undergoing trabeculectomy with mitomycin C between 2000 and 2008 under the care of a single glaucoma surgeon were included. Intraocular pressure, visual acuity and visual field outcomes were assessed from data prospectively collected into a surgical outcome database.

Results

One hundred and three patients were eligible for inclusion. The posttrabeculectomy group mean IOP varied between 11.3 and 13.3mmHg between 1 and 7 years. At year 5, 85.2% had an IOP < 16mmHg and 96.3% had an IOP < 21mmHg. The number completing a reliable visual field exam decreased significantly year on year, however the change in mean MD for the group as a whole and for individual patients remained stable. 28 patients experienced a significant reduction in acuity defined as 2 or more lines of Snellen, although this was not due to glaucoma surgery in the majority. The only pre-operative determinant for a significant reduction in VA was the pre-operative MD (-27.00dB (n=21) compared with -24.79dB (n=63; p=0.029).

Conclusion

Trabeculectomy is a successsful method of controlling intraocular pressure in the short to medium term in patients with advanced glaucoma.

Introduction

Currently intraocular pressure (IOP) is the only modifiable risk factor in patients with glaucoma and several studies have demonstrated reduced visual field progression when IOP has been lowered. [START_REF] Odberg | Visual field prognosis in advanced glaucoma[END_REF][START_REF] Mikelberg | The rate of progression of scotomas in glaucoma[END_REF][START_REF] Grant | Why do some people go blind from glaucoma?[END_REF] Glaucoma patients with advanced visual field loss represent a difficult group to manage as presentation with advanced visual field loss is a risk factor for further progression and blindness and therefore a very low IOP may be required to avoid further visual field loss. [START_REF] Odberg | Visual field prognosis in advanced glaucoma[END_REF][START_REF] Mikelberg | The rate of progression of scotomas in glaucoma[END_REF][START_REF] Grant | Why do some people go blind from glaucoma?[END_REF][START_REF] Wilson | Risk factors for rate of progression of glaucomatous visual field loss: a computer-based analysis[END_REF][START_REF] Hattenhauer | The probability of blindness from open-angle glaucoma[END_REF] The use of Mitomycin C (MMC) in trabeculectomy surgery has been shown to reduce the risk of failure in certain groups. [START_REF] Wilkins | Intra-operative mitomycin C for glaucoma surgery[END_REF] In addition the success of modern glaucoma surgery has been further enhanced by the use of postoperative bleb manipulation. [START_REF] Taube | Trabeculectomy with an active postoperative regimen: results and resource utilization[END_REF][START_REF] King | Frequency of bleb manipulations after trabeculectomy surgery[END_REF][START_REF] Marquardt | Intensified postoperative care versus conventional follow-up: a retrospective long-term analysis of 177 trabeculectomies[END_REF] The safety of modern glaucoma surgery using intraoperative techniques such as tight closure of the scleral flap and the anterior chamber maintainer to reduce the risk of hypotony have been encapsulated in the concept of the safe trabeculectomy. [START_REF] Gale | Medium-term outcomes of safe surgery system trabeculectomies[END_REF][START_REF] Stalmans | Safe trabeculectomy technique: long term outcome[END_REF] In this study we assess outcome of trabeculectomy surgery combined with MMC in patients with advanced glaucoma to document success in terms of IOP and visual outcomes. In addition we quantify the post-operative interventions undertaken to achieve these outcomes.

Methods

All trabeculectomies and phaco-trabeculectomies carried out by a single surgical team between October 2000 and July 2008 were reviewed. Data on all trabeculetomies was entered into a surgical database at the time of surgery and updated at each follow-up visit. Patients who had severe pre-operative glaucomatous visual field defects, defined as an MD greater than 20dB and patients who had poor acuity secondary to glaucoma which precluded formal visual field assessment were identified. Demographic and clinical information including any post-operative interventions was available from the database. All identified cases were included with no specific exclusion criteria. Data on all patients were included until the last recorded appointment which was considered the end of their follow-up.

An intervention was defined as any procedure or process undertaken after trabeculectomy surgery aimed at enhancing the success of the surgical outcome.

These were bleb massage, suture removal, 5-fluorourcil (5-FU) injection and needling in combination with 5-FU injection. Bleb massage was performed by applying digital pressure to the globe through the upper eyelid behind the posterior lip of the scleral flap.

Statistical analysis was undertaken using SPSS V.14.0. The tests for statistically significant differences between variables were performed using the 2 test or Fisher exact test for discrete variables, Student's t-test for parametric data and the Mann-Whitney U test for non-parametric data. Two-tailed p values <0.05 were taken to indicate significance.

The success of surgical outcome was defined as unqualified when no additional medication was required or qualified when additional medication was required to achieve the specific IOP definition. Success was also defined at two IOP cut-off points (IOP <21 mm Hg and IOP <16 mm Hg). Furthermore sustained hypotony <6mmHg for greater than 6 months was considered a failure. Visual acuity was measured on a Snellen chart and a reduction of 2 or more lines was considered significant.

The safe trabeculectomy approach was used. [START_REF] Stalmans | Safe trabeculectomy technique: long term outcome[END_REF] The dosage and exposure time of MMC was varied for patients according to the various preoperative and peroperative risk factors for surgical failure. The final dosage decision was taken once scleral exposure had been achieved. The dosage of MMC was defined as the product of the MMC concentration and the application time.

Results

In total 103 eyes were identified for the study (Table 1), second eyes that were eligible were not included. Twenty one patients were unable to complete a satisfactory pre-operative visual field test, the majority of whom [START_REF] Casson | Long-term effect of cataract surgery on intraocular pressure after trabeculectomy: extracapsular extraction versus phacoemulsification[END_REF]

IOP reduction

Intra-ocular pressure reduction achieved at 1 year was significantly below preoperative IOP (12.4mmHg from 25.7mmHg, p<0.0005) and was maintained throughout the duration of follow-up. The majority of patients achieved a successful outcome, although 35 (33.7%) required drops at the end of their follow-up to achieve the target IOP (mean number of drops 2.2, SD 1.08, range 1-4). This represented a significant reduction in the number of drops required. When viewing the group as a whole a mean number of 0.74 drops per-patient were required compared to an average of 2.8 pre-operatively (figure 1).

IOP (mmHg)(

There was a reduction in the number of patients achieving an unqualified success year on year, declining from 82% and 88% (<16mmHg and <21mmHg respectively) at year 1 (n=88) to 52% and 56% at year 5 (n=27). However, there was no real reduction in qualified success numbers which remained relatively static at roughly 85% and 95% for the duration (for IOPs <16mmHg and <21mmHg respectively). Figure 2 (a-d). Only one patient developed sustained hypotony (<6mmHg for greater than 6 months).

Subgroup survival analysis demonstrated a significantly better survival of those with primary open angle glaucoma for both IOP outcomes ≤21mmHg (log rank = 0.002) and ≤16mmHg (log rank <0.0005). Figure 2 (e,f). Further subgroup analysis of survival revealed no difference in outcome for patients having their surgery performed by either a consultant or glaucoma fellow trainee, for combined procedures compared to trabeculectomy or for patients who had their cataract surgery following their trabeculectomy compared to those that did not.

Visual field changes

Numbers completing a reliable visual field exam decreased significantly year on year. Some attrition was due to the fact that a number of patients had [START_REF] Fraser | Risk factors for late presentation in chronic glaucoma[END_REF] remained stable with no statistically significant decline evident.

Visual acuity changes

Pre-operative acuities ranged from 6/6 to perception of light, with a median acuity of 6/12. Post-operative median acuity remained unchanged with a range of 6/6 to no perception of light. 28 patients experienced a significant reduction in acuity defined as 2 or more lines of Snellen. However, it should be noted only a minority of these were due to the surgery itself (table 3).

No form of intervention was found to be associated with a significant reduction in acuity.

Cataract Surgery

The development or removal of clinically significant cataract may affect both VA and visual field parameters. At the time of their final follow-up a total of 63% of patients were pseudophakic. 24 (23%) were pseudophakes pre-trabeculectomy, 12 (12%) underwent phaco-trabeculectomy surgery, 28 (27%) had cataract surgery after their drainage procedure and 39 (37%) remained phakic (in 4 cases surgery was declined despite being indicated). There was no statistically significant difference in post-trabeculectomy MD between those that had cataract surgery after their drainage procedure (MD -25.55 (SD 3.091, n=20) and those that did not (MD -25.25 (SD 4.629, n=39) p=0.943 (Mann Whitney)). Furthermore similar proportions (20-30%) of each group (cataract surgery or no cataract surgery) experienced a decline in VA with little difference between the two.

Bleb Manipulations

In total 83 (79.8%) patients had some form of bleb manipulation, the majority of which had an episode of bleb massage (64%), Table 4 There was no statistically significant correlation between intervention and final MD or final VA. Looking at the group as a whole, any intervention was more likely if the operating surgeon was a trainee (p 0.006). Interventions were not associated with the concentration of MMC used although bleb manipulation was more likely with a lower dose (product of concentration with exposure time) of MMC (0.374 mg/ml/min vs 0.645 mg/ml/min, p 0.002). Patients receiving a 5-FU injection were significantly younger than those not doing so and also had significantly more advanced VF defects pre-op (MD -27.14dB vs -24.14dB, p<0.005). Indeed this was the only intervention associated with more advanced disease defined by the visual field defect.

Patients achieving an unqualified success (<16mmHg and <21mmHg) were statistically less likely to have had any intervention, a 5-FU injection or a needling episode (p 0.005, 0.006, <0.0005 and p 0.005, 0.014, <0.0005 respectively).

Final IOP success was otherwise not related to any form of intervention.

Discussion

For patients with advanced glaucoma target IOP is likely to be very low, introducing an additional challenge in this group of patients. Surgery has several potential advantages over medical management, including the magnitude of mean diurnal IOP lowering and the extent of any diurnal spiking of IOP. [START_REF] Konstas | 24-hour intraocular pressure control with maximum medical therapy compared with surgery in patients with advanced open-angle glaucoma[END_REF] Currently there are no reports of medium to long-term outcomes following MMC trabeculectomy in patients with advanced glaucoma. In this group of patients most likely to go blind from progressive disease such information would greatly inform management.

Intra-ocular pressure control

In our cohort IOP reduction was maintained significantly below pre-operation levels up to 6 years after surgery. Two previous studies have demonstrated adequate short-term IOP control between 3-12 months post-trabeculectomy with MMC in patients with advanced disease (MD -24.8 +/-4.1dB and -27.94 +/-2.7 respectively). [START_REF] Topouzis | Risk of sudden visual loss following filtration surgery in end-stage glaucoma[END_REF][START_REF] Law | Severe loss of central vision in patients with advanced glaucoma undergoing trabeculectomy[END_REF] However this is the first report to demonstrate sufficient IOP lowering in the medium to long-term in patients with advanced disease and are consistent with those of Beckers et al for trabeculectomy with MMC in a nonadvanced disease cohort. [START_REF] Beckers | Five-year results of trabeculectomy with mitomycin C[END_REF] At 5 years he demonstrated a 60% success rate, defined as a final IOP <15mmHg. In our study 51.9% achieved a final IOP of ≤16mmHg, rising to 85.2% when those also on topical medication were included.

There is a significant decrease in the number of patients able to maintain their IOP <16mmHg without the use of additional topical medication and we found a third of patients required drops to maintain target pressure. One may have expected this to be higher in this cohort with advanced disease as their target IOP would be as low as possible. It is therefore likely that medications were reintroduced earlier and at lower IOPs than would have been the case if the patients had had less advanced disease. Subset analysis of the outcomes revealed that patients undergoing surgery for primary open angle glaucoma were more likely to have a successful outcome, this is not surprising as many of the patients in the other group will have had risk factors for failure such as previous conjunctival incisional surgery, uvetis etc.. Furthermore, no difference was found in outcomes between consultants and trainees for this surgery, although more interventions were required to achieve this in the fellow group. This may reflect that although glaucoma fellows undergo extensive surgical exposure they are still surgeons in training and therefore require some additional interventions to achieve similar results.

The influence of cataract surgery on the outcomes of glaucoma surgery remains unresolved. [START_REF] Casson | Phacoemulsification with intraocular lens implantation after trabeculectomy[END_REF][START_REF] Casson | Long-term effect of cataract surgery on intraocular pressure after trabeculectomy: extracapsular extraction versus phacoemulsification[END_REF][START_REF] Rebolleda | Phacoemulsification in eyes with functioning filtering blebs: a prospective study[END_REF] In our cohort, sub-group analysis indicated that cataract surgery following trabeculectomy had no effect on survival.

Change in visual field

Our study failed to demonstrate a decline in visual field over a time period of 5 years in those completing visual field assessment. This should be viewed with some caution. Although the median MD remained constant at roughly -25.0dB

the numbers completing the field test declined year on year. This loss of follow up may be expected with time. However it may also represent the fact that some

Bleb manipulations

A high proportion of our patients (79.6%) had some form of manipulation postoperatively. In our attempt to minimise the potential risks associated with high dose MMC we tended to err on the side of caution when applying MMC. As a consequence some patients on the lower MMC doses required additional intervention (bleb manipulation) to manage post-operative bleb scarring. Another contributory factor could be the need to achieve low target IOPs and consequent intensive bleb manipulation to achieve this. However, our intervention rates were similar to a previous study of mixed visual field loss subjects suggesting patients with more advanced disease are no more likely to have any intervention postoperatively compared to those with less advanced disease. [START_REF] King | Frequency of bleb manipulations after trabeculectomy surgery[END_REF] Topouzis' prospective study on eyes with advanced glaucoma undergoing trabeculectomy augmented with MMC had similarly high intervention rates with 9/21 patients (43%) undergoing a 5 FU injection and 10/21 (48%) requiring argon suturelysis during a 3 month follow-up period. [START_REF] Topouzis | Risk of sudden visual loss following filtration surgery in end-stage glaucoma[END_REF] each clinic visit to overcome the possible error associated with visual acuity fluctuation. However, this study adds to the existing literature on the effectiveness of trabeculectomy with MMC in a cohort of patients with advanced glaucoma. IOP remained controlled over a period of up to 6 years albeit with the requirement of increasing medications with time. The visual acuity underwent further decline post-operation although unexplained loss of vision was rare.

Copyright Licence Statement

The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a wordwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article to be published in the British Journal of Ophthalmology and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence. Survival outcomes between primary open angle glaucoma and secondary glaucoma for IOP >21mmHg (e,f).

- 2 Humphrey

 2 visual fields pre-operation but only 10-2 fields post-operation. Postoperation there was no statistically significant deterioration in MD. Both the change in mean MD of the group as a whole and the change in individual patients' MDs for each of the time periods (years 1 (n=58),3 (n=29) and 5 (n=14))

Figure 1 :

 1 Figure 1: Plot demonstrating mean IOP and mean number of drops used for each year of follow up.

Figure 2 :

 2 Figure 2: Kaplan-Meier survival curves demonstrating qualified and unqualified survival outcomes for defined success IOPs of 21mmHg and 16mmHg (a-d). Survival outcomes between primary open angle glaucoma and secondary glaucoma for IOP >21mmHg (e,f).

Table 1 .

 1 Patient demographics

	had poor

*

21 Patients unable to complete a reliable VF test POAG-primary open angle glaucoma, PXS-pseudoexfoliation syndrome, PDSpigment dispersion syndrome, NTG-normal tension glaucoma, CACG-chronic angle closure glaucoma, ** Other (Congenital 1, postpenetrating keratoplasty 3, traumatic 1, rubeotic 3).

Table 2 .

 2 Change in IOP (

	number)	Mean	SD	Median	Range
	Presenting (87)	31.5	11.5	30.0	14-60
	Pre-operative (103)	25.8	8.0	24.0	13-55
	Day1 (102)	14.3 *	8.1	13.5	1-40
	Month 3 (102)	11.3 *	4.8	10.0	2-28
	Year 1 (87)	12.3 *	5.2	12.0	2-30
	Year 2 (73)	13.1 *	5.6	12.0	5-40
	Year 3 (59)	12.9 *	4.3	13.0	2-22
	Year 4 (38)	12.3 *	3.8	12.0	6-22
	Year 5 (26)	12.5 *	5.0	12.0	6-30
	Year 6 (14)	11.5 *	3.6	11.0	4-18
	Year 7 (3)	13.3 ^	3.5	13.0	10-17

* p<0.0005, ^p=0.02 compared with pre-op IOP)

Table 3 .

 3 Reasons for decline in VA.

	Pathology	Number
	Progressive glaucoma	7
	No specific cause (presumed fluctuation in VA)	5
	Cataract-declined surgery	4
	CRVO	2
	Corneal decompensation	2
	Dry AMD	2
	Progressive glaucoma plus cataract	2
	Macroaneurysm (leaking)	1
	Uncontrolled IOP followed by pthisis	1
	Extinction	1
	Dry AMD and progressive glaucoma	1

The only pre-operative determinant for a significant reduction in VA was the pre-operative MD (-27.00dB (n=21) compared with -24.79dB (n=63))

indicating those patients with more advanced field defects were more likely to experience a reduction in VA post-operatively (p=0.029). No association with VA loss was demonstrated between sex, surgeon, surgical approach, dose of MMC, number of pre-operative drops, any post-operative bleb manipulation or age at the time of surgery.

Table 4 .

 4 Frequency and timing (in days) for different types of postoperative bleb manipulations.

	. The first intervention

  of the patients previously able to reliably complete a visual field examination were subsequently unable, possibly as a result of disease progression or poorer

acuity. Additionally a number of patients had only 10:2 Humphrey visual fields performed post-operatively making direct comparison to the pre-operative field test impossible. A similar attrition rate was demonstrated by Law et al where only 39% of patients with advanced glaucoma were able to complete a reliable field test 1 year post-trabeculectomy.

[START_REF] Law | Intermediate-term comparison of trabeculectomy with intraoperative mitomycin-C between Asian American and Caucasian glaucoma patients: a case-controlled comparison[END_REF]

Loss of vision

Worries exist as to the possibility of severe loss of central vision or "extinction" described as the sudden reduction of visual acuity after filtration surgery in endstage glaucoma, with no apparent ocular pathology to account for this decline. [START_REF] Costa | Loss of visual acuity after trabeculectomy[END_REF] In total 27% (28/104) experienced a significant reduction in VA defined by a loss of 2 or more lines of Snellen acuity. Cataract formation was in the order of 63% which is comparable to that of the AGIS study (78% risk) and could not, by itself explain the drop in acuity. [START_REF]The Advanced Glaucoma Intervention Study: 8. Risk of cataract formation after trabeculectomy[END_REF] The only pre-operative factor found to be predictive for VA loss was more advanced visual field loss. Additionally we were unable to demonstrate any association between post-op intervention and VA loss. This is significant as this is one of the first studies specifically assessing the impact of post-operative bleb manipulation on final VA. Importantly all but one had ocular pathology which explained this loss of acuity with a minority associated with the surgery. In the case where acuity was lost inexplicably (presumed wipeout) the VA dropped from 6/18 to HM post-operation. Estimates of the risk of severe loss of central vision range from rare to 15.4% in patients with advanced preoperative field defects. [START_REF] Topouzis | Risk of sudden visual loss following filtration surgery in end-stage glaucoma[END_REF][START_REF] Law | Severe loss of central vision in patients with advanced glaucoma undergoing trabeculectomy[END_REF][START_REF] Costa | Loss of visual acuity after trabeculectomy[END_REF][START_REF] Martinez | Risk of postoperative visual loss in advanced glaucoma[END_REF][START_REF] Aggarwal | Risk of sudden visual loss following trabeculectomy in advanced primary open-angle glaucoma[END_REF] Only two have concentrated specifically on trabeculectomy augmented with MMC. [START_REF] Topouzis | Risk of sudden visual loss following filtration surgery in end-stage glaucoma[END_REF][START_REF] Law | Severe loss of central vision in patients with advanced glaucoma undergoing trabeculectomy[END_REF] The larger of these studies suggests severe loss of central vision following surgery to be in the order of 6%, however none of these were attributed to extinction and of the 7 eyes (out of a total of 117) experiencing loss of central vision, the decline was attributed to hypotony maculopathy (3), uncontrolled elevated IOP (2), posterior subcapsular lens opacities (1) and inflammatory reaction (1). [START_REF] Law | Severe loss of central vision in patients with advanced glaucoma undergoing trabeculectomy[END_REF] Similarly Topouzis' study of 21 consecutive patients with end-stage glaucoma undergoing MMC augmented trabeculectomy found no unexplained loss of vision although follow-up was for only 3 months. [START_REF] Topouzis | Risk of sudden visual loss following filtration surgery in end-stage glaucoma[END_REF] Five patients had no identifiable cause for visual acuity loss. It is presumed that fluctuation in Snellen acuity accounted for their loss although this assumption cannot be proven. The extent of the loss in all these cases were 2 Snellen lines only.

The information in this study may be extrapolated to managing patients initially presenting with advanced disease. A relatively large number of patients continue to present with advanced glaucoma, which tends to be associated with social deprivation in the UK. [START_REF] Fraser | Risk factors for late presentation in chronic glaucoma[END_REF][START_REF] Fraser | Deprivation and late presentation of glaucoma: case-control study[END_REF] A recent systematic review suggested that evidence from several studies supported the use of primary surgery over primary medical treatment in patients presenting with advanced glaucoma. [START_REF] Burr | Medical versus surgical interventions for open angle glaucoma[END_REF] This is further supported from the results of CIGTS which indicated that patients with severe disease at presentation had slower visual field loss when randomised to the surgery group. [START_REF] Musch | Visual field progression in the Collaborative Initial Glaucoma Treatment Study the impact of treatment and other baseline factors[END_REF] Furthermore primary surgery would avoid the effects of long term antiglaucoma therapy which may affect the outcomes of glaucoma surgery. [START_REF] Broadway | Adverse effects of topical antiglaucomatous medications on the conjunctiva[END_REF][START_REF] Broadway | Adverse effects of topical antiglaucoma medication. II. The outcome of filtration surgery[END_REF] Limitations of this study include the loss of numbers, particularly those able to complete a reliable visual field test during the follow-up period and refraction at


Karen Grønskov
email: karengrønskovkag@kennedy.dk

Karen Brøndum-Nielsen

Alma Dedic

Helle Hjalgrim

A nonsense mutation in FMR1 causing fragile X syndrome

Keywords:

Fragile X syndrome is a common cause of inherited intellectual disability. It is caused by lack of the FMR1 gene product FMRP. The most frequent cause is an expansion of a CGG repeat located in the 5' UTR of FMR1. Alleles with 200 or more repeats become hypermethylated and transcriptionally silent. Only few patients with intragenic point mutations in FMR1 have been reported and to-day routine analysis of patients referred for fragile X syndrome includes solely analysis for repeat expansion and methylation status. We identified a substitution in exon 2 of FMR1, c.80C>A, causing a nonsense mutation p.Ser27X, in a patient with classical clinical symptoms of fragile X syndrome.

The mother who carried the mutation in heterozygous form presented with mild intellectual impairment. We conclude that further studies including Western blot and DNA sequence analysis of the FMR1 gene should be performed in patients with typical symptoms of fragile X syndrome, in whom no CGG repeat expansion is detected.

INTRODUCTION

Fragile X syndrome is a frequent form of inherited intellectual disability affecting approximately 1 in 4000 males (reviewed by Chonchaiya et al. [START_REF] Chonchaiya | Fragile X: a family of disorders[END_REF] ). In the vast majority of patients the syndrome is caused by an expansion of a CGG repeat located in the 5'UTR of the FMR1 gene localized to Xq27.3. The gene was cloned in 1991 and comprises 17 coding exons. The protein, FMRP, is an RNA binding protein of 632 amino acids. [START_REF] Verkerk | Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome[END_REF] Only four point mutations in FMR1 have been reported: a missense mutation p.Ile304Asn, a 1-bp deletion c.373delA in exon5 resulting in a frameshift and premature truncation of the protein, a 2-bp change g.23714GG>TA spanning the intron/exon boundary of exon 2, and a missense mutation p.Arg138Gln. [START_REF] Collins | Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males[END_REF][START_REF] Deboulle | A point mutation in the FMR-1 gene associated with fragile X mental retardation[END_REF][START_REF] Lugenbeel | Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome[END_REF] Deletions of the CGG repeat and flanking sequences have been reported several times. [START_REF] Coffee | Mosaic FMR1 deletion causes fragile X syndrome and can lead to molecular misdiagnosis: a case report and review of the literature[END_REF]

MATERIALS AND METHODS

Patient

The patient, a 35 year old mentally retarded male, was referred to a tertiary epilepsy centre due to difficulties in treatment of epilepsy with absences, generalised tonic-clonic seizures and myoclonias. As a further diagnostic evaluation he was tested for fragile X syndrome. Phenotypically he had classical fragile X syndrome with elongated face, high, broad forehead, low set large ears, prognathia and enlarged testes. Neurological examination showed hypotonia and hypermobility, with hyperextensible joints. He had no active language except a few repeated words used out of context, and showed autistic features with little eye contact, discomfort upon physical contact, perseveration and limited interests. He was described by his mother as showing late motor development in childhood and having need for speech therapy, and special day-care and schooling.

Epilepsy debuted at age 4 years.

The mother presented with mild to moderate intellectual disability. She reported special education needs and carried out sheltered work. She was married and capable of maintaining a household. Physical examination showed a relatively unremarkable phenotype, but her behaviour included hypermotor activity and many automatisms. She was capable of a simple conversation.

Molecular biology procedures

Genomic DNA was extracted from peripheral blood lymphocytes (EDTA stabilized) using standard procedures.

Lymphobalstoid cell lines were established from the patient and his mother using Ebstein Barr virus transformation.

Southern blot analysis was performed as described. [START_REF] Gronskov | Deletion of all CGG repeats plus flanking sequences in FMR1 does not abolish gene expression[END_REF] Briefly, 9 µg of high molecular genomic DNA was digested with Pst1 and 6 µg DNA was digested with EcoR1 plus EagI and size separated on an agarose gel. Standard blotting technique was performed to transfer DNA to nitrocellulose filter, which was hybridized with radioactive labeled pPX6 probe. After washing the filter was analyzed using a Cyclone from Perkin Elmer (Waltham, Massachusetts, USA). PCR analysis of the FMR1 CGG repeat was performed using the fragile X kit from Abbott Molecular (Illinois, USA) following the manufacturer's instructions.

Western blot analysis was performed as described. [START_REF] Gronskov | Deletion of all CGG repeats plus flanking sequences in FMR1 does not abolish gene expression[END_REF] Mutational analysis of FMR1 was performed by direct DNA sequencing of PCR products of coding exons and at least 20 bp of flanking sequences. Primers and PCR conditions are available upon request. Sequencing was performed using BigDye v3.1 terminator chemistry, and an ABI3130XL genetic analyzer and using the Seqscape program for analysis (Applied Biosystems, Foster City, CA, USA). X-inactivation analysis was performed using the polymorphic CAG repeat in the androgen receptor locus as described. [START_REF] Gronskov | Deletion of all CGG repeats plus flanking sequences in FMR1 does not abolish gene expression[END_REF]

RESULTS

The patient and his mother are shown in figure 1.

Southern blot analyses of FRAXA (EcoRI+EagI and PstI digestion respectively)

showed band patterns corresponding to a repeat number in the normal range (Figure 2A and Figure 2B). PCR analysis showed an allele size corresponding to 29 repeats.

However, based on the classic clinical fragile X appearance we performed a Western blot using an anti-FMRP antibody, which showed that no FMRP was expressed (Figure 2C).We also performed Southern blot analysis and PCR of FRAXA of the mother of the patient; this showed a normal band pattern and repeat number of 29 and 33 with no indication of a full mutation. Subsequent DNA sequencing revealed a sequence variation in exon 2 of FMR1 c.80C>A resulting in a nonsense mutation p.Ser27X.

Verification of the sequence variation was performed on a second PCR product, and the variation was seen in both directions. The mother was found to be heterozygous for the mutation. We investigated X-inactivation pattern using a polymorphic CAG repeat in the androgen receptor locus and found an equal distribution of active and inactive Xchromosomes in blood (data not shown)..

DISCUSSION

We report a nonsense mutation in FMR1 in a patient with classic fragile X syndrome and his carrier mother with mild intellectual impairment. The fragile X syndrome can be considered semi-dominant with manifestations in female carriers of a full mutation depending on the proportion of active versus inactive X chromosomes carrying the mutation. Older studies showed that phenotypical normal females have a skewed X inactivation pattern in favor of the inactive X carrying the mutation. [START_REF] Martinez | Skewed X inactivation of the normal allele in fully mutated female carriers determines the levels of FMRP in blood and the fragile X phenotype[END_REF] Since the X inactivation in the mother showed an equal distribution, some degree of intellectual impairment would be expected. However, this study was performed in blood and therefore is an approximation since we do not know the pattern in brain.

Surprisingly few point mutations have been found in the FMR1 gene. Two of these are truncating mutations in the N-terminal half of the gene and supposedly lead to nonsense mediated decay of the mRNA. These two mutations caused classical fragile X syndrome. [START_REF] Lugenbeel | Intragenic loss of function mutations demonstrate the primary role of FMR1 in fragile X syndrome[END_REF] Another patient had a missense mutation in one of the KH domains and had a more severe phenotype. [START_REF] Deboulle | A point mutation in the FMR-1 gene associated with fragile X mental retardation[END_REF] This could be due to a gain-of-function effect; however, in this family a glucokinase defect was also present which might have contributed to the phenotype. The observed low frequency of point mutations in FMR1 might be because FMR1 is not routinely screened for point mutations, but only investigated for the common CGG expansion; another reason could be that point mutations in this gene give atypical or even lethal phenotypes. More than 1100 patients have been screened for point mutations in FMR1, [START_REF] Collins | Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males[END_REF][START_REF] Castellvi-Bel | Single-strand conformation polymorphism analysis in the FMR1 gene[END_REF][START_REF] Chiurazzi | No apparent involvement of the FMR1 gene in five patients with phenotypic manifestations of the fragile X syndrome[END_REF][START_REF] Collins | Array-based FMR1 sequencing and deletion analysis in patients with a fragile X syndrome-like phenotype[END_REF][START_REF] Gronskov | Mutational analysis of the FMR1 gene in 118 mentally retarded males suspected of fragile X syndrome: absence of prevalent mutations[END_REF][START_REF] Reyniers | Severe mental retardation and macroorchidism without mutation in the FMR1 gene[END_REF][START_REF] Shinahara | Single-strand conformation polymorphism analysis of the FMR1 gene in autistic and mentally retarded children in Japan[END_REF][START_REF] Wang | Novel point mutation within intron 10 of FMR-1 gene causing fragile X syndrome[END_REF] however only one missense mutation of questionable pathogeneity was identified. [START_REF] Collins | Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males[END_REF] The individuals investigated in these studies represent a very heterogenous group of patients with developmental delay, indicating that FMR1 mutations are not a common cause for mental impairment. To our knowledge, a mutational screening of FMR1 has not been performed in a large cohort of patients with typical fragile X syndrome negative for repeat expansion. Therefore, the frequency of point mutations in FMR1 in classical fragile X syndrome is unknown. The patient presented in this study shows a typical fragile X phenotype, and his mother shows a phenotype comparable to a female with a full mutation. We therefore suggest that in patients with clinical fragile X syndrome and no CGG expansion, expanded molecular diagnosis should be considered including Western blot analysis and DNA sequencing.

TITLES AND LEGENDS TO FIGURES

Facial appearance of the a) patient and b) his mother. Note the elongated face, high broad forehead and prognathia in patient (a). Figure 2 A. Southern blot analysis using EcoRI+EagI digestion and B. Southern blot analysis using PstI digestion. P: patient; M: mother; FM ♀: full mutated female; PM ♀: premutated female; FM ♂: full mutated male; PM ♂: premutated male; N ♂ normal male; N ♀: normal female. C. Western blot analysis using an anti-FMRP antibody. Patient (P); FM ♂: full mutated male; N ♂ normal male.

  Figure 2

CONFLICT OF INTEREST

The authors declare no conflict of interest.