Dirk Ahrens

MD, MPH Jean-François Chenot
email: jchenot@gwdg.de

Gesa Behrens

Thomas Grimmsmann

Michael M Kochen

Behrens

Appropriateness of treatment recommendations for PPI in hospital discharge letters

teaching and research institutions in France or abroad, or from public or private research centers.

PPIs are indicated for the treatment of gastro-oesophageal reflux disease (GERD) [START_REF] Moayyedi | Gastro-oesophageal reflux disease[END_REF], peptic ulcer [START_REF] Leontiadis | Proton pump inhibitor treatment for acute peptic ulcer bleeding[END_REF] and, in combination with two suitable antibiotics, for the eradication of Helicobacter pylori infection. After eradication, continuation of PPI is not necessary [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF]. PPIs also are recommended for prevention of NSAID and aspirin induced ulcers in high risk patients [START_REF] Dubois | Guidelines for the appropriate use of nonsteroidal anti-inflammatory drugs, cyclo-oxygenase-2-specific inhibitors and proton pump inhibitors in patients requiring chronic anti-inflammatory therapy[END_REF][START_REF] Chan | Review article: prevention of non-steroidal anti-inflammatory drug gastrointestinal complications--review and recommendations based on risk assessment[END_REF][START_REF] Lanza | Guidelines for prevention of NSAID-related ulcer complications[END_REF] (Table 1) and treatment of gastritis. In intensive care, PPIs are indicated for stress ulcer prophylaxis in patients with risk of bleeding [START_REF] Quenot | When should stress ulcer prophylaxis be used in the ICU?[END_REF]. The use of PPI for patients with Barrett-Oesophagus is controversial and prevention of carcinoma induction has not yet been demonstrated [START_REF] Armstrong | Canadian Consensus Conference on the management of gastroesophageal reflux disease in adults -update 2004[END_REF]. A Cochrane Review reported that PPI could be effective in a small proportion of patients with dyspepsia, but studies showed a significant heterogeneity. Some guidelines recommend testing for H. pylori and eradication if necessary. Alternatively, an empirical PPI-treatment could be tried for 4-8 weeks [START_REF] Talley | Guidelines for the management of dyspepsia[END_REF]. Also, there is no clear evidence to support the assumption that PPIs prevent bleeding and promote quicker healing after ligation in patients with liver cirrhosis and oesophageal varices [START_REF] Lodato | Proton pump inhibitors in cirrhosis: tradition or evidence based practice?[END_REF]. Some authors recommend ulcer prophylaxis for patients on a combination of aspirin and clopidogrel [START_REF] Ng | Upper gastrointestinal bleeding in patients with aspirin and clopidogrel co-therapy[END_REF] but there has been some concerns about interaction of clopidogrel and proton pump inhibitors reducing cardiovascular protection and increasing arteriosclerotic complications [13;14].

Prescription of PPIs without clear indications has been frequently observed in many

countries in hospitals [START_REF] Saad | Proton pump inhibitors: a survey of prescribing in an Irish general hospital[END_REF][START_REF] Niklasson | Overuse of acid suppressive therapy in hospitalised patients with pulmonary diseases[END_REF][START_REF] Scagliarini | Inadequate use of acidsuppressive therapy in hospitalized patients and its implications for general practice[END_REF][START_REF] Strid | Overuse of acid suppressant drugs in patients with chronic renal failure[END_REF][START_REF] Zink | Long-term use of acid suppression started inappropriately during hospitalization[END_REF] and primary care [START_REF] Hungin | Long-term prescribing of proton pump inhibitors in general practice[END_REF] alike. Reported rates of nonindicated prescriptions on general medical wards ranged from 40% to 81% [START_REF] Saad | Proton pump inhibitors: a survey of prescribing in an Irish general hospital[END_REF][START_REF] Niklasson | Overuse of acid suppressive therapy in hospitalised patients with pulmonary diseases[END_REF][START_REF] Scagliarini | Inadequate use of acidsuppressive therapy in hospitalized patients and its implications for general practice[END_REF][START_REF] Strid | Overuse of acid suppressant drugs in patients with chronic renal failure[END_REF][START_REF] Zink | Long-term use of acid suppression started inappropriately during hospitalization[END_REF].

Inadequate acid-suppressive medication is often continued after discharge for long time [19;21].

Although PPIs are generally considered safe it has been shown that long term use might be associated with hip fractures [22;23], pseudomembranous colitis [START_REF] Dial | Use of gastric acid-suppressive agents and the risk of community-acquired Clostridium difficile-associated disease[END_REF] and Two raters (DA and GB) assessed all clinical information available in the discharge letter (including co-medication) justifying a recommendation for continuous treatment with PPIs. Indication was rated as adequate, inadequate or uncertain (Table 1). Adequate use of PPI was defined according to approved indications (as written in the official product information

Results

In the participating practices a total of 2951 patients discharged from hospitals were identified in the respective time period. Practice nurses identified 681 (23%) hospital discharge letters with PPI recommendations. The patient flow is shown in figure 1. Of the participating patients, 382 (57%) were female; the mean age was 70.7 years (SD ± 13.7). Demographic data and relevant medication of patients discharged with PPI is shown in table 2. In 96 (14.2%) of 209 patients with documented upper gastrointestinal endoscopy H. pylori testing was performed (44 tested positive). 17 patients had no pathologic findings on endoscopy and did not take any ulcer inducing medication.

In 371 (54.5%) of all discharge letters no information justifying the recommendation for continuous PPI recommendation could be identified. In 12.7% indication was uncertain, and in 32.7% we found an evidence based indication for PPI medication.

The most common indication for adequate PPI use was NSAID-prophylaxis in high risk-patients, followed by endoscopically proven gastritis. In patients without adequate indication most commonly no reason for PPI-administration could be found in the discharge letters, followed by ulcer prophylaxis in patients taking low dose aspirin alone (Table 3).

Factors associated with lower risk of inadequate recommendation for PPI in univariate analysis were endoscopy (OR 0.18 CI 95 0.12-0.16), testing for H. pylori (OR 0.1 CI 95 0.06-0.18), NSAID (OR 0.15 CI 95 0.08-0.25) or oral anticoagulation (OR 0.4 CI 95 0.22-0.69). No association was found with age, gender, polypharmacy (defined as more then 5 concomitant medications) and hospital type (Table 2 Guidelines recommend peptic ulcer prophylaxis only for intermediate to high risk patients. Without risk factors no peptic ulcer prevention is necessary in patients using NSAID or low-dose ASA [5;7]. In addition there is evidence that concomitant use of PPIs might reduce the cardiovascular protection by aspirin [START_REF] Wurtz | The antiplatelet effect of aspirin is reduced by proton pump inhibitors in patients with coronary artery disease[END_REF]. Concerns also exist about the interaction of PPI and clopidogrel, increasing arteriosclerotic complications [13;14]. Hence we rated ulcer prophylaxis in the combination of aspirin and clopidogrel as an uncertain indication, but it should be considered that the relating studies were published after the study sampling period. Steroids without concomitant NSAID therapy do not significantly increase the risk of peptic ulcers [START_REF] Piper | Corticosteroid use and peptic ulcer disease: role of nonsteroidal anti-inflammatory drugs[END_REF]. For oral anticoagulation in therapeutic range without history of ulcer/ bleeding or concomitant NSAID treatment no ulcer prophylaxis is necessary [START_REF] Petty | Frequency of major complications of aspirin, warfarin, and intravenous heparin for secondary stroke prevention. A population-based study[END_REF].

Stress ulcer prophylaxis is recommended only for high risk patients at intensive care wards. For these patients, respiratory failure requiring mechanical ventilation and coagulopathy have been identified as strong independent risk factors [START_REF] Cook | Risk factors for gastrointestinal bleeding in critically ill patients. Canadian Critical Care Trials Group[END_REF]. Aside these factors, American Society of Health Service guidelines note sepsis and specific illnesses as spinal cord injury as determining factors for stress ulcer prophylaxis [START_REF]ASHP Commission on Therapeutics and approved by the ASHP Board of Directors on November 14, 1998[END_REF].

Patients from ICU wards have been excluded from our study.

Diagnosis of gastritis is based on the histological examination of the gastric mucosa.

Macroscopic signs like erythema or erosion are of very limited value in the evaluation of gastritis and helicobacter pylori infection [31;32]. We considered recommendation of continuous PPI therapy based only on visual findings of macroscopic erythema or erosion to be inadequate, if there were no contraindications for biopsy and no biopsy was taken.

We assume that low dose aspirin or NSAIDs in low risk patients, steroid therapy or oral anticoagulant treatment might have been a frequent trigger for inappropriate prescriptions (Table 3). For more than one third of inappropriate PPI prescriptions we found no explaination. Although endoscopy, testing for H. pylori and comedication with NSAID or oral anticoagulation were factors decreasing significantly the risk of inadequate PPI recommendation more than half of these patients had no identifiable indication for PPI prescription. It is noticeable that 17 patients in our sample underwent endoscopy and were subsequently prescribed PPIs although no pathology was found and no ulcerogenic medication was administered.

The high rates of inappropriate PPI prescriptions in hospitals observed in our study are consistent with previous reports. Two Swedish studies found 59-81% of hospitalised patients receiving acid suppression therapy without appropriate indication [16;18], an Italian study 41,5% [START_REF] Scagliarini | Inadequate use of acidsuppressive therapy in hospitalized patients and its implications for general practice[END_REF] and 68%. [START_REF] Parente | Hospital use of acidsuppressive medications and its fall-out on prescribing in general practice: a 1-month survey[END_REF] A one-day-survey at an Irish hospital showed 30% of patients on PPI-medication; 71% of them were started in hospital and 33% had no evidence-based indication [START_REF] Saad | Proton pump inhibitors: a survey of prescribing in an Irish general hospital[END_REF]. An American study found 60% of medically unfounded PPI treatment started in hospital [START_REF] Zink | Long-term use of acid suppression started inappropriately during hospitalization[END_REF]. After discharge 46-80% of patients were still on PPIs after 3 months and 50% after 6 months [19;21]. Inappropriate recommendation of PPI is an important issue mainly for two reasons.

First, administration of unnecessary medication leads to polypharmacy and could cause side-effects and pharmacological interactions. PPI use has been found to have a significant association with community acquired pneumonia [START_REF] Laheij | Risk of community-acquired pneumonia and use of gastric acid-suppressive drugs[END_REF] and Clostridium difficile associated diarrhoea [START_REF] Dial | Use of gastric acid-suppressive agents and the risk of community-acquired Clostridium difficile-associated disease[END_REF] Long term PPI therapy has been suspected to be associated with increased risk of hip fractures [22;23]. PPI are a major burden for the national health care budget. It is noticeable, that more than two thirds of letters recommended pantoprazole which was much more expensive than generic omeprazole at the time of the study. At equipotent doses proton pump inhibitors are considered to be equally efficient in inhibition of gastric acid secretion [33;34]. It has been shown previously that hospitals significantly influence drug prescription in primary care [START_REF] Grimmsmann | The influence of hospitalisation on drug prescription in primary care--a large-scale follow-up study[END_REF]. Moreover, in Germany many hospitals receive PPIs at no or minimal cost and manufacturers expect that continued prescribing in primary care will reimburse them for this initial "loss" [START_REF] Kochen | Proton pump inhibitors -informations on rational pharmacotherapy in primary care [Protonenpumpenhemmer-Informationen zur rationalen Arzneitherapie in der hausärztlichen Praxis[END_REF]

INDICATIONS rated as ADEQUATE

Gastroesophageal reflux disease [START_REF] Moayyedi | Gastro-oesophageal reflux disease[END_REF] Treatment and recurrent prophylaxis of peptic ulcer [3;37] Eradication of Helicobacter pylori [START_REF] Malfertheiner | Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report[END_REF] Pathologic hypersecretory conditions (e.g. Zollinger-Ellison-Syndrome) [START_REF] Wilcox | Treatment strategies for Zollinger-Ellison syndrome[END_REF] Histological proven diagnosis of gastritis [31;32] Prevention of medication induced ulcers: [START_REF] Dubois | Guidelines for the appropriate use of nonsteroidal anti-inflammatory drugs, cyclo-oxygenase-2-specific inhibitors and proton pump inhibitors in patients requiring chronic anti-inflammatory therapy[END_REF][START_REF] Chan | Review article: prevention of non-steroidal anti-inflammatory drug gastrointestinal complications--review and recommendations based on risk assessment[END_REF][START_REF] Lanza | Guidelines for prevention of NSAID-related ulcer complications[END_REF] -NSAID at patients >65 years -NSAID and Corticosteroid -NSAID and Warfarin /Coumadin -NSAID and patient history of ulcer/ GI bleeding -Aspirin and Corticosteroid -Aspirin and Warfarin /Coumadin -Aspirin and NSAID

INDICATIONS rated as UNCERTAIN

Dyspepsia

Barrett-Oesophagus [START_REF] Armstrong | Canadian Consensus Conference on the management of gastroesophageal reflux disease in adults -update 2004[END_REF] Oesophageal varices [10;11] Ulcer prophylaxis with clopidogrel and low dose aspirin [13;14] Patient underwent upper gastrointestinal endoscopy and biopsy, result outstanding at discharge [31;32] History of gastritis, no endoscopy, no further information Anaemia, no endoscopy NSAID= Non-steroidal anti-inflammatory drug

Introduction

  Proton pump inhibitors (PPIs) are the most potent medications available to reduce gastric acid secretion. Their use is widespread and increasing and annual sales of PPIs worldwide have surpassed $ 25 billion. Prescription of PPI in Germany rose from 44 Million DDD in 1993 to 1674 million DDD in 2008 (+3805%) with an associated costs of 540 million Euros per year. The reasons for this are unclear and cannot be explained solely by increased morbidity, new indications [1] or a decrease in alternative medication.

Methods

  as pneumonia.[START_REF] Laheij | Risk of community-acquired pneumonia and use of gastric acid-suppressive drugs[END_REF] In addition the cost of unnecessary medication burdens the national health budget.The aim of this study was to analyse the appropriateness of PPI treatment recommendations in patients discharged from hospital in a large German county. This cross sectional observational study was conducted in 35 primary care practices in the state of Mecklenburg-Vorpommern (MV), North-Eastern Germany.Recruitement of practices:We invited all 933 registered GP practices in MV to participate in the study. Addresses were obtained by the Association of Statutory Health Insurance Physicians (Kassenaerztliche Vereinigung). A total of 97 GPs agreed to join the study and 35 practises were included in this study (Figure1) The sample was stratified by area; two practices from each of 12 rural districts and 6 major towns in MV were randomly selected.Identification of patients:Patients included in this study were members of the AOK (Allgemeine Ortskrankenkasse) MV, the largest statutory health insurance covering 27% of the population in MV. All patients from the participating practices > 18 years of age discharged from hospital between July 1 2006 and June 30 2007 were identified from insurance records. In patients with multiple hospital admission only the first discharge was included. Patients with intensive care treatment were excluded. Data collection: Practice nurses received an instruction containing the names of all PPI preparations available in Germany. They screened discharge letters of all identified patients for PPIs in the discharge medication. Letters recommending PPIs were copied, anonymized and sent to the study centre.

  ) and indications supported by clinical guidelines and scientific literature. If PPI recommendation was rated as inadequate, we analysed the discharge letter to assess what might have triggered the decision. Differences in judgment between the authors were resolved by discussion.The study was approved by the ethics committee of the medical school of the university of Göttingen.Statistical analysis:We use simple descriptive statistics. To explore factors associated with inadequate recommendation of PPI, we conducted univariate analyses comparing patients who received adequate PPI recommendation with those not. In a second step, we performed logistic regression analysis to calculate the probability of receiving an inadequate PPI recommendation. We excluded patients with uncertain indication from the regression model. Covariates retained in the final model were selected with the score procedure. Goodness of fit was assessed with the Hosmer Lemshow-test. The software package SAS 9.2 was used for analysis.

Discussion

  Our study confirms that PPIs in hospitals are often given without a clear indication. In 54.5% of analysed discharge letters recommending PPI no appropriate indication justifying continuous prescription could be identified and in 12.8% indication was uncertain.

  Doctors might not be aware of existing guidelines and use PPI uncritically in good faith for stress ulcer prophylaxis without indication. Inappropriate assumptions about the risk of ulcer development during hospitalisation might be an explanation.

10 This

 10 is the first study in Germany to assess appropriateness of PPI recommendation in hospital discharge letters. Theoretically it is possible that practices failed to identify all discharge letters with PPI recommendation. However, it seems unlikely to have introduced a significant selection bias. Our assessment of appropriate drug recommendation is solely based on the available information in the discharge letter and numbers of inappropriate PPI recommendations might be slightly overestimated.Nevertheless discharge letters should provide sufficient information to the GP to understand the recommendation in case specific hospital drugs are to be continued in primary care.Hospitals should critically review their practice of recommending PPI in their discharge letters and clearly document the reason for continued PPI use after discharge. Likewise GPs should carefully assess the need for continuous prescription. The reasons why evidence-based clinical guidelines are obviously not observed merits further research.

Table 1 :

 1 Rating of indications for proton pump inhibitors

Table 2 :

 2 Baseline socio-demographic and clinical characteristics and univariate analysis of association with inadequate prescription of PPI (significant associations are bold)

			Comparison of
			inadequate with
	n = 681		adequate PPI
			recommendation
			n = 594*
	Age	71 (SD ±14)	n.s.
	Sex
	female	382 (57.0%) OR 0.87 (CI 95 0.6-1.2)
	male	299 (43.0%)
	Length of stay median (days)	9 (IQR 6-14)	n.s.
	Hospital category		n.s.
	primary / regional care	214 (31.4%)
	secondary dare centres	266 (39.0%)
	tertiary care centres	100 (14.7%)
	specialty care centres	101 (14.8%)
	Endoscopy	209 (30.7%) OR 0.18 (CI 95 0.12-0.16)
	Testing for H. pylori	96 (15.0%)	OR 0.1 (CI 95 0.06-0.18)
	Comedication
	none	16 (2.3%)	0.26 (CI 95 0.09-0.77)
	NSAID	85 (12.5%)	0.15 (CI 95 0.08-0.25)
	Aspirin (low dose)	243 (36.0%)	1.2 (CI 95 0.8-1.73)
	Coxib	23 (3.4%)	1.96 (CI 95 0.71-5.4)
	Corticoid	59 (8.7%)	0.66 (CI 95 0.38-1.1)
	Warfarin/ Coumadin	54 (7.9%)	0.4 (CI 95 0.22-0.69)
	Polypharmacy (> 5 substances)	520 (76.4%) OR 1.15 (CI 95 0.79-1.68)
	* Patients with indeterminate indication for PPI were excluded
	OR: odds ratio, CI95 95% confidence interval, SD = standard deviation, IQR interquartile range
	NSAID= Non-steroidal anti-inflammatory drug, PPI Proton pump inhibitor

Table 3 :

 3 Rating of indication-Results and categories (n = 681)

	Rating of
	indication for	Presumed reason for recommendation of continuous prescription of PPIs
	PPI
	Adequate	-NSAID in high-risk patients	90 (40.4%)
	documented	-Gastric or duodenal ulcer	46 (20.6%)
	indication	-GERD	45 (20.2%)
	n=223	-Proven gastritis with/ without helicobacter	37 (16.6%)
	(32.8 %)	-Macroscopic erosion or erythema, no biopsy because of oral	5 (2.2%)
		anticoagulation
	Documented	-Clopidogrel and low dose aspirin	43 (49.4%)
	indication	-Macroscopic gastric erythema or erosion, result outstanding at	13 (15.0%)
	uncertain	discharge
	n=87	-History of gastritis, no endoscopy, no further information	10 (11.5%)
	(12.8%)	-Oesophageal varices	10 (11.5%)
		-Barrett-Oesophagus	5 (5.7%)
		-Dyspepsia	4 (4.6%)
		-Anaemia, no endoscopy	2 (2.3%)
	No	-No reason for PPI found	140 (37.8%)
	documented	-Low-dose aspirin, no documented history of bleeding	108 (29.1%)
	adequate	-Macroscopic gastric erythema or erosion, no biopsy, no HP-testing	27 (7.3%)
	indication	-NSAID <65y, no history of bleeding	23 (6.2%)
	n=371	-Clopidogrel alone	23 (6.2%)
	(54.4%)	-Corticosteroid alone	21 (5.7%)
		-Oral anticoagulation	15 (4.0%)
		-Coxib alone	9 (2.4%)
		-PPI after completed HP-eradication, no ulcer	3 (0.8%)
		-Corticosteroid and oral anticoagulation	2 (0.5%)
	PPI = Proton pump inhibitor, NSAID= Non-steroidal anti-inflammatory drug,
	GERD = Gastroesophageal reflux disease

Table 4 :

 4 Recommended PPI / daily doses

	PPI Type	Pantoprazole	Esomeprazole	Omeprazole	Lansoprazole
	n=	487 (71.5%)	116 (17.0%)	77 (11.3%)	1 (0.2%)
	No dosage	4 (0.8%)	1 (0.9%)	3 (3.9%)
	20 mg	104 (21.3%)	60 (51.7%)	49 (63.6%)
	40 mg	312 (64.1%)	47 (40.5%)	20 (26%)	1 (100%)
	80 mg	65 (13.4%)	8 (6.9%)	5 (6.5%)
	120 mg	2 (0.4%)

European Journal of Clinical Pharmacology

Acknowledgements:

This study was financed by the German Ministry for Education and Science (BMBF); grant number 01GK0711. No other financial support was received. We are indebted to the AOK Statutory Health Insurance Mecklenburg-Vorpommern who helped to identify discharged patients.

Conflict of interests: None.


Steven J Lubbe

Cornelis Matijssen

Alan M Pittman

Philip Twiss

Bianca Olver

Amy Llyod

Mobshra Qureshi

Nathan Brown

Emma Nye

Gordon

Amy Lloyd

Gordon Stamp

Julian Blagg

Richard S Houlston
email: richard.houlston@icr.ac.uk

Evaluation of germline BMP4 mutation

Keywords: bone morphogenetic protein-4, BMP4, rare mutations, colorectal cancer bone morphogenetic protein-4, BMP4, rare mutations, colorectal cancer

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Transforming growth factor-β (TGF-β; OMIM 190180) signalling plays a key role in developmental biology, cell proliferation, differentiation and migration [START_REF] Xu | TGF-beta signaling alterations and susceptibility to colorectal cancer[END_REF]; and increasingly recognised to be important in cancer biology [START_REF] Tenesa | New insights into the aetiology of colorectal cancer from genome-wide association studies[END_REF]. Furthermore, germline mutations in the genes encoding the TGF-β signalling proteins, SMAD4 [START_REF] Howe | Mutations in the SMAD4/DPC4 gene in juvenile polyposis[END_REF]OMIM 600993) and BMPR1A [START_REF] Howe | Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis[END_REF]OMIM 601299) have been shown to cause juvenile polyposis, an autosomal dominant polyposis syndrome which confers a high risk of colorectal cancer (CRC).

Genome-wide association (GWA) studies have recently shown that common genetic variation in the gene encoding the TGF-β signalling protein BMP4 influences CRC risk [START_REF] Houlston | Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer[END_REF]. The bone morphogenetic protein-4 (BMP4; OMIM 112262; NM_001202.3) gene maps to 14q22.2 and is composed of 4 exons. Multiple BMP4 transcripts are generated [START_REF] Shore | The human bone morphogenetic protein 4 (BMP-4) gene: molecular structure and transcriptional regulation[END_REF], but only exons 3 and 4 are translated (van den [START_REF] Van Den Wijngaard | Genomic organization of the human bone morphogenetic protein-4 gene: molecular basis for multiple transcripts[END_REF], from which a homodimer of two 408 amino acid BMP4 polypeptides is formed. Six cysteine residues form the critical protein folding motif (referred to as the cysteine knot) while a seventh cysteine is responsible for homodimerisation of BMP4 [START_REF] Mcdonald | A structural superfamily of growth factors containing a cystine knot motif[END_REF]. This homodimer consisting of TGF-β1 prodomains and active domains [START_REF] Mcdonald | A structural superfamily of growth factors containing a cystine knot motif[END_REF] is proteolytically cleaved at two sites to generate the active ligand [START_REF] Aono | Potent ectopic boneinducing activity of bone morphogenetic protein-4/7 heterodimer[END_REF]. Mature BMP4 initiates TGF-β signalling by binding to type I (BMPR1A) and type II (BMPR2; OMIM 600799) serine or threonine kinase receptors, triggering intracellular SMAD-signalling [START_REF] Schmierer | TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility[END_REF].

It is increasingly being recognised that both common variants and coding mutations of the same gene can confer cancer susceptibility, exemplified by CDH1 (OMIM 192090) in colorectal cancer [START_REF] Houlston | Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer[END_REF][START_REF] Richards | Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer[END_REF][START_REF] Salahshor | A germline E-cadherin mutation in a family with gastric and colon cancer[END_REF]. To examine if rare germline mutations in the coding region of BMP4 cause CRC we analysed 504 genetically enriched CRC cases.

METHODS

Subjects

Five hundred and four CRC cases (298 male) were ascertained through the National Study of Colorectal Cancer (NSCCG) [START_REF] Penegar | National study of colorectal cancer genetics[END_REF] and the Royal Marsden Hospital NHS Trust Family History and DNA Registry (RMHNHST). All cases had histologically proven colorectal adenocarcinoma (International classification of diseases, 9th Revision [ICD9] codes 153 or 154) and none had previously been documented to have a diagnosis of a cancer syndrome known to be associated with increased CRC risk. To enhance our power to detect germline mutations, case selection was prioritised for early-age of onset (n=182 diagnosed <55 years, family history of CRC n=250) and microsatellite stable (MSS) disease (n=210). Samples from 524 healthy individuals collected through RMHNHST (219 males; mean age at sampling 58.0 years, SD 14.0), who did not have a personal history of malignancy at time of ascertainment served as a source of controls. Both cases and controls were UK residents and had self-reported European ancestry.

The study was conducted with informed consent and ethical approval (MREC 02/0/097 and RMHNHST-CCR1552) in accordance with the declaration of Helsinki.

Molecular analyses

Genomic DNA was salt-extracted from EDTA-venous blood samples [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF]. Amplification of genomic DNA was performed with 12.5ng DNA and PCR was carried out by use of Qiagen Multiplex Kit (QIAGEN Ltd, Crawley, UK). Sequencing was performed with Big Dye version 3.1 using ABI 3730xl semiautomated sequencers (Applied Biosystems, Foster City, USA) in accordance with the manufacturer's protocol. Sequence data was analysed using Mutation Surveyor (Soft Genetics, USA) and sequence changes were annotated against GenBank contig NC_000014 sequence data according to the nomenclature advocated by Human Genetic Variation (HGV; den Dunnen and Antonarakis, 2000; http://www.hgvs.org/).

To assess allelic imbalance at 14q22.2-BMP4 in the CRCs from cases carrying germline BMP4 mutations, DNA was extracted from microdissected formalin fixed paraffin embedded (FFPE) tumors using Qiagen DNA Mini kits (QIAGEN Ltd., Crawley, UK). Loss of heterozygosity was assessed by comparing peak heights of PCRamplified germline and tumor exon fragments encompassing mutations using ABI 3730xl semi-automated sequencers and Mutation Surveyor software.

Microsatellite instability (MSI) in CRCs was determined using BAT25 and BAT26 markers which are highly sensitive MSI markers [START_REF] Boland | A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer[END_REF], as previously described [START_REF] Penegar | National study of colorectal cancer genetics[END_REF]. Samples showing novel alleles at either or both markers were assigned as MSI (corresponding to MSI-high).

To assess promoter CpG island methylation of BMP4 (chr14:53,489,492,708 and 53,488,488,631) germline genomic DNA and tumor DNA from microdissected FFPE tumors were subjected to bisulfite conversion and were purified using the EpiTect Bisulfite kit (QIAGEN Ltd., Crawley, UK). PCR amplification of the putative BMP4 sequence was performed on eluted DNA and search for differential methylation conducted by Pyrosequencing technology (QIAGEN Ltd., Crawley, UK) using biotinylated oligonucleotide primers.

Details of all oligonucleotide primers used are shown in Supplementary Table 1.

Bioinformatic analyses

We applied two in silico algorithms, PolyPhen [START_REF] Ramensky | Human non-synonymous SNPs: server and survey[END_REF] http://genetics.bwh.harvard.edu/pph/) and SIFT [START_REF] Ng | Predicting deleterious amino acid substitutions[END_REF]; http://sift.jcvi.org/), to predict the putative effect of non-synonymous coding changes in BMP4 on expressed protein function. Protein sequence of BMP4 (NP_001193.2) was obtained from the NCBI Human RefSeq database [START_REF] Pruitt | NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins[END_REF]; http://www.ncbi.nlm.nih.gov/refseq/). PolyPhen scores were designated probably damaging (≥2.00), possibly damaging (1.50-1.99), potentially damaging (1.25-1.49), borderline (1.00-1.24), or benign (0.00-0.99) according to the classification proposed by Xi et al. 2004. SIFT scores were classified as intolerant (0.00-0.05), potentially intolerant (0.051-0.10), borderline (0.101-0.20), or tolerant (0.201-1.00) according to the classification proposed by [START_REF] Ng | Predicting deleterious amino acid substitutions[END_REF][START_REF] Ng | Predicting deleterious amino acid substitutions[END_REF]Xi et al., 2004. The effect of BMP4 mutations on the stability of BMP4, as well as the ability of BMP4 to interact with other proteins, was investigated by homology modelling using Molecular operating environment v2008.10 (MOE, Chemical Computing Group Inc. Montreal, Canada). The functionally inactive prodomain could not be modelled due to lack of crystallised structures of BMP4 and/or closely homologous proteins thus the structural analysis was limited to the active domain. BMP2 (NP_001191.1) shows the greatest similarity to BMP4 with a sequence identity of 90% and a similarity of 95% for the active domain. The active domains of BMP2 and BMP4 are also of similar length. Given the high sequence identity, similarity and length between the proteins, the effect of BMP4 mutation in the active domain can be derived directly from the known crystal structure of BMP2. This also allowed for the generation of a homology model of BMP4 based upon the BMP2 structure.

Two crystal structures of BMP2, 2goo [START_REF] Pinto | Discovery of potent, efficacious, and orally bioavailable inhibitors of blood coagulation factor Xa with neutral P1 moieties[END_REF] and 2h64 [START_REF] Weber | A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor[END_REF] were obtained from The Protein Databank [START_REF] Berman | The Protein Data Bank[END_REF]; http://www.pdb.org/pdb/home/home.do) which served as templates for generation of the homology models of BMP4. Homology models were generated using the default settings with the Amber99 forcefield and medium model refinement. An overlay of the BMP4 homology model and the BMP2 crystal structure showed that the backbones of both proteins align and that sidechains occupy the same orientation.

RESULTS

Complete sequence data of the coding regions of BMP4 and accompanying splice sites was obtained for all 504 CRC cases and 524 controls. One common non-synonymous single nucleotide polymorphism (nsSNP) rs17563T>C (p.V152A) was found in both cases and controls at similar frequency (minor allele frequencies 0.44 and 0.41 respectively).

Five missense mutations, p.E93G (g.6788A>G), p.R226W (g.8150C>T), p.R287H (g.8334G>A), p.W325C (g.8449G>T) and p.C373S (g.8592G>C), and one truncating mutation, p.R286X (g.8330C>T), were identified in single individuals among the 504 CRC cases screened (Table 1). Four missense mutations, p.S154F (n=2; g.7935C>T), p.T225A (n=2; g.8147A>G), p.R226W and p.I381V (g.8615A>G), were identified in the 524 controls (Table 1). Comparative amino acid sequence analysis revealed complete conservation of the amino acid sequence of BMP4 in eight species (human, chimpanzee, cow, rat, dog, chicken, frog, and zebrafish) at each of these positions (Figure 1). p.E96G is located in the functionally inactive prodomain of BMP4 and is predicted by SIFT and PolyPhen to be benign/tolerated. p.S154F and p.R226W are both predicted to be probably damaging or to affect the protein structure, with p.T225A predicted to be benign/tolerated. However, all three mutations map to the prodomain of BMP4 and are thus unlikely to have functional consequences a priori.

p.R287H was identified in a female case diagnosed with rectal CRC at age 42 (ICD9 code 154.1). The individual had concomitant colorectal adenomas at diagnosis. p.R287H is located in the prodomain adjacent to the proteolytic S1 cleavage site (-R-A-K-R-; amino acids 289-292). Cleavage of the S1 site is necessary for subsequent cleavage at the S2 site (-R-I-S-R-; amino acids 253-256) [START_REF] Cui | The activity and signaling range of mature BMP-4 is regulated by sequential cleavage at two sites within the prodomain of the precursor[END_REF]. A basic residue at position at amino acid 287 is prerequisite for site recognition/cleavage by furin [START_REF] Watanabe | Positional and additive effects of basic amino acids on processing of precursor proteins within the constitutive secretory pathway[END_REF]. Arginine and histidine are both basic amino acids and p.R287H is therefore unlikely to have any effect on the post-translational cleavage by furin. This mutation is predicted to be benign/tolerated by PolyPhen and SIFT (Table 1).

The nonsense mutation, p.R286X, was identified in a female CRC case diagnosed with sigmoid MSS CRC (ICD9 code 153.3) at age 42 who at the time of diagnosis had no first-degree relative affected with CRC. p.R286X localises to the N-terminal region of the TGF-β1 prodomain truncating the protein prior to the active domain (amino acids 292-408). Thus, BMP4 haploinsufficiency is likely to occur through nonsense-mediated RNA decay.

The missense mutations p.C373S and p.W325C map to the active domain of BMP4. p.C373S was identified in a female case diagnosed with sigmoid CRC (ICD9 code 153.3) at age 34. The case's father and brother who were diagnosed with colorectal adenomas at ages 67 and 36, respectively, have also been shown to carry the p.C373S mutation. In addition, one of the case's sisters, also a carrier of p.C373S, had hyperplastic polyps diagnosed at age of 35. The cysteine 373 residue is essential for the correct formation of the cysteine knot [START_REF] Mcdonald | A structural superfamily of growth factors containing a cystine knot motif[END_REF]. p.W325C was identified in a male familial CRC case diagnosed with rectal disease at age 62 (ICD9 code 154.1). The patient's brother had previously died of colon cancer (ICD9 code 1539.0) at the age 60. Both p.C373S and p.W325C are likely to be detrimental to the structure of BMP4 on the basis of PolyPhen and SIFT predictions (Table 1).

Only one missense mutation, p.I381V localising to the genomic region encoding the functional TGF-β domain was identified in a healthy control. This amino acid substitution is predicted to be benign/potentially intolerant by PolyPhen and SIFT (Table 1).

To further examine the impact of p.W325C, p.C373S and p.I381V mutations we modelled the effects of these amino acid substitutions on the structure of the BMP4 protein. Isoleucine 381 interacts with other residues due to its location within a lipophilic pocket, but the p.I381V mutation changes its lipophilic properties to a limited extent thereby maintaining its interactions with the other residues. This mutation is not, therefore, predicted to impact protein stability (Figure 2A). Two crystal structures (2goo and 2h64) show tryptophan 325 in two conformations. Both conformations show an edge-to-face interaction with tryptophan 322 but the tryptophan residue flips in orientation between the two conformations (Figure 2B). The p.W325C mutation reduces this edge-to-face interaction and is predicted to lower the stability of the protein. The p.C373S mutation (Figure 2C) is predicted to result in a less stable protein structure due as to the removal of a critical disulphide bond and cysteine knot motif.

Since BMP4 is known to associate with BMPR1A, protein-protein interactions were investigated between the BMP4-BMPR1A protein-complex. Tryptophan 325 of BMP4 is the only residue predicted to interact with BMPR1A as it is identical in the aligned sequence of BMP2 and BMP4. Therefore, the crystal structure of BMP2 was used as a model to analyse the effect of mutation on the protein-protein complex. Tryptophan 325 forms form a π-stacking interaction with arginine 88 and forms an edge-to-face interaction with phenylalanine 85 (Figure 2D). The mutation p.W325C is thus predicted to significantly reduce these interactions and result in reduced binding of BMPR1A to BMP4. Because multiple residues are involved in binding between both proteins it is difficult to predict if this variant would result in a complete loss of the formation of the BMP4-BMPR1A complex.

Archival tumor blocks were available on CRCs from p.R286X and p.C373S. Using these we examined tumor for loss of heterozygosity indicative of BMP4 having a tumor suppressor role. In both cases no evidence of differential allelotype was seen between germline and tumor DNA (data not shown). Using paired normal and tumor DNA samples we also failed to demonstrate differential methylation of the BMP4 promotor (Data not shown).

DISCUSSION

The BMP pathway is inactivated in a large proportion of sporadic CRC [START_REF] Kodach | The bone morphogenetic protein pathway is inactivated in the majority of sporadic colorectal cancers[END_REF]. Evidence for germline mutation in BMP4 having a role in CRC predisposition is provided by the study of inhibition of BMP4 signalling by transgenic expression of noggin in the mouse intestine. This leads to the formation of numerous ectopic crypt units, perpendicular to the crypt-villus axis, changes which typify the juvenile polyposis syndrome [START_REF] Haramis | De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine[END_REF].

In this study we have sought to establish a direct relationship between germline mutation in BMP4 and risk of CRC. It can be difficult to prove that a particular rare variant observed is etiologic unless those variants are strong candidates. BMP signalling inhibits intestinal stem cell self-renewal through suppression of Wnt-β-catenin signalling and disruption of this signalling pathway has strong candidacy for CRC predisposition [START_REF] He | BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling[END_REF]. Direct evidence for this assertion is provided by the fact that inactivating mutations in SMAD4 [START_REF] Howe | Mutations in the SMAD4/DPC4 gene in juvenile polyposis[END_REF] and BMPR1A [START_REF] Howe | Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis[END_REF], which, like BMP4 are members of the TGF-β superfamily cause the rare juvenile polyposis syndrome, which carries a very high risk of CRC. These data provide strong evidence that haploinsufficiency of BMP4 will increase CRC risk. Kryukov et al. have provided empiric evidence that about 70% of missense mutations present at population frequencies of 1% or less probably contribute to the phenotype in which they are first identified [START_REF] Kryukov | Most rare missense alleles are deleterious in humans: implications for complex disease and association studies[END_REF]. While we identified six mutations in both case and controls, three and five of the mutations in the cases and controls respectively map to the pro-domain of the expressed protein and are very unlikely to have any functional consequences. On the basis of detailed bioinformatics and protein homology modelling, only the three case mutations are likely to have functional consequences on the expressed protein. Sequence variations in the coding region of BMP4, aside from rs17563, are rare in both European [START_REF] Felder | Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs)[END_REF][START_REF] Weber | SIX2 and BMP4 mutations associate with anomalous kidney development[END_REF] and Chinese populations [START_REF] Suzuki | Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip[END_REF]. On the basis of collective data on over 1,300 healthy population controls screened in our study and previously reported data [START_REF] Bakrania | Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways[END_REF][START_REF] Felder | Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs)[END_REF][START_REF] Suzuki | Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip[END_REF][START_REF] Weber | SIX2 and BMP4 mutations associate with anomalous kidney development[END_REF] we identified three BMP4 mutations in our CRC cohort which are predicted to functionally deleterious on expressed BMP4 (none were identified in controls) would mean that there is more than a 90% probability that at least one of these variants contributes directly to the development of CRC.

Although in part speculative the demonstration that colorectal adenomas segregated with the p.C373S mutation in the one family and the fact that the case harboring p.W325C had a first-degree relative affected with CRC suggests that BMP4 mutations are likely to confer a moderate-high risk of CRC. In our study we did not observe loss of the wild-type allele in CRC from mutation carriers, providing little support for a classical tumor suppressor model of carcinogenesis. Although we were unable to demonstrate differential methylation of the putative promoter region of BMP4 we cannot fully exclude the possibility of inactivation of the wild-type allele through hypermethylation as a basis of tumor development. Accepting this caveat there exists the possibility that BMP4 mutations may impact on risk through a dominant negative effect as has previously been shown for SMAD4 mutations causing Juvenile polyposis [START_REF] Howe | Mutations in the SMAD4/DPC4 gene in juvenile polyposis[END_REF].

Bmp signalling is a determinant of dorsal closure in Drosophila Melanogaster [START_REF] Fernandez | Dpp signalling orchestrates dorsal closure by regulating cell shape changes both in the amnioserosa and in the epidermis[END_REF], and in concert with Shh, growth factors and Wnt7a coordinates growth and anteroposterior, proximodistal, and dorsoventral axes morphogenesis [START_REF] Basler | Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein[END_REF][START_REF] Kingsley | The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms[END_REF][START_REF] Tickle | Vertebrate limb development[END_REF]. While we did not document any associated extra-colonic clinical feature in the cases in which we identified BMP4 mutations, intriguingly a relationship between genetic susceptibility to clefting and cancer risk is supported in some epidemiological studies [START_REF] Bille | Cancer risk in persons with oral cleft--a populationbased study of 8,093 cases[END_REF][START_REF] Zhu | Do parents of children with congenital malformations have a higher cancer risk? A nationwide study in Denmark[END_REF]. Germline mutations in BMP4 have been suggested to be causal of cleft lip/palate [START_REF] Suzuki | Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip[END_REF], anomalies kidney development [START_REF] Weber | SIX2 and BMP4 mutations associate with anomalous kidney development[END_REF] and neural tube defects [START_REF] Felder | Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs)[END_REF]. Aside from one truncating mutation [START_REF] Suzuki | Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip[END_REF] all of the mutations on which these assertions rest are based on sequence variation predicted to have no functional consequence. Contiguous gene defects involving BMP4 have been implicated in etiology of eye, brain, and digit anomalies [START_REF] Bakrania | Mutations in BMP4 cause eye, brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways[END_REF] although carrier first-degree relatives of some of the affected individuals were shown to be phenotypically unaffected.

There is increasing recognition that much of the excess familial risk of CRC is a consequence of rare diseasecausing variants. The use of next generation sequencing technologies to screen CRC cases for causal variants will require enunciation of variants through bioinformatic methodologies. This study serves to illustrate the use of protein homology modelling methods for such analyses.

We have previously shown that SNPs close to the BMP antagonist, Gremlin-1 (GREM1; OMIM 603054) are associated with CRC risk [START_REF] Jaeger | Common genetic variants at the CRAC1 (HMPS) locus on chromosome 15q13.3 influence colorectal cancer risk[END_REF]. Our findings provide a strong rationale for evaluating additional members of the TGF-β superfamily of genes for coding mutations. Furthermore our finding provides a strong rationale for screening other genes identified by GWA studies is likely to identify additional germline mutations, which may cause CRC. Human Mutation

FIGURE LEGENDS

4

  Lubbe et al.

  Mutation in BMP4 and colorectal cancer risk 5

6

  Lubbe et al.

Figure 1 :

 1 Figure 1: Amino acid sequence alignment of vertebrate BMP4.

Figure 2 :

 2 Figure 2: Homology modelling of BMP4 using the structure of BMP2.

FIGURESFigure 1 :

 1 FIGURES

Figure 2 :Page

 2 Figure 2: Homology modelling of BMP4 using the structure of BMP2. A: Isoleucine 381 (green) within a lipophilic pocket (purple); B: The interactions formed by tryptophan 325 in two different conformations (purple and yellow) with tryptophan 322 and tyrosine 385 (grey); C: The disulphide bridge formed by cysteine 373 (white); D: The interacting residues with tryptophan 325 (white), BMP4 (grey) and BMPR1A (orange).

TABLE LEGENDS Table 1 :

 LEGENDS1 Predicted effects of non-synonymous coding changes in BMP4 on expressed protein function.g, position of mutation within genomic DNA (GenBank reference sequence NM_001202.3); N/A, Not applicable; p, position of mutation within protein (GenBank reference sequence NP_001193.2); PolyPhen, polymorphism phenotyping; SIFT, sorts intolerant from tolerant

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGEMENTS

Funding was provided by Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund and C309/A8274 awarded to the Cancer Research UK Centre for Cancer Therapeutics) and the European Union (CPRB LSHC- CT-2004-503465). We are grateful to the NCRN, clinicians and other hospital staff for supporting NSCCG. Finally, we are grateful to all the patients and individuals for their participation in this study.

Contract grant sponsor: Cancer Research UK (CRUK), and the European Union (EU); Contract grant number: C1298/A8362 supported by the Bobby Moore Fund and C309/A8274 awarded to the Cancer Research UK Centre for Cancer Therapeutics (CRUK). CPRB LSHC-CT-2004-503465 (EU).

SUPPLEMENTARY TABLE LEGENDS

Supplementary Table 1: Oligonucleotide primer pairs * Biotin label; F, forward primer; p, position of mutation within protein (GenBank reference sequence NP_001193.2); R, reverse primer; S, sequencing primer. All oligonucleotide primer pairs for BMP4 were designed using GenBank reference sequence NM_001202.3

TABLES


Javier Torres-Torronteras

Agustí Rodriguez-Palmero

Tomàs Pinós

Anna Accarino

Antoni Lluís Andreu

Guillem Pintos-Morell

Ramon Martí
email: rmarti@ir.vhebron.net

A novel nonstop mutation in TYMP does not induce nonstop mRNA decay in a

Keywords: TYMP, Nonstop mutation, Nonstop mediated decay, NSD, MNGIE, Neuropathy TYMP, Nonstop mutation, nonstop mediated decay, NSD, MNGIE, neuropathy

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

ABSTRACT:

The cellular quality control systems enable surveillance and selective degradation of nonsense, nonstop, and no-go mRNAs. In the case of nonstop mRNA, different mechanisms of nonstop-mediated decay (NSD) have been described for bacteria, yeast and mammals, but the molecular consequences of nonstop mutations have been examined in only few cases of human disease. We describe a novel homozygous nonstop mRNA mutation (c.1416delC) in the TYMP gene encoding thymidine phosphorylase, in a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In contrast to previous reports showing selective decay of pathogenic nonstop mRNAs, quantitative real-time PCR and 3'-RACE-RFLP analysis revealed unreduced nonstop mRNA levels in our patient and 2 heterozygous carriers of the mutation. The absence of thymidine phosphorylase protein in the homozygous patient, together with the partial decrease in levels of this protein in 2 carriers suggest that the main control system in this case resides at the translational or post-translational levels rather than through NSD. This is the first report showing an absence of NSD in a human disease, revealing that this surveillance mechanism has exceptions in vivo. ©2010 Wiley-Liss, Inc.

INTRODUCTION

Cells have developed surveillance systems to avert deleterious effects of premature stop, nonstop, or no-go mRNAs [START_REF] Isken | Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function[END_REF]. Several studies have described a translation-dependent surveillance step that detects and promotes degradation of mutant mRNA, which otherwise would result in formation of aberrant proteins. In mutations leading to mRNAs that lack in-frame stop codons (nonstop mRNAs), different mechanisms have been described for bacteria, yeast, and mammals [START_REF] Akimitsu | Messenger RNA surveillance systems monitoring proper translation termination[END_REF]. In eubacteria and yeast, stalled ribosomes at the 3'end of nonstop mRNA trigger degradation of the messenger, an action mediated by several factors (nonstop-mediated decay, NSD). In mammalian cells, translation repression, sparing the nonstop mRNA, has been observed [START_REF] Akimitsu | Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells[END_REF]. However, studies of nonstop mRNAs in human disease have reported considerable reductions in steady state nonstop mRNA levels in all cases [START_REF] Ameri | A nonstop mutation in the factor (F)X gene of a severely haemorrhagic patient with complete absence of coagulation FX[END_REF][START_REF] Chatr-Aryamontri | Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia[END_REF][START_REF] Lualdi | Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms[END_REF], indicating that NSD has occurred.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the nuclear gene TYMP, encoding thymidine phosphorylase (TP) [START_REF] Nishino | Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder[END_REF]. This enzyme catalyzes a key step in degradation of thymidine (dThd) and deoxyuridine (dUrd), which reach micromolar concentrations in MNGIE patients, and are undetectable in controls (Marti, et al., 2004;[START_REF] Marti | Late-onset MNGIE due to partial loss of thymidine phosphorylase activity[END_REF]. The main clinical features of MNGIE are gastrointestinal dysmotility, cachexia, peripheral neuropathy, progressive external ophthalmoplegia, diffuse leukoencephalopathy, and mitochondrial dysfunction (Hirano, et al., 2004). However, cases of mild or incomplete clinical symptoms have been reported [START_REF] Bedlack | MNGIE neuropathy: five cases mimicking chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Gamez | Phenotypic variability in a Spanish family with MNGIE[END_REF][START_REF] Martin | Lack of gastrointestinal symptoms in a 60-year-old patient with MNGIE[END_REF][START_REF] Needham | Mitochondrial disease mimicking Charcot-Marie Tooth disease[END_REF][START_REF] Szigeti | MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation[END_REF].

To date, around 60 different TYMP mutations have been associated with MNGIE [START_REF] Giordano | Gastrointestinal dysmotility in mitochondrial neurogastrointestinal encephalomyopathy is caused by mitochondrial DNA depletion[END_REF][START_REF] Massa | Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity[END_REF][START_REF] Poulton | Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1)[END_REF]. More than 50% are missense mutations, followed by splice site mutations (~20%), and frameshifts generating nonstop mRNA (~10%). Nonsense mutations or frameshifts generating premature stop codons (~10%), and mutations causing amino acid duplications or deletions (~4%) are less common. The consequences of TYMP mutations on transcripts have been experimentally characterized for some splice mutations [START_REF] Nishino | Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder[END_REF][START_REF] Szigeti | MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation[END_REF]Taanman, et al., 2009), but such studies have never been performed on nonstop mRNA mutations in MNGIE. We report the case of a MNGIE patient with marked peripheral neuropathy, harboring a novel nonstop mRNA mutation. Unlike the reported features of other nonstop mutations in human disease, we found that the nonstop mRNA is stable and coexists with wild-type mRNA at similar levels in carriers.

MATERIALS AND METHODS

Subjects

Informed consent for participation was obtained from all subjects included. A 16-year-old female was visited in the emergency department for weakness that had dramatically exacerbated in the previous 2 weeks, with hypoesthesia and hyperalgesia in hands and feet. Her parents were second cousins, and her mother had psoriatic arthritis and mild bilateral ptosis; her sister was asymptomatic. The pregnancy, neonatal period, and early neurological development were normal. She had a history of vomiting, flatulence, and abdominal pain after meals since the age of 7 years. She had been diagnosed of aorto-mesenteric compression syndrome that was surgically treated, with no improvement. She began to lose weight and was admitted to another hospital for suspected anorexia nervosa. The patient gradually developed generalized weakness, with difficulty standing up from a low chair and sitting up in bed, but she had an almost normal lifestyle, excluding sports. Her weakness had worsened before her first visit to our hospital, affecting predominantly the right arm and leg, and progressed to tetraparesis of distal predominance in a few weeks. She was able to walk only a few steps without help and became dependent for some everyday tasks with her hands.

Physical examination showed cachexia (35 kg), short stature (154 cm), generalized muscle atrophy, and higharched feet. Neurological study revealed normal cognitive function, upgaze ophthalmoparesis, asymmetric generalized weakness with distal predominance, and absence of deep tendon reflexes. There was distal sensory loss for all modalities and hyperalgesia in the distal legs. She had a steppage gait and was unable to stand up from the floor without help. Fundoscopy findings were normal. Laboratory exams showed high plasma lactate concentrations in 2 determinations (4.2 and 2.6 mmol/L) and CSF protein of 3.29 g/L (normal 0.2-0.5) with albumino-cytologic dissociation. Neurophysiologic studies demonstrated demyelinating, axonal polyneuropathy. These later findings prompted treatment with intravenous immunoglobulin administration, with no improvement. Cranial MRI showed diffuse leukoencephalopathy, sparing the U fibers and corpus callosum. Abdominal ultrasound examination and echocardiography showed no significant abnormalities. Gastrointestinal evaluation showed hypomotility in the distal esophagus, with normal lower esophageal sphincter. Gastric emptying scintigraphy study showed mild gastroparesis with 26% retention of radiolabeled meal at completion of the study (normal <10%). The small bowel was diffusely dilated and intestinal manometry showed neuropathic dysmotility with bacterial overgrowth. TP dysfunction was then assessed on the basis of suspected MNGIE.

Biochemical and Molecular Studies

Anticoagulated blood was collected from the proband, her parents, and her sister, and from 2 healthy controls, age-and sex-matched to the father (C1) and mother (C2). Plasma dThd and dUrd concentrations and TP activity in buffy coat were measured as reported (Marti, et al., 2004), with minor changes. Buffy coat DNA was isolated and TYMP coding regions and intronic boundaries were sequenced [START_REF] Nishino | Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder[END_REF]. Total RNA was extracted from buffy coat (Trizol, Invitrogen), and total random-primed cDNA (High-capacity cDNA reverse transcription kit, Applied Biosystems) and poly(A) RNA-derived cDNA (by 3'-RACE with the FirstChoice RLM-RACE kit, Applied Biosystems) were obtained. One random primed cDNA and 2 different 3'-RACE cDNAs from the same RNA extracts were used for TYMP mRNA real-time PCR quantification (Hs00157317_m1, Applied Biosystems). Results were normalized to beta-glucuronidase (GUSB) mRNA levels (human GUSB endogenous control probe 4333767F, Applied Biosystems). As a representative control of low TYMP expression, poly(A) RNA-derived cDNA, obtained from a control skeletal muscle biopsy following the above detailed procedure was included in the real-time PCR analysis, because skeletal muscle is konwn to express TYMP very poorly [START_REF] Yoshimura | Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticulocytes and tumors[END_REF].

For TYMP mRNA sequencing and PCR-RFLP analysis, 3'-RACE cDNA was PCR-amplified with specific forward TYMP primer (cDNA-TYMP-F1, AGGCCCGCCAGACTTAAGGG), and reverse primer annealing the 3'-RACE adapter sequence (GCGAGCACAGAATTAATACGACT), followed by semi-nested PCR (forward, cDNA-TYMP-F1; reverse cDNA-TYMP-R1, CGCGGCAAAGGAGCTTTATT). The PCR product was sequenced using internal forward primer (cDNA-TYMP-F2, AGCAGGAGGAGCTGCTGGCG), and BssHIIdigested for RFLP analysis. The digestion products were electrophoresed and stained with ethidium bromide, and bands were quantified (Quantity One, Bio-Rad). The wild-type 574-bp amplicon has a unique BssHII target resulting in 2 bands (502-bp+72-bp). This target disappears with the c.1393G>A change, cis-linked to the c.1416delC mutation.

Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_001113755.1, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

Western-blot

Four µg of buffy coat protein extract, obtained as described (Marti, et al., 2004), were electrophoresed in 10% SDS-PAGE, transferred to PVDF membrane, and incubated with anti-TP (Calbiochem) and anti-β-actin (Sigma-Aldrich) mouse monoclonal antibodies, and then with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Bands were visualized (LumiGlo chemiluminiscent kit, Cell Signaling Technology) and quantified using Quantity One software. All TP-to-β-actin ratios were referred to the corresponding ratio obtained for the C2 sample.

Statistics

The association between the number of mutated alleles and TP activity, TP protein levels, percentage of mutated RNA and mRNA levels was tested with the nonparametric Spearman correlation test. For statistical purposes, undetectable values were considered as zero.

RESULTS

Biochemical and Genetic Studies

The patient's TP activity was severely reduced, and plasma dThd and dUrd concentrations were increased, confirming the diagnosis of MNGIE (Table 1). TYMP gene sequencing revealed a homozygous single-nucleotide deletion in the last coding segment of exon 10 (c.1416delC; Ref Seq NM_001113755.1), predicting a frameshift that leads to a nonstop mRNA (p.F473SfsX41). Both parents and the sister were heterozygous mutation carriers. Analysis of the sequences revealed that the c.1416delC change was cis-linked to two previously reported nonpathogenic polymorphisms: c.1393G>A [START_REF] Martin | Lack of gastrointestinal symptoms in a 60-year-old patient with MNGIE[END_REF], located in exon 10, 23 bases upstream of c.1416delC and absent in both parents' alleles lacking the deletion, and c.972C>T (rs131804), in exon 8, present in both alleles from the father but only in the c.1416delC-mutated allele of the mother (Figure 1). Buffy coat TP activity in the parents and sister was moderately reduced (62%-76% of control values). Plasma dThd and dUrd were undetectable in all carriers (Table 1).

TYMP mRNA and TP Protein Levels

Several studies have reported decay of nonstop mRNA species in other human diseases caused by nonstop mRNA mutations [START_REF] Ameri | A nonstop mutation in the factor (F)X gene of a severely haemorrhagic patient with complete absence of coagulation FX[END_REF][START_REF] Chatr-Aryamontri | Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia[END_REF][START_REF] Lualdi | Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms[END_REF][START_REF] Temperley | Investigation of a pathogenic mtDNA microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria[END_REF]; hence, we investigated whether the c.1416delC mutation results in NSD. Quantitative real-time PCR was used to assess mRNA levels in buffy coat of the patient, parents, and 2 healthy controls. As is shown in Table 1, levels of TYMP poly(A) mRNA (analyzed from the 3'-RACE cDNA product) were similar in the patient (homozygous for the mutation), both parents, and both controls. Nor were differences detected when randomprimed cDNA instead of 3'-RACE cDNA was used as template. A healthy control's skeletal muscle, tissue known to express TYMP poorly [START_REF] Yoshimura | Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticulocytes and tumors[END_REF], expectedly showed very low poly(A) TYMP mRNA levels (around 4% -8 % of those observed in buffy coat from controls, mutation carriers and patient).

Specific quantification of mutated and wild-type species by 3'-RACE-nested PCR-RFLP analysis of the c.1393G>A polymorphism (cis-linked to the mutated allele) revealed that more than 50% of the carriers' TYMP mRNA was mutant species (Figure 1, Table 1). This finding was consistent with the cDNA sequencing results, which showed coexistence of mutant and wild-type TYMP mRNA in both parents (Figure 1). Despite the similar levels of TYMP mRNA in the patient, parents, and controls, TP protein levels were undetectable in the patient and partially reduced in the carriers as compared to controls (Figure 1, Table 1), in accordance with the TP activity results.

DISCUSSION

The number of TYMP mutations associated with MNGIE has continuously increased since the genetic cause of this disorder was discovered [START_REF] Nishino | Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder[END_REF][START_REF] Poulton | Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1)[END_REF]. Although the clinical picture of MNGIE has been well defined (Hirano, et al., 2004), cases with missing or reduced symptoms are not rare, including presentations with predominant neuropathy and reduced or subclinical gastrointestinal involvement [START_REF] Bedlack | MNGIE neuropathy: five cases mimicking chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Gamez | Phenotypic variability in a Spanish family with MNGIE[END_REF]Hirano, et al., 2004;[START_REF] Martin | Lack of gastrointestinal symptoms in a 60-year-old patient with MNGIE[END_REF][START_REF] Needham | Mitochondrial disease mimicking Charcot-Marie Tooth disease[END_REF][START_REF] Szigeti | MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation[END_REF]. Genotype differences do not account for these variations, as the only genotype-phenotype correlation observed in MNGIE was reported in patients with late-onset disease, harboring p.R202T, p.V208M, p.L285P, p.G311R or p.E379K mutations, which are associated with less severe TP dysfunction [START_REF] Marti | Late-onset MNGIE due to partial loss of thymidine phosphorylase activity[END_REF][START_REF] Massa | Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity[END_REF]. In the case reported here, subacute worsening of weakness established peripheral neuropathy as the predominant clinical feature. This fact, together with the strikingly high CSF protein level, led us to consider a diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP) at one point. Nonetheless, TP dysfunction ruled out CIDP and confirmed MNGIE. This case further illustrates variants in the clinical presentation of this disease, and the need to test TP dysfunction to rule out MNGIE in patients whose clinical features resemble other conditions [START_REF] Bedlack | MNGIE neuropathy: five cases mimicking chronic inflammatory demyelinating polyneuropathy[END_REF].

We found a novel single-nucleotide deletion in the TYMP region encoding the C-terminal fraction of the protein. This change generates a frameshift that eliminates the termination codon, with no additional stop codons within the 3'-UTR (nonstop mRNA mutation). At least 5 different nonstop mRNA mutations have been associated with MNGIE [START_REF] Poulton | Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1)[END_REF], but the stability or steady-state levels of the nonstop mRNA species were not investigated. Study of the molecular consequences of the novel mutation reported here has provided singular results. Previous reports found that nonstop mRNA mutations associated with Diamond-Blackfan anemia [START_REF] Chatr-Aryamontri | Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia[END_REF], mucopolysaccharidosis II [START_REF] Lualdi | Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms[END_REF] and FX-coagulation deficiency [START_REF] Ameri | A nonstop mutation in the factor (F)X gene of a severely haemorrhagic patient with complete absence of coagulation FX[END_REF] result in pronounced reduction of steady-state levels of mutant mRNA. Similarly, translationdependent deadenylation decay was observed in nonstop mitochondrial mRNA as a consequence of a microdeletion [START_REF] Temperley | Investigation of a pathogenic mtDNA microdeletion reveals a translation-dependent deadenylation decay pathway in human mitochondria[END_REF]. In contrast, TYMP mRNA levels were not reduced in the patient presented herein (homozygous for the c.1416delC mutation) or her parents, heterozygous carriers. Consistently, RFLP analysis showed similar amounts of mutant and wild-type transcripts in the carriers. Therefore, we conclude that c.1416delC nonstop mRNA molecules are as stable as those transcribed from the wild-type alleles in this family. The protein product was absent in the patient and reduced to ~50% in the carriers, as compared to the amounts detected in the controls, strongly suggesting that translation of mutant transcript is repressed and/or its protein product is not stable.

Cells surveillance systems result in post-transcription specific degradation of mRNAs harboring nonstop, premature stop, or no-go mRNA mutations [START_REF] Isken | Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function[END_REF]. Different NSD mechanisms have been described in bacteria, yeast and mammals [START_REF] Akimitsu | Messenger RNA surveillance systems monitoring proper translation termination[END_REF]. All studies analyzing nonstop transcripts in human genetic disease have shown drastically reduced steady-state levels of nonstop mRNAs, but the case reported here demonstrates that this mechanism has exceptions. Since we only analyzed mRNA and protein from buffy coat samples, we cannot rule out reduced nonstop mRNA levels in other tissues. However, the lack of NSD in human platelets and leukocytes, where TYMP is highly expressed, indicates that, at least for this particular mutation, the main control system to avoid additional deleterious effects of an aberrant protein happens at the translational or post-translational levels rather than through NSD.

The reasons for the lack of NSD for this particular mutation are unknown. In nonsense-mediated decay, another mechanism that selectively eliminates mRNAs with premature stop codons, degradation occurs or not, depending on the relative position of the premature stop codon with respect to the exon-exon junctions of mRNA [START_REF] Isken | Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function[END_REF]. Similarly, unknown molecular restrictions might determine the occurrence or not of NSD, but this mechanism remains to be characterized. We speculate that this could account for the lack of NSD in the mutation studied herein. [START_REF] Akimitsu | Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells[END_REF] examined translation of nonstop mRNA in vitro in mammalian cells and found no decreases in steady-state nonstop mRNA levels or reductions in protein stability. Instead, they observed that translation of nonstop mRNA is repressed, a mechanism that better fits with the observations reported here.

In summary, we report the first case of a patient with a pathogenic nonstop mRNA mutation and unreduced levels of nonstop mRNA, in contrast to previous reports which always show selective mRNA decay associated with this type of mutation in human disease. Determination of the reasons for these different responses to pathogenic nonstop mRNA mutations and the mechanisms involved will require further investigation.

Formatted

  Lack of NSD in a nonstop mRNA mutation 5

  Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_001113755.1, according to journal guidelines (www.hgvs.org/mutnomen). Panel A: Scheme of the TYMP gene. Boxes represent exons (white: 5'UTR and 3'UTR; grey: coding sequence). Intron lengths are not in scale. The 3'-end of the exon 10 (uppercase) plus a portion of flanking DNA (lowercase) are expanded, showing the wildtype and mutant (c.1416delC) sequences. The deleted 1416 C is labeled with an asterisk. The encoded amino acids are indicated in one-letter code, and the native stop codon is framed. The frame-shifted codons in the mutated sequence are underlined, showing the disruption of the native TAA stop codon, with no additional stop codons in the transcript. Panel B: pedigree of the family; the arrow indicates the patient. c.972C>T and c.1393G>A are previously reported, non-pathogenic polymorphisms. c.1416delC (framed) is the pathogenic mutation. Panel C: electropherograms showing the cDNA sequences from buffy coat polyA+ TYMP mRNA, obtained from a healthy control, showing the wild-type sequence (C1), the patient showing the c.1416delC mRNA (II:1) and a mutation carrier showing the coexistence of both mRNA species (I:2). A similar result was obtained for the other carrier analyzed (I:1; not shown). Panel D: PCR-RFLP analysis of cDNA obtained from buffy coat polyA+ TYMP mRNA. The wild type amplicon (574 bp) harbors a target for BssHII, which products of 502 bp and 72 bp (not shown); the c.1393G>A polymorphism (cis-linked to the c.1416delC mutation) destroys the restriction target. U, undigested sample; D, digested sample. Panel E: representative western-blot showing the TP and β-actin bands from buffy coat homogenates

TABLE 1 TYMP mRNA 2

 1 Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_001113755.1, according to journal guidelines (www.hgvs.org/mutnomen). 2 Real-time PCR relative quantification of 3 different cDNAs obtained from two 3'-RACE reverse transcriptions (a and b) and one random primed reverse transcription (c) of the same buffy coat total RNA. The results indicate mean (minimum and maximum) of 3 replicates from each cDNA, normalized to GUSB mRNA levels and referred to the value obtained for control 2, used as calibrator. 3 Percentage of the mutant band (574 bp, Figure1) over the total product (574 bp +502 bp bands) obtained from BssHII digestion of the 3'-RACE -seminested PCR product of buffy coat RNA (see methods).4 Relative quantification of the TP protein bands obtained by western blot, normalized by β-actin and referred to the ratio obtained for control 2, used as calibrator; results represent the mean (SD) of 3 different western blots of the same buffy coat homogenate. 5 Buffy coat TP activity in nmol thymine/hour/mg protein.6 Plasma dThd and dUrd in µmol/L (lower limit of detection, 0.05 µmol/L). The number of mutated alleles (2 for the patient, 1 for the carriers, 0 for the controls) correlated positively with the percentage of mutated RNA (P=0.005), and negatively with TP activity (P=0.008) and TP protein (P=0.014), but did not correlate with 3'-RACE primed (P=0.306) or random primed (P=0.760) cDNA levels. wt: wild-type UND: undetectable. ND: not determined.

	Deleted: ¶
	Genotype 1 c.1416delC / c.1416delC c.1416delC / wt wt / c.1416delC wt / c.1416delC wt / wt wt / wt wt / wt 1 Formatted Table 3'-RACE cDNA (a) 3'-RACE cDNA (b) Random primed cDNA (c) % mutant mRNA 3 TP protein 4 TP activity 5 dThd 6 dUrd 6 Patient (II-1) 1.08 (0.87 -1.32) 1.20 (1.03 -1.40) 0.94 (0.48 -1,81) 100 UND 3.0 9.0 10.0 Father (I-1) 0.95 (0.88 -1.03) 0.71 (0.54 -0.95) 0,79 (0,70 -0.89) 63 0.28 (0.08) 602 UND UND Mother (I-2) 1.07 (0.85 -1.35) 1.23 (1.02 -1.49) 1.12 (1.01 -1.24) 79 0.38 (0.06) 733 UND UND Sister (II-2) ND ND ND ND ND 613 UND UND Control 1 0.90 (0.74 -1.08) 0.74 (0.62 -0.88) 0.94 (0.78 -1.13) UND 0.69 (0.08) 920 UND UND Control 2 1.00 (0.92 -1.08) 1.00 (0.81 -1.24) 1.00 (0.85 -1.18) UND 1.00 1,017 UND UND Control muscle 0.04 (0.04 -0.05) 0.08 (0.04 -0.19) ND ND ND ND ND ND Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Formatted Deleted: TYMP mRNA 2 ¶ 1.08 (0.87-1.32) ¶ Deleted: 1.07 (0.85-1.35) ¶ Deleted: 0.90 (0.74-1.08) ¶ Deleted: Deleted: Deleted: Deleted: Deleted: ND Formatted	... [11] ... [8] ... [2] ... [14] ... [9] ... [15] ... [6] ... [3] ... [7] ... [17] ... [13] ... [18] ... [4] ... [19] ... [5] ... [20] ... [10] ... [16] ... [21] ... [1] ... [12]
	Formatted	... [22]
	Deleted: Reference sequence: GenBank # NM_001113755.1 ... [23]

John Wiley & Sons, Inc.Human Mutation

ACKNOWLEDGMENTS

We would like to thank the patient and the family that participated in the study, and Celine Cavallo for English Language assistance. This work was supported by the Instituto de Salud Carlos III (PS09/01591).