MD K Allegaert
email: karel.allegaert@uz.kuleuven.ac.

G Naulaers

Haemodynamics of intravenous paracetamol in neonates

Keywords: developmental pharmacology, paracetamol, haemodynamics, newborn developmental pharmacology, paracetamol, haemodynamics, newborn

Introduction: Haemodynamics of intravenous (iv) paracetamol in adult intensive care were recently 3 published. We therefore wanted to explore haemodynamics of iv paracetamol in neonates. 4

Methods: Retrospective, pooled analysis of heart rate (bpm) and blood pressure (mean, systolic, 5 diastolic) observations collected during iv paracetamol pharmacokinetic studies in neonates. Heart 6 rate and blood pressure were recorded just before and 30, 60, 120, 180, 240, 300 and 360 minutes 7 after iv paracetamol (paired, ANOVA). Clinical characteristics in hypotensive (mean mmHg < 8 gestational age, weeks) cases were compared with controls (Mann Whitney U). 9

) bpm (paired p<0.05, ANOVA p=0.36). There 11 were no changes in systolic and diastolic pressure, but mean arterial pressure decreased from 46 (7) 12 to 43(8) mmHg at 60 minutes (paired p<0.05, ANOVA p=0.75). Eight neonates developed 13 hypotension. These cases had lower pre-administration arterial pressure (38 vs 47 mmHg, p<0.05). 14 Conclusions: In a setting of open label administration to alleviate (procedural) pain, haemodynamic 15 effects of iv paracetamol in neonates remained modest. We do suggest to consider impaired 16 haemodynamics to be a relative contra-indication for iv paracetamol in neonates. 17 18 19 20 21 22 23 24 25 Page 2 of 22 European Journal of Clinical Pharmacology F o r P e e r R e v i e w neonates underwent minor, painful procedures (e.g. insertion of peripheral arterial or venous line, 47 insertion of central venous line, placement of chest tube) or as additional therapy in neonates on 48 opioids. The decision to prescribe propacetamol or any other analgesic was made by the attending 49

improves prediction of concentration compared to enteral formulations by elimination of absorption 29 variability. In recent years, data on iv paracetamol pharmacokinetics were described [1][2][3]. In 30 contrast, data on the pharmacodynamics of this formulation in neonates are unreported. 31 de Maat et al. recently published their observations with iv paracetamol in adult medium and 32 intensive care [4]. The authors illustrated that iv paracetamol affects haemodynamics (i.c. induces 33 hypotension) in critically ill adults. They hereby confirmed other reports on haemodynamic (side)-34 effects of this drug in critically ill adults [5][6][7]. Since clinical characteristics, co-morbidity and 35 pharmacodynamics may be different in adults compared to neonates, we decided to quantify 36 haemodynamics following iv paracetamol in neonates, based on pooled data analysis of observations 37 collected during a reported pharmacokinetic study [1] and an ongoing study on pharmacokinetics 38 and -dynamics of iv paracetamol (PARANEO, NCT 00969176). 39

Methods 40

Available cohorts 41

Single dose pharmacokinetic study in neonates [1] 42 F o r P e e r R e v i e w 4 neonatologist. Exclusion criteria were major congenital malformations or birth asphyxia (Apgar < 4 at 50 5 minutes). In this study, 30 patients were included. In the first 15 patients, 20 mg (equal to 10 mg 51 paracetamol)/kg was administered, in the next 15 patients, 40 mg (equal to 20 mg paracetamol)/kg. 52

Mean birth weight was 2111 (SD 1094) g, mean gestational age (GA) was 33.8 (SD 3.9) weeks. Mean 53 postnatal age was 12.7 (SD 6.4) h. 10 neonates were term, 20 were preterm (<37 weeks GA), of 54 whom 10 <32 weeks GA [1]. 55

Repeated dose PK/PD study (PARANEO, NCT 00969176, www.clinicaltrials.gov). 56

Interim analysis of a still ongoing study on pharmacokinetics, -dynamics and safety of repeated iv 57 paracetamol administration in the 42 neonates currently included. Indications for initiation of iv 58 paracetamol were medical or surgical painful conditions [8]. Mean weight was 2580 (SD 890) g, mean 59 gestational age (GA) 35.5 (SD 4.4) weeks, mean postnatal age 3 (range 1-27) days. Twenty-two 60 neonates were term, 20 preterm (<37 weeks GA), of whom 4 < 32 weeks GA. Primary outcome of this 61 ongoing study are iv paracetamol pharmacokinetics, but pharmacodynamics -including 62 haemodynamics -are also collected. The dosing regimen is based on a loading dose (20 mg/kg 63 paracetamol), followed by an age dependent maintenance dose (5 to 10 mg/kg/6h) [2]. 64

2.Haemodynamics 65

Individual patient files were analysed for data on blood pressure [mean, systolic, diastolic (mmHg)] 66 and heart rate (beats/min) from just before the first administration of iv paracetamol until 6 hours 67 after iv paracatamol administration. We hereby focused on hemodynamic effects of the first dose. 68

Besides absolute values (mmHg), any mean arterial blood pressure < GA for a given GA (e.g. neonate 69 of 30 weeks should have a mean arterial blood pressure of at least 30 mmHg) was recorded since this 70 is the most frequently used, 'bedside' indicator of hypotension in neonates [8] The pooled dataset consisted of haemodynamics in 72 patients and both preterm (n=40) and term 101 (n=32) neonates were included. Baseline mean, systolic and diastolic blood pressure before iv 102 paracetamol were 46 (SD 7), 55 (SD 10) and 38 (7) mmHg, baseline heart rate was 145 (SD 20) bpm. 103 Significant correlations between age and mean blood pressure (r= -0.39, p<0.01) or heart rate 104 (r=0.28, p<0.05) were documented, reflecting the relative lower mean arterial pressure and higher 105 heart rate in the smallest neonates. 106

Consecutive observations (mean, SD) on heart rate and mean, systolic and diastolic blood pressure 107 are provided in table 1. There was a significant decrease (paired analysis) in heart rate 30 (-7 bpm), 108 60 (-4 bpm) and 120 (-8 bpm) (all at least p<0.05) minutes following iv paracetamol with a 109 subsequent trend towards normalization but still remaining significantly lower (all p < 0.05) up to 360 110 minutes afterwards. Using ANOVA, there were no significant differences in the consecutive 111 measurements (p = 0.36). Mean arterial blood pressure displayed a minor, statistically significant 112 decrease (-3 mmHg) at 60 minutes, with subsequent return to baseline values, without alterations in 113 systolic or diastolic blood pressure. Similarly, there were no significant differences in mean arterial 114 pressures using ANOVA (p=0.75). 115

Still 18 observations in 8 (9 %) neonates were below the threshold of normal mean arterial blood 116 pressure, defined as below the mmHg for the given age at inclusion. When clinical characteristics of 117 hypotensive (n=8) were compared with normotensive (n=64) cases, there was no significant 118 difference in age (35 vs 36 weeks) or weight (2455 vs 2425 g), but baseline mean arterial pressure (38 119 vs 47 mmHg, p<0.05) was already lower in cases who subsequently developed hypotension. Baseline 120 heart rate was not significantly different (154 vs 142 bpm). 121 Based on prospectively collected observations in 72 neonates, we observed a clinical modest 125 decrease in heart rate (7bpm) and mean arterial blood pressure (3mmHg) following iv paracetamol 126 administration. A minority (9 %) developed hypotension. Interestingly, these neonates already had 127 significantly lower blood pressure before paracetamol administration. 128

Effective management of pain should be based on systematic assessment of pain, followed by 129 titrated administration of the most appropriated analgesic(s) with subsequent re-assessment. In any 130 patient, the administration of an analgesic is a balanced decision based on perceived benefits (e.g. 131 agitation, pain, stress) and risks (e.g. hypotension, bowel paresis, hypersensitivity, intolerance, 132 withdrawal, neuro-apoptose) [9]. When we focus on the haemodynamics of analgesics or sedatives 133 in neonates, there are observations on effect of midazolam (+30 bpm, -3 mmHg) [10], fentanyl (-13 134 bpm, -3 mmHg) [START_REF] Jacqz-Aigrain | Placebo-controlled trial of midazolam sedation in mechanically ventilated newborn babies[END_REF], fentanyl and midazolam (-19 bpm, -12 mmHg) [START_REF] Guinsburg | Physiological, hormonal, and behavioral responses to a single fentanyl dose in intubated and ventilated preterm neonates[END_REF], propofol (-9 bpm, -14 135 mmHg) [START_REF] Burtin | Hypotension with midazolam and fentanyl in the newborn[END_REF] or morphine (-8 bpm, -3 mmHg) [START_REF] Vanderhaegen | Cerebral and systemic hemodynamic effects of intravenous bolus administration of propofol in neonates[END_REF][START_REF] Sabatino | Hemodynamic effects of intravenous morphine infusion in ventilated preterm babies[END_REF] on heart rate and mean arterial blood pressure. 136 Pre-existing low mean arterial pressure was as risk factor for subsequent hypotension following 137 morphine administration [START_REF] Sabatino | Hemodynamic effects of intravenous morphine infusion in ventilated preterm babies[END_REF]. 138

The present observations following iv paracetamol in neonates are likely the phenotypic result of 139 both improved analgesia and procedural (initial indication for iv paracetamol) related effects and one 140 should take into account that paracetamol was administered unblinded and the pooled analysis was 141 retrospective. Taking these weaknesses into account, we suggest that -compared to other analgesics 142 or sedatives, haemodynamic changes following iv paracetamol are limited, but not absent [START_REF] Jacqz-Aigrain | Placebo-controlled trial of midazolam sedation in mechanically ventilated newborn babies[END_REF][START_REF] Guinsburg | Physiological, hormonal, and behavioral responses to a single fentanyl dose in intubated and ventilated preterm neonates[END_REF][START_REF] Burtin | Hypotension with midazolam and fentanyl in the newborn[END_REF][START_REF] Vanderhaegen | Cerebral and systemic hemodynamic effects of intravenous bolus administration of propofol in neonates[END_REF][START_REF] Sabatino | Hemodynamic effects of intravenous morphine infusion in ventilated preterm babies[END_REF][START_REF] Simons | Pain control: opioid dosing, population kinetics and side-effects[END_REF]. It 143 seems cautious to consider preadministration impaired haemodynamics a relative contra-indication 144 to initiate iv paracetamol. 145

We can't but speculate why haemodynamics of iv paracetamol seems to be different in neonates 146 [5,7]. Hersch et al. [7] suggested that (traumatic) brain injury and pyrexia/hyperthermia are risk 147 factors while de Maat et al. [5] were unable to unveil risk factors in medium-and intensive care adult 148 Introduction: Haemodynamics of intravenous (iv) paracetamol in adult intensive care were recently 3 published. We therefore wanted to evaluate haemodynamics of iv paracetamol in neonates. 4

F o r P e e r R e v i e w

Methods: Pooled analysis of data on heart rate (bpm) and blood pressure (mean, systolic, diastolic) 5 collected during iv paracetamol pharmacokinetic studies in neonates. Heart rate and blood pressure 6 were recorded just before and 30, 60, 120, 180, 240, 300 and 360 minutes after iv paracetamol 7 (paired, ANOVA). Clinical characteristics in hypotensive (mean mmHg < gestational age, weeks) cases 8 were compared with controls (Mann Whitney U). 9

Results: Based on observations in 72 neonates, heart rate decreased from 145 (SD 20) to 138 (21), 10 141(20), 137(20), 137(22), 140(20), 139(20) and 140(21) bpm (paired p<0.05, ANOVA p=0.36). [START_REF] Jacqz-Aigrain | Placebo-controlled trial of midazolam sedation in mechanically ventilated newborn babies[END_REF] were no changes in systolic and diastolic pressure, but mean arterial pressure decreased from 46 (7) 12 to 43(8) mmHg at 60 minutes (paired p<0.05, ANOVA p=0.75). Eight neonates developed 13 hypotension. These cases had lower pre-administration arterial pressure (38 vs 47 mmHg, p<0.05). 14 Conclusions: Although these data were collected following unblinded administration to alleviate 15 (procedural) pain, its seems that haemodynamic effects iv paracetamol are modest (-8 bpm, -3 16 mmHg mean arterial pressure). We suggest to consider impaired haemodynamics a relative contra-17 indication for iv paracetamol in neonates. 18 were described [2,3,4]. In contrast, data on the pharmacodynamics of this formulation in neonates 31 are unreported. 32 de Maat et al. recently published their observations with iv paracetamol in adult medium and 33 intensive care [5]. The authors illustrated that iv paracetamol affects haemodynamics (i.c. induces 34 hypotension) in critically ill adults. They hereby confirmed other reports on the haemodynamic 35 (side)-effects of this drug in critically ill adults [6,7]. Since clinical characteristics, co-morbidity and 36 pharmacodynamics may be different in adults compared to neonates, we decided to quantify 37 haemodynamics following iv paracetamol in neonates, based on pooled data of observations 38 collected during a reported pharmacokinetic study [2] and an ongoing study on pharmacokinetics 39 and -dynamics of iv paracetamol (PARANEO, NCT 00969176). 40 neonatologist. Exclusion criteria were major congenital malformations or severe birth asphyxia 51 (Apgar < 4 at 5 minutes). In this study, 30 patients were included. In the first 15 patients, 20 mg 52 (equal to 10 mg paracetamol)/kg was administered, in the next 15 patients, 40 mg (equal to 20 mg 53 paracetamol)/kg was administered. Mean birth weight was 2111 (SD 1094) g, mean gestational age 54 (GA) was 33.8 (SD 3.9) weeks. Mean postnatal age at inclusion was 12.7 (SD 6.4) h. 10 neonates were 55 term, 20 were preterm (<37 weeks GA), of whom 10 <32 weeks GA [2]. 56

Methods 41

Available cohorts 42

Study 2: repeated dose PK/PD study (PARANEO, NCT 00969176, www.clinicaltrials.gov). 57 Interim analysis of a still ongoing study on pharmacokinetics, -dynamics and safety of repeated 58 administration of iv paracetamol in the first 42 included neonates. Indications for initiation of 59 intravenous paracetamol were either medical or surgical painful conditions in line with guidelines of 60 the unit [8]. Mean weight was 2580 (SD 890) g, mean gestational age (GA) was 35.5 (SD 4.4) weeks. 61

Mean postnatal age (days) was 3 (range 1-27) days, 22 neonates were term, 20 were preterm (<37 62 weeks GA), of whom 4 < 32 weeks GA. Primary outcome of this ongoing study are iv paracetamol 63 pharmacokinetics in neonates, but pharmacodynamics -including haemodynamics -are also 64 collected. The dosing regimen is based on a loading dose (20 mg/kg paracetamol), followed by an age 65 dependent maintenance dose (5 to 10 mg/kg/6h) [3]. 66

2.Hemodynamics 67

Individual patient files were analysed for data on blood pressure [mean, systolic, diastolic (mmHg)] 68 and heart rate (beats/min) from just before the first administration of iv paracetamol until 6 hours 69 after initiation of iv paracatamol. We hereby focused on hemodynamic effects of the first dose. 70

Besides absolute values (mmHg), any decrease in any mean arterial blood pressure < GA in mmHg for 71 a given GA (weeks, e.g. neonate of 30 weeks should have a mean arterial blood pressure of at least Based on prospectively collected observations in 72 neonates, we observed a clinical very modest 126 decrease in heart rate (-7 bpm) and mean arterial blood pressure (-3 mmHg) following initiation of iv 127 paracetamol. However, a minority of neonates (9 %) developed hypotension. Interestingly, these 128 cases already had significantly lower blood pressures before administration of paracetamol. 129

Effective management of pain should be based on systematic assessment of pain, followed by 130 titrated administration of the most appropriated analgesic(s) with subsequent re-assessment. In any 131 patient -also in neonates -the administration of an analgesic is a balanced decision based on 132 perceived benefits (e.g. agitation, pain, stress) and risks (e.g. hypotension, bowel paresis, 133 hypersensitivity, intolerance, withdrawal, neuro-apoptose) [10]. 134

When we focus on the haemdynamic impact of analgesics or sedatives in neonates, there are 135 documented observations on effects of e.g. midazolam (+30 bpm, -3 mmHg) [START_REF] Jacqz-Aigrain | Placebo-controlled trial of midazolam sedation in mechanically ventilated newborn babies[END_REF], fentanyl (-13 bpm, -136 3 mmHg) [START_REF] Guinsburg | Physiological, hormonal, and behavioral responses to a single fentanyl dose in intubated and ventilated preterm neonates[END_REF], fentanyl and midazolam (-19 bpm, -12 mmHg) [START_REF] Burtin | Hypotension with midazolam and fentanyl in the newborn[END_REF], propofol (-9 bpm, -14 mmHg) [START_REF] Vanderhaegen | Cerebral and systemic hemodynamic effects of intravenous bolus administration of propofol in neonates[END_REF] 137 or morphine (-8 bpm, -3 mmHg) [START_REF] Sabatino | Hemodynamic effects of intravenous morphine infusion in ventilated preterm babies[END_REF][START_REF] Simons | Pain control: opioid dosing, population kinetics and side-effects[END_REF] on heart rate and mean arterial blood pressure. Pre-existing 138 low mean arterial pressure was as risk factor for subsequent hypotension following morphine 139 administration [START_REF] Simons | Pain control: opioid dosing, population kinetics and side-effects[END_REF]. 140

Compared to the impact of other analgesics or sedatives in neonates, haemodynamic changes 141 following iv paracetamol are limited, but not absent. 142 143 144 Obviously, these changes following iv paracetamol at least in part likely represent improved 145 analgesia, but it seems cautious to consider preadministration impaired haemodynamics as a relative 146 contra-indication to initiate intravenous paracetamol. 147 We can't but speculate why the extent of the impact of intravenous paracetamol on the 148 haemodynamics seems to be different in neonates compared to the observations in adult intensive 149 care setting. Hersch et al. [7] suggested that (traumatic) brain injury and pyrexia/hyperthermia are 150 specific risk factors while de Maat et al. [5] were unable to unveil specific risk factors in their 151 medium-and intensive care adult patients. At least, it was hereby reillustrated that focused studies 152 on the pharmacokinetics and -dynamics of analgesics -including intravenous paracetamol -in early 153 life are needed and feasible using appropriated methodology [1]. 154

  least, it was illustrated that focused studies on pharmacokinetics and -dynamics in 149 neonates are relevant [16R, Kopelman BI, Anand KJ, et al (1998) Physiological, hormonal, and behavioral responses to a single fentanyl dose in intubated and ventilated preterm neonates. J Pediatr 132:954-959 12. Burtin P, Daoud P, Jacqz-Aigrain E, Mussat P, Moriette G (1991) Hypotension with midazolam and fentanyl in the newborn. Lancet 337(8756):1545-1546 13. Vanderhaegen J, Naulaers G, Van Huffel S, Vanhole C, Allegaert K (2010) Cerebral and systemic hemodynamic effects of intravenous bolus administration of propofol in neonates. Neonatology 98:57-63 14. Sabatino G, Quartulli L, Di Fabio S, Ramenghi LA (1997) Hemodynamic effects of intravenous morphine infusion in ventilated preterm babies. Early Hum Dev 47:263-270 15. Simons SH, Anand KJ (2006) Pain control: opioid dosing, population kinetics and side-effects. Semin Fetal Neonatal Med 11:260-267 16. De Cock RF, Piana C, Krekels EH, et al (2010) The role of population PK-PD modelling in paediatric clinical research. Eur J Clin Pharmacol Mar 26, online available (PMID 20340012)

  prediction of concentration and effect compared to enteral formulations 29 by elimination of absorption variability. In recent years, data on iv paracetamol pharmacokinetics 30

Study 1 :

 1 single dose pharmacokinetic study in neonates [2decision to prescribe propacetamol or any other analgesic was made by the attending 50

3 .(

 3 analysis and statistics 75For statistical analysis, Medcalc® (Mariakerke, Belgium) was used. From the individual data, mean 76 values and standard deviation were calculated when normal distribution was documented 77 (Kolmogorov-Smirnov test), otherwise reported by median and range. 78Baseline values of haemodynamics were recorded just before initiation of iv paracetamol 79 administration. Correlations (spearman rank) between baseline values (heart rate, mean arterial 80 blood pressure) and clinical characteristics (weight, age) were investigated. Compared to baseline 81 haemodynamics, changes in parameters up to 6 h after iv paracetamol were collected. From the 82 individual data, mean values over the whole group were calculated. Paired Wilcoxon was 83 subsequently used to analyze changes compared to baseline (bpm, mmHg). One-way analysis of 84 variance (ANOVA) was used to test the difference between the means of the consecutive 85 measurements. Finally, incidence of hypotension was calculated, and characteristics of neonates who 86 developed hypotension (any time) following iv paracetamol were compared with normotensive cases 87

Table 1

 1 Haemodynamics of iv paracetamol in 72 neonates. Heart rate and blood pressure (mean, systolic, diastolic) reported by mean and standard deviation for all consecutive time intervals.

		Pre	30 min	60 min	120 min	180 min	240 min	300 min	360 min
	Heart rate (bpm)	145 (20)	138 (21)	141 (20)	137 (20)	137 (22)	140 (20)	139 (20)	140 (21)
	Blood pressure
	Mean (mmHg)	46 (7)	45 (7)	43 (8)	45 (8)	44 (7)	45 (8)	45 (8)	45 (8)
	Systolic (mmHg)	55 (10)	52 (8)	51 (9)	53 (10)	51 (9)	53 (10)	51 (9)	52 (9)
	Diastolic (mmHg)	38 (7)	39 (7)	38 (8)	39 (8)	38 (7)	39 (8)	37 (7)	39 (8)

Paracetamol (acetaminophen) is the most frequently prescribed drug for pain or fever, also in

neonates and can be administered by oral, rectal or intravenous (iv) route. An iv formulation likely

propacetamol (Prodafalgan®, Bristol Myers Squibb, Braine l'Alleud, Belgium) was administered.

Propacetamol is iv prodrug of paracetamol but necessitates plasma esterase activity. This esterase

function is already at adult level of activity in early life[3]. Propacetamol was administered when

based on intra-arterial measurements.

analysis and statistics

European Journal of Clinical Pharmacology

Paracetamol (acetaminophen) is the most often prescribed drug for mild to moderate pain or fever in

children, including neonates and can be administered by oral, rectal or intravenous (iv) route. An iv

function is already at adult level of activity in early life[4]. Propacetamol was administered when

neonates underwent minor, painful procedures (i.e. insertion of peripheral arterial or venous line,

insertion of central venous line, placement of chest tube) or as additional therapy in neonates on

mmHg) was recorded. This is the most frequently used, 'bedside' definition of clinical hypotension

Acknowledgements 152 Clinical research supported by the Fund for Scientific Research, Flanders (Belgium) (FWO Vlaanderen) 153 by a Fundamental Clinical Investigatorship (1800209N). 154 Acknowledgements 156 The clinical research of Karel Allegaert is supported by the Fund for Scientific Research, Flanders 157 (Belgium) (F.W.O. Vlaanderen) by a Fundamental Clinical Investigatorship (1800209N). 158

Systolic (mmHg) 55 (10) 52 ( 8) 51 ( 9) 53 ( 10) 51 ( 9) 53 ( 10) 51 ( 9) 52 ( 9) Diastolic (mmHg) 38 ( 7) 39 ( 7) 38 ( 8) 39 ( 8) 38 ( 7) 39 ( 8) 37 ( 7) 39 (8)


Michal Konecny
email: mkonecny@ousa.sk

Miriam Milly

Katarina Zavodna

Eva Weismanova

Jaroslava Gregorova

Iveta Mlkva

Denisa Ilencikova

Juraj Kausitz

Zdena Bartosova

Konecny Michal

Milly Miriam

Zavodna Katarina

Weismanova Eva

Gregorova Jaroslava

Mlkva Iveta

Ilencikova Denisa

Kausitz Juraj

Bartosova Zdena

Comprehensive genetic characterization of hereditary breast/ovarian cancer families from Slovakia

Keywords: Hereditary breast ovarian cancer, BRCA1, BRCA2, mutation, haplotype, MLPA, uncertain variant

come

INTRODUCTION

Each year, approximately 2000 of newly diagnosed breast and 450 of ovarian cancer cases appear in Slovakia.

The average risk of breast cancer in general Slovak population of women is 4-5% and of ovarian cancer 2% [START_REF] Ondrusova | Comprehensive analysis of incidence and mortality of malignant tumours in the Slovak Republic[END_REF]. About 5% of breast or ovarian carcinomas show Mendelian, monogenic, autosomal-dominant inheritance represented by hereditary breast and ovarian cancer syndrome (HBOC), for which two major associated genes (BRCA1, BRCA2) have been identified [START_REF] Antoniou | Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies[END_REF].

The BRCA1 gene was localized on chromosome 17q21. It contains 24 exons and encodes a 208 kDa protein composed of 1863 amino acids [START_REF] Miki | A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1[END_REF]. The second breast cancer susceptibility gene, the BRCA2 was localized to chromosome 13q12.3 and consists of 27 exons encoding a 380 kDa protein composed of 3418 amino acids [START_REF] Wooster | Localization of a breast cancer susceptibility gene BRCA2, to chromosome 13q12-13[END_REF].

The BRCA proteins are mainly involved in the repair of DNA double-strand breaks (DSB) and in homologous recombination (HR). About 85% of causal germline alterations in the BRCA1/2 are frame-shift or nonsense mutations, which lead to the truncation of the protein. The particular mutations differ in distribution depending on ethnicity and geographic location. Some authors believe that missense variants and single nucleotide polymorphisms (SNP) in high penetrance genes may represent additional HBOC risk factors. Even haplotypes may play a more important role than an individual SNP. BRCA1 is hypothesized to be a locus under recombination inhibition, and very few haplotypes have been described here [START_REF] Cox | Haplotype analysis of common variants in the BRCA1 gene and risk of sporadic breast cancer[END_REF]. Frosk et al. [START_REF] Frosk | The use of ancestral haplotypes in the molecular diagnosis of familial breast cancer[END_REF] in their recent study identified eight distinct haplotypes in BRCA1, mostly derivates of two main lineages. In BRCA2, 17 distinct haplotypes were reported, indicating more complex polymorphic pattern in this gene.

The structure of BRCA1 gene exhibiting high density (42%) of intragenic Alu repeated regions [START_REF] Welcsh | BRCA1 and BRCA2 and the genetics of breast and ovarian cancer[END_REF] together with the existence of BRCA1 pseudogene located 30 kb upstream of BRCA1 is increasing the likelihood of formation of the large genomic rearrangements (LGRs) by homologous recombination. The proportion of LGRs among the BRCA1 mutations in HBOC families seems to be population-dependent, varying from 2.1% in a series of Spanish breast/ovarian high-risk families [START_REF] De La Hoya | Genomic rearrangements at the BRCA1 locus in Spanish families with breast/ovarian cancer[END_REF] to 36% in the Dutch patients [START_REF] Petrij-Bosch | BRCA genomic deletions are major founder mutations in Dutch breast cancer patients[END_REF]. Some authors refuse the so called polygenic model for HBOC, in which the risk of breast/ovarian cancer is not influenced by only single high penetrance gene, but rather by a cumulative effect of the variations of several genes with moderate risk. Generally, the products of HBOC moderate risk genes cooperate with BRCA proteins through participation in common protein complexes and intracellular processes, such as DNA repair, transcription, cell cycle control (e.g. ATM, ATR, CHEK2, STK11/LBK1, p53, RAD50, RAD51, BARD1, BAP1, BACH1, CtIP, HDAC1/2, MSH2/6, MLH1, PCNA, PTEN, RNA polymerase II and others) [START_REF] Narod | BRCA1 and BRCA2: 1994 and beyond[END_REF].

Our study was primarily focused on the evidence (analysis) of genomic germline mutations in BRCA1/2 genes and other selected HBOC risk genes, such as TP53 and CHEK2*1100delC. In the second step, we aimed to improve clinical interpretation of the role of uncertain missense variants and SNP haplotypes in Slovak HBOC families.

MATERIAL AND METHODS

Samples and families

All analysed individuals signed informed consent and were referred for HBOC genetic analysis on the basis of family history of breast/ovarian cancer. All tested families were classified into the groups A and B due to their family history and each group was discriminated into the 4 subgroups according to the number of affected cases and the age at diagnosis (Tab. 1). The analysed families and data were collected since 2002 if one of these indication criteria occurred within the family history:

1. Presence of at least 2 patients with diagnosed breast or ovarian cancer among the direct relatives and at least one case diagnosed before age of 45.

2. Presence of bilateral breast or ovarian cancer among the direct relatives diagnosed at any age.

3. Occurence of duplex breast and ovarian cancer in at least one patient diagnosed at any age.

4. Presence of sporadic breast or ovarian cancer diagnosed at age under 35 years.

5.

Presence of at least one case of male breast cancer diagnosed at any age.

Genomic DNA was extracted from peripheral blood samples using QIAamp Mini Kit (Qiagen, Germany).

PCR amplification

In a primarily used approach, we analysed a whole coding region of the BRCA genes using combination of PCR BRCA2 was analysed in 52 PCR fragments, 20 covering coding exons 2-26 (exon 5 and 6 in one fragment) with exception of large exons, which were sub-divided into smaller fragments (exon 10 into 5, exon 11 into 20, exon 14 and 18 into 2 each, and exon 27 into 3 fragments). Size of all BRCA PCR fragments varied from 162 bp to 450 bp. The fragments with atypical mobility or with an atypical number of bands in the SSCP analysis were further sequenced. Primer sequences used for PCR amplification were similar to those published by Friedman et al. [START_REF] Friedman | Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families[END_REF], but were mostly redesigned using OligoCalc [12].

Each 25 μl PCR reaction consisted of following reagents: 1x PCR buffer EXT, 1.5 mM MgCl 2 , 0.2 mM dNTP mix, 0.6 U EXT polymerase (all Finnzymes), 12.5 µM of each primer and 150 ng of DNA. The PCR cycling was performed using thermal profile: 94°C/4 min, 94°C/30 sec, 53-65°C/35-50 sec, 72°C/45-80 sec, 72°C/7 min, 4°C/pause, for 35-45 cycles. In the nested PCR, 20x dilution of large PCR fragments was prepared and a 3 μl aliquot was used in the re-amplification of sub-fragments.

Fragment analysis

Fragment analysis using dye labelled primers was performed for the detection of most relevant frame-shift mutations: c.5266dupC in exon 20, c.68_69delAG in exon 2 of BRCA1 gene and c.1100delC in CHEK2 gene.

Primers were labelled with FAM, or VIC.

Allelic discrimination

Allelic discrimination analysis using TaqMan probes was used for detection of mutation p.Cys61Gly

MLPA analysis

Multiplex ligation-dependent probe amplification (MLPA) kit SALSA P002B and SALSA P087 (MRC-Holland) were used for BRCA1 analysis and SALSA P045B (MRC-Holland) for BRCA2 and partial CHEK2 analysis (mutation 1100delC included), according to the manufacturer's protocol.

Sequencing analysis

In a novel approach to BRCA1 analysis, the exons 3, 11, 13, 16 and 18 were directly sequenced without using any pre-screening method. Exon 11 of BRCA1 was divided into 7 large fragments (347 to 778 bp) and all were directly sequenced. PCR amplifications for direct sequencing were performed in 25 μl PCR reaction volume, using 1x AmpliTaq Gold Mastermix, 12.5 µM of each primer and 100 ng of DNA in the thermal profile: 95°C/5 min., 95°C/30 sec., 58°C/35-45 sec., 72°C/45-55 sec., 72°C/7 min., 4°C/pause, 30-40 cycles.

Hot spot exons 5-8 of TP53 gene were also analysed using direct sequencing of two large PCR fragments using primers designed according to the same rules as described previously.

PCR fragments were purified with ExoSAP-IT (USB Corp.) according to the manufacturer's protocol.

Subsequently, the sequencing reaction was prepared using 0.2x Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems), 0.75x Sequencing Buffer (Applied Biosystems), 2 µM primer and 10-20 ng of PCR product. Sequencing PCR analysis was performed with the following thermal profile: 96°C/1 min., 96°C/10 sec., 50°C/5 sec., 60°C/3 min., 4°C/pause for 25 cycles. Afterwards, the samples underwent postsequencing purification via columns (Sigma-Aldrich) according to the manufacturer´s protocol.

Protein prediction analysis

Potential clinical effect of variants with unknown significance (UVs) was evaluated by analyses of the severity of the amino acid changes and their conservation across species. These analyses were performed using prediction analysis web tools Align-GVGD [13], PMut [14] and PolyPhen [15].

Nomenclature

Identified mutations were compared with human BRCA reference sequences, at the cDNA level with NM 007294.3 and U 43746.1. In the case of intronic variations, genomic DNA reference sequences NG 005905.2 and U 94788.1 were used. The HUGO-approved systematic nomenclature, for the description of sequence variants in DNA and protein sequences published by den Dunnen and Paalman [START_REF] Den Dunnen | Standardizing mutation nomenclature: why bother?[END_REF] was used. Exonic frame-shift and all intronic mutations were assigned at the nucleotide level, while exonic missense and nonsense mutations were marked at the protein level using HUGO-approved systematic nomenclature.

Novel mutations

The presence of all identified mutations, uncertain variants and polymorphisms was examined in Breast Cancer Information Core database [START_REF]Breast Cancer Information Core database[END_REF], MutDb [18], HGMD database [19] and Swiss-Prot database [START_REF]Swiss-Prot database[END_REF]. Variants assigned as novel were not present in any of these databases.

RESULTS

Molecular genetic analysis of 585 HBOC families was performed using different approaches (SSCP, fragment analysis, allelic discrimination and direct sequencing and MLPA). Within this set, we identified 100 families with the presence of clearly deleterious mutations (17.1%). Majority of these 100 families carried mutations in BRCA1 (88%), remaining 12% in BRCA2 gene. Furthermore, the analysis of TP53 exons 5-8 in 64 BRCA negative families and the mutation c.1100delC of CHEK2 gene in 159 families, did not reveal any causal mutation.

Pathogenic germline BRCA1 mutations

Pathogenic mutations in BRCA1 were detected by DNA sequencing in 14.5% (85/585) of analysed families (Table 2). Altogether, 25 different BRCA1 mutations were identified, of which 21 caused a premature stop codone (84%), 15 were frameshifts and 6 nonsense mutations. Furthermore, 2 splice-site and 2 missense mutations were detected. The majority of mutations occurred in the largest exon 11 (54%).

The most frequently detected BRCA1 mutations were c.5266dupC in 32 families (38%); p.Cys61Gly in 14 families (17%); c.68_69delAG and c.3700_3704del5 each in 4 families (5%), and finally c.843_846del4 and c.4243delG in 3 families each (4%). Altogether, mutations identified in exons 2, 5, 11 and 20 represented 86% of all BRCA1 mutation-positive Slovak HBOC families (73 out of 85). Mutations c.1166delG, c.1938_1947del10 and c.80+4del4 were previously reported only in Slovak HBOC kindreds [START_REF] Konecny | The spectrum and incidence of BRCA1 pathogenic mutations in slovak breast/ovarian cancer families[END_REF][START_REF] Cierniková | The novel exon 11 mutation of BRCA1 gene in a high-risk family[END_REF][START_REF] Konecny | Identification of a novel mutations BRCA1*c.80+3del4 and BRCA2*c.6589delA in Slovak HBOC families[END_REF].

The highest detection rate of BRCA1 causal mutations (33%) was observed in families with the presence of duplex breast and ovarian carcinoma in one patient (6 of 18), followed by 33% in the group of families with the presence of breast and ovarian cancer (25 of 75), 29% in families with presence of bilateral ovarian cancer (2 of 7), 13% in families with presence of bilateral breat cancer (11 of 85), 12% in families with presence of unilateral ovarian cancer (6 of 50), and 10% in families with presence of unilateral breast cancer only ( The presence of LGRs was analysed in 61 HBOC families negative for BRCA1/2 pathogenic small mutations. The MLPA analysis of both BRCA genes and fragments of CHEK2 gene (promoter, exon 9, exon 10/c.1100delC) was performed simultaneously. Altogether, we identified 3 families positive for the presence of BRCA1 gene LGRs (5%), while no mutation was detected in BRCA2. Two type of LGRs were present, a deletion of exon 21 and 22 and a deletion of exons 1 through 24. The breakpoints of the detected LGRs were not determined.

Pathogenic germline BRCA2 mutations

Causal BRCA2 mutations were identified in 12 out of 104 families (11.5%) (Table 2). Nine different BRCA2 mutations were detected, 6 frameshifts, 2 nonsense mutations and 1 missense mutation of the initial Met codon.

Almost all mutations cause a premature stop codon (91%). The majority of mutations occurred in the largest exon 11 (56%). Mutation c.6589delA was previously reported only in Slovak population [START_REF] Konecny | Identification of a novel mutations BRCA1*c.80+3del4 and BRCA2*c.6589delA in Slovak HBOC families[END_REF].

The detection rate 10% of BRCA2 causal mutations was observed in families with diagnosed breast cancer only (6 of 59), 9% in families with presence of bilateral breat cancer (2 of 22). In families with the presence of breast and ovarian cancer (4 of 14), the detection rate was determined to 29%. However, in these cases the presence of male breast cancer, prostate cancer and colorectal cancer was observed as well. No causal mutations were detected in 5 families with the occurrence of duplex breast and ovarian carcinoma in one patient, 3 families with unilateral ovarian cancer and 1 family with bilateral ovarian cancer. Mutation c.9098dupA was detected in family with the presence of male breast cancer.

The mean age at the time of cancer diagnosis of the BRCA2-positive patients was 46 years (33 -59), while in the subgroup with the unilateral breast cancer diagnosis age was 52.7 (37 -59). Two positive patients with bilateral breast cancer were diagnosed at mean age of 42.5 years [START_REF] Ruffner | Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity[END_REF]52) and one patient with ovarian cancer at the age of 49 years.

BRCA1/2 variants with uncertain clinical effect

In 117 HBOC families (out of 585) we identified 19 different types of UVs in BRCA1 gene (20%). In 19 cases UVs were associated with the presence of pathogenic mutation. The most UVs (84%) were missense substitutions [START_REF] Den Dunnen | Standardizing mutation nomenclature: why bother?[END_REF], 16% intronic variants (1 substitution and 1 insertion) and 1 was an in-frame deletion. The spectrum of detected variants is reported in Table 3. Focused on the localization of UVs in BRCA1 gene, we observed that 79% of the variants were situated in exon 11 (92 of 117). The most frequently detected UV in BRCA1 gene was p.Gln356Arg, found in 68 families (58%), however, in 16 cases it was associated with a one of the known pathogenic mutations (p.Cys61Gly, c.5266dupC, c.3700_3704del5, p.Arg1751X). The second most frequent variant was p.Met1652Ile identified in 14 families (12%) with no detected association to pathogenic mutations. Uncertain variant p.Ser1040Asn was identified in 11 families (9.4%) and associated with two pathogenic mutations (c.1166delG, p.Leu974X). We have identified three previously unreported novel variants p. Asn630Asp, p. Asn916Thr and p.Thr1246Asn. Variant p.Thr1246Asn was detected in association with mutation c.4065_4068del4 in two families.

BRCA2 UVs were identified in 17 (16%) out of 104 analysed families. Altogether, 8 different UV types were observed; 7 missense and 1 intronic variant. The most frequently detected UV was c.1909+12delT found in 7 families, in 1 case associated with mutation p.Ser1882X. Variant p.Thr1912Met was found in 4 families, and was not associated with the pathogenic mutation. All other UVs were observed only in one family. Uncertain variant p.Ser2052Leu was identified as novel, not yet published, however, segregation analysis did not reveale any association with breast/ovarian cancer. Missense UVs in the BRCA2 were mostly localized in exon 11 (71%).

Prediction analysis of BRCA UVs was based on the comparison of the substituted amino acids and predicted proteins using three software packages: Align-GVGD, Pmut, and Polyphen. Results of the prediction analysis are shown in Table 4. Summarizing prediction results, variants p.Glu1346Gly, p.Ser1512Ile in BRCA1 and p.Trp2626Cys, p.Val2908Gly in BRCA2 were predicted as being most probably pathogenic in all used prediction approaches. Uncertain variants p.Arg841Trp in BRCA1 and p.Gly2274Val in BRCA2 were predicted as possible pathogenic. The haplotype consisting of the 8 SNPs: p.Ser694Ser, p.Leu771Leu, p.Pro871Leu, p.Glu1038Gly, p.Lys1183Arg in exon 11, p.Ser1436Ser in exon 13, p.Ser1613Gly in exon 16 and c.5152+66G/A behind exon the wild-type haplotype without any BRCA1 SNP. The spectrum of identified assumed BRCA1 haplotypes is shown in Table 5.

BRCA

Other HBOC risk genes -TP53, CHEK2*1100delC analysis

The samples from the 61 HBOC BRCA negative families were analysed for the presence of germline mutations in exons 5-8 of TP53 gene. By performing sequencing analysis, no pathogenic mutation was detected. Despite this fact, we identified 6 different variants: 5 intronic variants and one silent variant in exon 6. Silent alteration p.Arg213Arg (c.836G>A) was identified in 2 families. Altogether, detected variants were distributed in 40% of families (24 of 60). Analysis of the CHEK2 gene using fragment analysis and MLPA analysis was focused on the most frequently reported mutation c.1100delC in exon 10 and revealed no mutation in the 159 BRCA negative families.

DISCUSSION

Molecular-genetic analysis of BRCA1/2 genes in 585 Slovak HBOC families carried out since 2002 revealed 100 mutation positive families (17.1%). Eighty five and 12 families carried pathogenic mutations in BRCA1 and BRCA2, respectively that were detected by DNA sequencing and 3 families carried LGRs in BRCA1 detected by MLPA. Comparing to other studies, especially from the Central European region, e.g. to Czech population with detection rate ranging from 19.8% [START_REF] Wagner | BRCA1-related breast cancer in Austrian breast and ovarian cancer families: specific BRCA1 mutations and pathological characteristics[END_REF] to 29.1% [START_REF] Machackova | Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer[END_REF], the detection rate in Slovak HBOC population is lower.

Several factors, such as composition of families, number of families, selection criteria or used screening techniques might contribute to this inequality between historically associated populations. The detection rate comparable to ours was reported in the Sicilian population 16% [START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF], or in Spain 17.1% [START_REF] Llort | Low frequency of recurrent BRCA1 and BRCA2 mutations in Spain[END_REF].

The highest detection rate of BRCA1 pathogenic mutations was observed in families with the presence of both breast and ovarian cancer (33%) and in families with duplex ovarian and breast carcinoma diagnosed in one patient (33%). The lowest rate of only 10% was present in families with unilateral breast cancer. According to the reported data and also comparing with other studies [START_REF] Machackova | Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer[END_REF][START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF][START_REF] Krajc | Five recurrent BRCA1/2 mutations are responsible for cancer predisposition in the majority of Slovenian breast cancer families[END_REF][START_REF] Miramar | Genetic analysis of BRCA1 and BRCA2 in breast/ovarian cancer families from Aragon (Spain): two novel truncating mutations and a large genomic deletion in BRCA1[END_REF], the presence of ovarian cancer in association with breast cancer serves as a potential predictive factor for BRCA1 mutation positivity and represents the strongest indication. On the other hand, the presence of bilateral ovarian (17%) or breast (13%) cancer is still stronger criterion for selection as the familiar unilateral cancer. The highest BRCA2 mutation detection rate of 29% was observed in families carrying a wide range of carcinomas (breast, ovarian, colorectal, prostate, uterus) and a male breast cancer. Exclusive presence of bilateral breast cancer (9%) and unilateral breast cancer (10%) also shown association with several mutations, however, the exclusive presence of ovarian cancer does not seem to be an indication. Surprisingly, the mean age of patients at ovarian cancer diagnosis in BRCA1-negative cases was 45 years , but in BRCA1 positive cases was nearly 50 years (unilateral ovarian cancer -51.2, duplex breast and ovarian cancer -49.2 and bilateral ovarian cancer -52 years). This observation is indicating that the presence of BRCA1 pathogenic mutations is more likely in the patients with breast cancer at 40 years and ovarian cancer at relatively higher age of 50 years. Thus, sporadic ovarian cancer cases diagnosed at very young age (under 40 years) probably should not be indicated to BRCA analysis because their cancers may be caused by the environmental and lifestyle risk factors (smoking, contraceptives etc.).

According to our data, the BRCA1 pathogenic mutations in Slovak HBOC population may be divided into following groups:



Frequent mutations considered as Slavic founder: p.Cys61Gly and c.5266dupC. Other pathogenic mutations (27%) identified only in one or two families fall in a group of Slovak HBOC low frequency BRCA1 mutations, though some of them were detected in neighbouring populations as well.



Machackova et al. [START_REF] Machackova | Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer[END_REF] identified mutations p.Cys39Arg, p.Gln563X, c.2488_2497dup10, c.3700_3704del5 and p.Gln1447X in Czech population, which is probably the closest to the Slovak population. Mutations c.4065_4068del4 and p.Arg1443X were found in Sweden [START_REF] Bergman | A high frequency of germline BRCA1/2 mutations in western Sweden detected with complementary screening techniques[END_REF], p.Arg1443X in the Sicilian population [START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF] as well as in the French population as a founder mutation [START_REF] Vezina | Molecular and genealogical characterization of the R1443X BRCA1 mutation in high-risk French-Canadian breast/ovarian cancer families[END_REF]. Other Slovak low frequency BRCA1 pathogenic mutations were not occuring in any European HBOC report.

All detected missense causal mutations were localized in a highly conserved structure of C 3 HC 4 RING domain of BRCA1, which has been described as Zn 2+ binding site of RING domain for dimerization with BARD1 [START_REF] Ruffner | Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity[END_REF].

Highly conserved residues C39, H41, C61, and C64 are localized in the regions where mutations p.Cys39Arg, p.Cys61Gly were observed. The small deletion c.80+4del4 results in the skipping of exon 2 and subsequent generation of an aberrant translational start in exon 5.

Interestingly, a minimum of nonsense mutations was identified in exon 11 [START_REF] Konecny | The spectrum and incidence of BRCA1 pathogenic mutations in slovak breast/ovarian cancer families[END_REF][START_REF] Machackova | Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer[END_REF][START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF][START_REF] Krajc | Five recurrent BRCA1/2 mutations are responsible for cancer predisposition in the majority of Slovenian breast cancer families[END_REF][START_REF] Miramar | Genetic analysis of BRCA1 and BRCA2 in breast/ovarian cancer families from Aragon (Spain): two novel truncating mutations and a large genomic deletion in BRCA1[END_REF] in comparison to other types of mutations in this exon and no causal missense mutations were present in this exon due to the lack of highly conserved domain in this region. However, it can not be excluded that some of the frequently occurring missense variants with uncertain clinical significance localized mainly in exon 11 (and similarly in exon 16), might have some effect on the protein function and thus play a role in the development of HBOC phenotype.

Altogether, 9 different BRCA2 mutation types in 12 families were identified in the set of 104 HBOC families (11.5%). Generally, the most of the pathogenic mutations in this gene in our series are localized in exon 11, similarly to many other reports [START_REF] Bergman | A high frequency of germline BRCA1/2 mutations in western Sweden detected with complementary screening techniques[END_REF][START_REF] Pohlreich | High proportion of recurrent germline mutations in the BRCA1 gene in breast and ovarian cancer patients from the Prague area[END_REF][START_REF] Kataki | Identification of germline BRCA1 and BRCA2 genetic alterations in Greek breast cancer moderate-risk and low-risk individualscorrelation with clinicopathological data[END_REF]. Interestingly, 44% of BRCA2 pathogenic mutations detected in our cohort of patients have also been reported in Czech HBOC population [START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF][START_REF] Foretova | BRCA1 and BRCA2 mutations in women with familial or early-onset breast/ovarian cancer in the Czech Republic[END_REF] confirming the genetic relationship of these two neighbouring populations.

It seems that

LGRs do not represent frequent mutational events in Slovak HBOC families (5%). Compared to other studies, our LGR detection rate was similar to the one in Czech 5.8% [START_REF] Vasickova | High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic[END_REF] or German population 5.3% [START_REF] Hartmann | Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families[END_REF]. Deletion of exons 21-22 in BRCA1 gene was previously reported in Czech population [START_REF] Vasickova | High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic[END_REF]. This deletion affects the C-terminal BRCT domain of BRCA1 protein and result in the skipping of 43 amino acids from the position 1760 to 1802. This loss of a part of the conservative BRCT domain is suggested to have a cancer susceptibility effect. Deletion of exons 1 through 24 represents a very rare event and was previously reported in one Spain [START_REF] De La Hoya | Genomic rearrangements at the BRCA1 locus in Spanish families with breast/ovarian cancer[END_REF] and one German family [START_REF] Engert | MLPA screening in the BRCA1 gene from 1,506 German hereditary breast cancer cases: novel deletions, frequent involvement of exon 17, and occurrence in single early-onset cases[END_REF]. Identifying the second family with the same deletion of BRCA1 allele living in the same geographical area raises the question whether these two mutations are exactly identical, what can be answered after the determiantion of breakpoints. Alternatively, testing of families relationship and haplotype analysis of family members may reveal wether this mutation is family specific or represents a new Slovak founder mutation.

Mutation detection rate of BRCA1/2 uncertain variants in Slovak HBOC families is relatively high, 23% (133 families), in contrast to recent studies reporting substantially lower frequencies: 6.8% in Spanish population [START_REF] Díez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF],

5.7% in Sicilian population [START_REF] Russo | BRCA1 genetic testing in 106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses[END_REF], 5.1% in Indonesian population [START_REF] Purnomosari | BRCA1 and BRCA2 germline mutation analysis in the Indonesian population[END_REF], only 1.5 % in both, New Zealand and Australian populations [START_REF] Tesoriero | Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families[END_REF]; and 0.86% in US HBOC families [START_REF] Pal | BRCA1 and BRCA2 mutations account for a large proportion of ovarian carcinoma cases[END_REF]. A big deviation in the detection rates may reflect sensitivity of detection techniques used in the different studies. In some studies, the identification of mutations in exon 11 was based on the performance of techniques such as SSCP, PTT, DGGE, CSGE [START_REF] Díez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF] or DHPLC [START_REF] Miramar | Genetic analysis of BRCA1 and BRCA2 in breast/ovarian cancer families from Aragon (Spain): two novel truncating mutations and a large genomic deletion in BRCA1[END_REF][START_REF] Purnomosari | BRCA1 and BRCA2 germline mutation analysis in the Indonesian population[END_REF]. We assume that employment of direct sequencing for analysis of large exons in our study resulted into the identification of a broader spectrum of missense UVs.

However, interpretation of uncertain variants still remains problematic. Prediction of the effect of UVs in functional analysis studies and in the prediction software may generate controversial results. Complex evaluation of missense UVs should consist of the combination of several approaches based on, but not limited to cosegregation analysis of UVs with disease in a family, loss of heterozygosity in tumors, determination of the frequency of variant in unaffected controls, in silico prediction analysis, functional assays [START_REF] Chenevix-Trench | Genetic and histopathologic evaluation of BRCA1 and BRCA2 DNA sequence variants of unknown clinical significance[END_REF]. In our study, we analysed the BRCA UVs using prediction software [13][14][15], next by studying the association of UVs with pathogenic mutation and number of entries in the BIC database and finally, if possible, by performing a cosegregation analyses of UVs with the disease. Taking in account the results of our examinations, we suggest that BRCA2 variants p.Gly2274Val, p.Trp2626Cys and p.Val2908Gly, and BRCA1 variants p.Glu1346Gly and p.Ser1512Ile are very likely pathogenic. Interestingly, relatively high portion of variants in BRCA2 gene are predicted as possible pathogenic (Table 4). However, these predictions should be confirmed by the segregation analysis of oncological disease within the affected family and other type of analyses in the future.

It is believed that the explanation of discrepancy between the frequency of hereditary breast/ovarian cancer families and detected BRCA mutations may lie in a polygenic heredity with many non-BRCA moderate risk susceptibility genes, although the existence of an autosomal recessive mendelian allele cannot be excluded [START_REF] Pharoah | Polygenic susceptibility to breast cancer and implications for prevention[END_REF][START_REF] Antoniou | The BOADICEA model of genetic susceptibility to breast and ovarian cancer[END_REF]. In this sense, breast cancer represents a complex genetic disease. Moderate risk candidate genes, which presumably alter breast cancer predisposition, may be found in a variety of pathways, ranging from the detoxification of environmental carcinogens to the steroid hormone metabolism and the DNA damage repair [START_REF] Syamala | Influence of germline polymorphisms of GSTT1, GSTM1, and GSTP1 in familial versus sporadic breast cancer susceptibility and survival[END_REF]. On the other hand, we suggest that many features of BRCA genes such as promoter sequences, epigenetic changes, microRNA, uncertain variants, intronic splice variants or SNP haplotypes may represent additional factors that might seriously alter the risk of breast/ovarian cancer onset. We hypothesize that so-called "BRCA intra-gene modifier factors" should be more investigated and may bring alternatives to polygenic models. SNP haplotypes in BRCA genes are mostly determined by the presence of a set of SNPs, which are grouped on one allele and inherited in a linkage. Haplotypes may play a more important role than individual SNPs and may alter the risk of breast/ovarian cancer. Identification of such haplotypes depends on the analysis of large number of samples to obtain sufficient amount of data. BRCA1 gene is a relatively optimal example since this gene is hypothesized to be a locus under recombination inhibition with very few haplotypes described. In fact, only one haplotype block and two major haplotypes have been shown to exist in Caucasians [START_REF] Cox | Haplotype analysis of common variants in the BRCA1 gene and risk of sporadic breast cancer[END_REF]. However, in BRCA2 gene the situation concerning haplotypes is more complex, reflecting the existence of a large number of variations

[49].
We observed the main haplotypes of BRCA1 gene in Slovak HBOC families, their homozygous or heterozygous status and we tried to interpret their possible clinical effect and/or identify a possible pathogenic haplotype. The most frequent haplotype (31.4%) seen in Slovak HBOC families consists of SNP variants: p. Interestingly, the pathogenic mutation p.Cys61Gly was in all 13 cases associated with the same haplotype with uncertain variant p.Gln356Arg. The significance of this observation and our assumptions may be supported by future investigations involving more functional and segregation analyses.

CONCLUSIONS

The analysis of BRCA1 and BRCA2 genes in 585 Slovak HBOC families revealed the presence of deleterious mutations in 100 families, representing a prevalence of 17.1%. In total, [START_REF] Machackova | Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer[END_REF] In silico prediction analysis complemented with segregation analysis may represent the model of standard diagnostic protocol for interpretation of exonic missense uncertain variants. Haplotypes of BRCA1 gene may represent potential modifiers of cancer risk in the future, as a part of the concept of BRCA intra-gene modifier factors. This approach should be based on the complex genomic analysis of both genes (promoter, epigenetic, intronic, microRNA, UVs, SNP haplotypes). Finally, we conclude that the analysis of other HBOC risk genes TP53 and CHEK2*c.1100delC is not efficient in Slovak HBOC families. This is the first comprehensive study of molecular-genetic features of HBOC syndrome in Slovak families which includes deleterious mutations, variants with uncertain clinical significance and SNP haplotypes.

  (c.181T>G) in exon 5 of BRCA1. The PCR amplification was mixed using 1x TaqMan universal PCR master mix (Applied Biosystems), 1x TaqMan Assay Mix (Applied Biosystems) and 20 ng of DNA in 20 μl volume on the ABI 7500 Real-Time analyser (Applied Bosystems) with thermal conditions 95°C/10 min., 92°C/15 sec., 60°C/1 min., 35 cycles. The positive and negative control samples were always analysed, thus the Ct and post-PCR fluorescence of normal and mutant allele were determined.

  35 of 350). The mean age at the time of cancer diagnosis in patients positive for BRCA1 mutation was 42.7 years (range 21-75). Mean age at diagnosis in the subgroup with the unilateral breast cancer was 40.7 (range 21-66), with the bilateral breast cancer 39.7 (range 30-46), with the unilateral ovarian cancer 51.2 (range 40-75), and with the duplex breast and ovarian cancer 47.5 (49, 50, 41 and 50 years). Bilateral ovarian cancer was diagnosed only in one BRCA1 positive patient (52 years).

  SNPs and BRCA1 haplotypes Single nucleotide polymorphisms (SNPs) in BRCA1 gene are mainly localized together on one allele and thus generate the SNP haplotype. The most frequently identified BRCA1 SNPs were p.Pro871Leu detected in 277 families (47%), p.Glu1038Gly in 276 families and p.Lys1183Arg in 275 families out of 585 analysed HBOC families. Altogether, 15 different BRCA1 SNPs were identified, of which 6 were silent, 5 missense and 4 intronic substitutions. The silent change p.Pro1562Pro in exon 16 was detected as novel, not yet reported. Major part (40%) of detected SNPs were localized in exon 11. The most frequently identified BRCA2 SNPs were p.His372Asn observed in 83 families (80%), c.7806-14T>C in 54 families (52%) and p.Lys1132Lys in 53 families (51%) out of 104 analysed HBOC families. Altogether, 16 different BRCA2 SNPs were detected, 7 silent, 6 missense and 2 intronic substitutions and 1 frame-shift variant. The silent change p.Asp980Asp in exon 11 was detected as novel, not yet reported. The most of identified SNPs were situated in exon 11 (50%) and in exon 10 (19%).

  Mean age of the BRCA1-positive patients was 43 years, ranging from 21 to 75 years. Mean age at the time of diagnosis was higher in BRCA1-negative cases (44 years, 21-70) than in BRCA1-positive cases (40 years, 21-66).

  p.Lys1183Arg in exon 11, p.Ser1436Ser in exon 13, p.Ser1613Gly in exon 16 and intronic variants c.4987-68G>A in intron 16, c.5152+66G>A in intron 18. The presence of a set of variants on both alleles (in homozygous stage) or the presence of two different SNP haplotypes (each in heterozygous stage) may importantly alter the biological pathways dependent on the BRCA1 protein.

  amplification, SSCP analysis and direct sequencing. BRCA1 was analysed in 38 PCR fragments: 21 of them covering coding exons 2-24, with exception of exon 11. Remaining 17 fragments covered exon 11; and were amplified by the nested PCR from two large fragments (11/5F with size of 2059 bp, and 11/3F of 1846 bp).

  and 12 different BRCA1 and BRCA2 mutations, respectively, that were detected by DNA sequencing and 2 LGRs in BRCA1 gene were found by We observed that besides the familial occurrence of the cancer disease, other important clinical criterion for BRCA1 analysis is the presence of a breast cancer diagnosed around the age of 40, in association with the presence of ovarian cancer diagnosed around 50 years of age. Conversely, for BRCA2 analysis, the diagnosis of breast cancer diagnosed at older age, around age of 50, or the presence of breast cancer in association with broader spectrum of other carcinomas (prostate, colorectum, ovaria, uterus) was clinically most indicative.

	MLPA.. Mutations c.5266dupC detected in 38%; p.Cys61Gly in 17%; c.68_69delAG, c.3700_3704del5 each in
	5% and finally c.843_846del4, c.4243delG each in 4% of families represent high-frequency Slovak BRCA1
	pathogenic mutations. Altogether, these five BRCA1 mutations were present in 73% of all BRCA1 positive
	HBOC families.

Table 1 .

 1 The distribution of breast/ovarian cancer families in the groups due to family history.

	Family history	Families Characterization	Number of families
	A. Strong	A1	At least 2 cases of breast or ovarian cancer but 1 under the age of 45, the rest under the age of 65 years	248	338

Table 2 . The spectrum of BRCA1/2 pathogenic mutations in Slovak HBOC families.

 2 Abbreviations: BIC, Breast information core; aa, amino acid; Fam., number of families; Sam., number of samples; del, deletion; dup, duplication; *, novel mutation; IVS, Intron Variable Site; FS, frame-shift; IS, intronic splicing; MS, missense; NS, nonsense; mut., mutation.

	No Approved nomenclature	BIC traditional nomenclature	Mutation type; predicted aa effect	Exon	Fam. World appearance
			BRCA1 pathogenic mutations
	1	c.68_69delAG	185delAG	FS; p.Glu23ValfsX16	2	4	Ashkenazi
	2	c.80+4del4	199+4delAGTC	IS, exon 2 skipping	2/3	1	Only in Slovakia
	3	c.115T>C	234T>C	MS; p.Cys39Arg	3	1	Central/ Eastern EU
	4	c.181T>G	300T>G	MS; p.Cys61Gly	5	14	EU and USA
	5	c.843_846del4	962delCTCA	FS; p.Ser281SerfsX15	11	3	Austria
	6	c.1166delG	1285delG	FS; p.Ser389MetfsX4	11	1	Only in Slovakia
	7	c.1687C>T	1806C>T	NS; p.Gln563X	11	1	Czech, Austrian, German
	8	c.1938_1947del10	2057del10CAGTGAAGAG FS; p.Ser646ArgfsX2	11	2	Only in Slovakia
	9	c.1953_1956del4	2072delGAAA	FS; p.Lys651LysfsX48	11	1	Western EU, USA
	10	c.2068delA	2187delA	FS; p.Lys690LysfsX9	11	2	German
	11	c.2488_2497dup10	2607dupAAGTATCCAT	FS; p.Lys833LysfsX2	11	1	Czech
	12	c.2921T A	3040T>A	NS; p.Leu974X	11	2	Australian
	13	c.3016_3019del4	3135delCATT	FS; p.His1006GlnfsX16	11	1	Austria
	14	c.3018_3021del4	3137delTTCA	FS; p.His1006GlnfsX16	11	1	Western Europe
	15	c.3700_3704del5	3819delGATAA	FS; p.Val1234GlnfsX7	11	4	Central/Eastern EU, Poland
	16	c.3770_3771del2	3889delAG	FS; p.Glu1257GlyfsX8	11	1	Central/Eastern EU
	17	c.4065_4068del4	4184delTCAA	FS; p.Asp1355LysfsX9	11	2	Norwegian, British, French/
							Canadian, Central EU
	18 c.4243delG	4362delG	FS; p.Glu1415LysfsX3	13	3	Slovak, German
	19	c.4327C>T	4446C>T	NS; p.Arg1443X	13	1	France, French/Canadian;
							Finland
	20 c.4339C>T	4458C>T	NS; p.Gln1447X	13	1	African, Czech
	21 c.4986+4A>T	5105+4A>T	IS, exon 16 skipping	16/17	2	Central-Eastern Europe
	22	c.5084_5085del2	5203delTT	NS; p.Phe1695CysfsX2	18	1	Ashkenazi, Central EU
	23	c.5251C>T	5370C>T	NS; p.Arg1751X	20	2	Finland, Greece, Western EU
	24	c.5266dupC	5385dupC	FS; p.Gln1756ProfsX73	20	32	Founder in EU, Ashkenazi
	25	c.5511G>A	5630G>A	NS; p.Trp1837X	24	1	Czech, French
			BRCA2 pathogenic mutations
	1	c.3G>A	231G>A	MS; p.Met1Ile	2	1	Western EU
	2	c.1408insG	1636insG	FS; p.Glu470GlufsX5	10	1	Not specified
	3	c.2806_2809del4	3034delAAAC	FS; p.Lys936LysfsX23	11	1	Western EU, Spanish, Czech
	4	c.3076A>T	3304A>T	NS; p.Lys1026X	11	2	Czech
	5	c.5645C>A	5873C>A	NS; p.Ser1882X	11	2	Western, Central EU
	6	c.5946delT	6174delT	FS; p.Ser1982ArgfsX21	11	1	Ashkenazi
	7	c.6589delA	6817delA	FS; p.Thr2197LeuX7	11	1	Only in Slovakia
	8	c.9098dupA	9326insA	FS; p.Thr3033AsnfsX9	23	1	Ashkenazi, Hungarian, USA,
							Spanich, Western EU
	9	c.9403delC	9631delC	FS; p.Leu3135PhefsX27	25	2	Polish, Czech

Table 3 . The spectrum of the variants of uncertain clinical significance identified in BRCA1 and BRCA2

 3

	genes in Slovak HBOC families. Abbreviations: aa, amino acid; nt, nucleotide; del, deletion; ins, insertion; *,
	novel mutation; FS, frame-shift; IS, intronic splicing; MS, missense.
	No Approved nomen. (aa)	Approved nomen. (nt)	Mutation type	Exon	Families	Association with pathogenic mutation
			BRCA1

Table 4 . Detected missense uncertain variants in BRCA1/2 genes. Characterization of substituted aminoacids and protein prediction.

 4 Abbreviations: aa, amino acid; mol. size, molecular size; sec. struc., secondary structure; BIC, Breast Cancer information core; neu, neutral; path, pathogenic.

		Wild-type aa	Mutant aa				Times
	UVs	Charge	Mol. size	Charge	Mol. size	PolyPhen	AGVGD	PMut	reported (BIC)
			BRCA1
	p.Gln356Arg acide, hydrophile	146.15	positive, basic	174.2	probable 2.08	C35	0.20 neu (5)	92x
	p.Arg496His	positive, alkaline	174.2	basic, aromatic	155.16	benign 0.75	C25	0.46 neu (0)	90x

Table 5 . The spectrum of probable haplotypes represented by mutations identified in BRCA1 gene in Slovak HBOC families.

 5

	Probable BRCA1 haplotypes


Steffen Kunsch

Volker Gross

Albrecht Neesse

Christoph Nell

Keywan Sohrabi

Prof.Dr Thomas M Gress
email: gress@med.uni-marburg.de

Volker Ellenrieder

Ulrich Köhler

A K Sohrabi

U Koehler

A pilot study of a novel approach to detect nocturnal respiratory symptoms in Gastro-Oesophageal Reflux Disease (GERD)

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Background and aims: Increased prevalence of respiratory symptoms has been commonly reported in patients with gastroesophageal reflux disease (GERD). Here, we introduce a novel Lung-Sound-Monitoring device that allows simultaneous measurement of both nocturnal respiratory symptoms and episodes of acidic and biliary refluxes. Methods: Nocturnal respiratory symptoms (coughing and wheezing) were continuously recorded in 20 healthy subjects and 30 reflux patients with respiratory symptoms in parallel to combined pH-monitoring and Bilitec measurement. Results: Analysis could be completed in 20 healthy subjects and in 25 patients with reflux. A clear temporal correlation to reflux phases was detected in 49% of coughing and 41% of wheezing events, respectively. Moreover, 89% of the coughing and 100% of the wheezing events succeeded reflux episodes. Finally, the technique was capable of assessing a statistically significant difference between patients and controls regarding the occurrence of these symptoms. Conclusions:

Our pilot study establishes the Lung-Sound-Monitoring system as a unique tool to objectively measure the temporal correlation between gastroesophageal reflux and the appearance of respiratory symptoms. It represents a useful technique to identify patients with respiratory symptoms due to reflux and therefore allows to determine and quantify the impact of therapeutic interventions such as antireflux therapy on respiratory symptoms. Gastro-oesophageal reflux disease (GERD) is caused by reflux of gastric acids and duodenal components such as bile acids into the oesophagus and represents one of the most common disorders in the industrialized world [START_REF] Moayyedi | Review article: gastro-oesophageal reflux disease--the extent of the problem[END_REF]. Interestingly, an increased prevalence of respiratory symptoms such as chronic coughing or bronchial asthma have been reported in association with GERD [START_REF] Vakil | The Montreal definition and classification of gastroesophageal reflux disease: a global evidencebased consensus[END_REF][START_REF] Havemann | The association between gastrooesophageal reflux disease and asthma: a systematic review[END_REF][START_REF] Sontag | Prevalence of oesophagitis in asthmatics[END_REF][START_REF] Ford | Cough in the community: a cross sectional survey and the relationship to gastrointestinal symptoms[END_REF][START_REF] Nordenstedt | The relation between gastroesophageal reflux and respiratory symptoms in a population-based study: the Nord-Trondelag health survey[END_REF][START_REF] Irwin | Gastroesophageal reflux and chronic cough[END_REF], in particular during the sleeping period [START_REF] Fass | Review article: supraoesophageal manifestations of gastro-oesophageal reflux disease and the role of night-time gastro-oesophageal reflux[END_REF][START_REF] Gislason | Respiratory symptoms and nocturnal gastroesophageal reflux: a population-based study of young adults in three European countries[END_REF][START_REF] Sontag | Asthmatics have more nocturnal gasping and reflux symptoms than nonasthmatics, and they are related to bedtime eating[END_REF]. However, an etiologic relationship of these two disease entities has not been corroborated [START_REF] Galmiche | Review article: respiratory manifestations of gastro-oesophageal reflux disease[END_REF]. Oesophageal-bronchial microaspiration and vagus nerve stimulation by refluxate with subsequent bronchial obstruction and hyperresponsiveness are pathophysiological mechanisms that have been hypothesized to be the cause of respiratory symptoms in GERD patients [START_REF] Tuchman | Comparison of airway responses following tracheal or esophageal acidification in the cat[END_REF][START_REF] Jack | Simultaneous tracheal and oesophageal pH measurements in asthmatic patients with gastro-oesophageal reflux[END_REF][START_REF] Wu | Effects of esophageal acid perfusion on cough responsiveness in patients with bronchial asthma[END_REF][START_REF] Singh | Effect of intraesophageal acid instillation on airway reactivity in patients with asthma[END_REF][START_REF] Vincent | Gastro-oesophageal reflux prevalence and relationship with bronchial reactivity in asthma[END_REF][START_REF] Ing | Pathogenesis of chronic persistent cough associated with gastroesophageal reflux[END_REF]. These concepts are further supported by clinical observations showing that respiratory symptoms alleviate or even disappear after surgical or medical therapy of GERD [START_REF] Kiljander | Gastroesophageal reflux in asthmatics: A double-blind, placebo-controlled crossover study with omeprazole[END_REF][START_REF] Field | Does medical antireflux therapy improve asthma in asthmatics with gastroesophageal reflux?: a critical review of the literature[END_REF][START_REF] Littner | Effects of 24 weeks of lansoprazole therapy on asthma symptoms, exacerbations, quality of life, and pulmonary function in adult asthmatic patients with acid reflux symptoms[END_REF][START_REF] Field | The effects of antireflux surgery on asthmatics with gastroesophageal reflux[END_REF]. In contrast, there is also evidence that chronic coughing and wheezing caused by any type of bronchopulmonal disease can precipitate reflux events by increasing intraabdominal pressure, thus consecutively triggering passage of gastric contents into the oesophagus [START_REF] Moote | Increase in gastroesophageal reflux during methacholine-induced bronchospasm[END_REF]. Controversially, respiratory symptoms of a large percentage of patients with chronic coughing or asthma do not respond favourably to proton pump inhibitor therapy [18;23-28].

Since the suspected mechanisms of disease as described above are diverse there is no level A evidence to support any type of treatment regimen, thus management of patients with suspected reflux-induced respiratory symptoms remains largely empirical [11;29]. Consequently it is of major importance to develop an approach and technology reliably allowing to measure and quantify the occurrence of respiratory symptoms in association with the occurrence of acidic and biliary gastro-oesophageal reflux in a standardized and objective way. This is necessary to identify subgroups of symptoms, which should help to determine the pathophysiological mechanism responsible for this association. Furthermore it would allow to identify patients that will benefit from medical or surgical treatment, and to monitor treatment efficacy. A large number of studies have investigated this potential association [30;31], however, methodological limitations mainly concerning the accurate and objective detection of respiratory symptoms reduce their validity. For instance, the use of event buttons or patient diaries for the recording of coughs is highly subjective and not possible during the sleeping period, when the majority of reflux-associated respiratory symptoms are supposed to occur [START_REF] Paterson | Combined ambulatory esophageal manometry and dual-probe pH-metry in evaluation of patients with chronic unexplained cough[END_REF]. Therefore, a new technical device is required that combines objective detection of respiratory symptoms with simultaneous measurement of reflux episodes.

Here, we introduce an outpatient Lung-Sound-Monitoring device capable to continuously record respiratory symptoms throughout the night and to measure acid (pH-monitoring) and biliary dominated (Bilitec Measurement) reflux events in parallel.

For the first time, the two distinct respiratory symptoms "coughing" and "wheezing" can be objectively distinguished and precisely determined in temporal association with different types of gastro-oesophageal reflux.

The presented pilot study was conducted to proof the feasibility of this novel approach combining Lung-Sound-Monitoring with simultaneous measurement of acidic and biliary reflux in patients with respiratory symptoms.

Materials and Methods

Evaluation of healthy subjects

Lung-Sound-Monitoring was performed in 20 healthy subjects prior to the recruitment of GERD patients to prove the feasibility and to evaluate normal frequency of nocturnal respiratory symptoms like coughing or wheezing. Individuals with the history of clinical symptoms of GERD, chronic pulmonary diseases or regular medication of any type were excluded. To evaluate a potential effect of the oesophageal catheter on the frequency of respiratory symptoms Lung-Sound-Monitoring was performed twice in 8 out of the 20 healthy subjects. Throughout the first night the participants were evaluated without an oesophageal catheter, in the following night Lung-Sound Monitoring was repeated with an oesophageal pH/Bilitec catheter in place.

Patients and study protocol:

30 patients were enrolled in this pilot study. All patients were referred to the outpatients department of the University of Marburg for suspected reflux disease. The pattern of clinical symptoms was evaluated with a questionnaire, specifically designed by our group [START_REF] Kunsch | Impact of pantoprazole on duodeno-gastro-esophageal reflux (DGER)[END_REF]. The symptoms heartburn, regurgitation, chest pain, dysphagia, nocturnal coughing, wheezing, hoarseness and vomiting were graded according to severity into 1: not existing, 2: mild, 3: moderate and 4: severe. Only patients with typical clinical symptoms of a reflux disease and suspected respiratory symptoms like wheezing, nocturnal coughing or hoarseness within the last six month were enrolled. Patients with chronic pulmonary diseases (e.g. asthma or chronic obstructive bronchitis) or previous GI-surgery, such as antireflux surgery or gastric resection were excluded. Standardised clinical work-up of patients included combined oesophageal 24h pH-measurement and Bilitec2000 in combination with

Lung-Sound-Monitoring

Following CORSA criteria for standardisation of lung sound measurement [START_REF] Sun | Estimation of analogue prefiltering characteristics for CORSA standardisation[END_REF] we developed a mobile device for long-time recording of lung sounds [START_REF] Gross | Mobile nocturnal long-term monitoring of wheezing and cough[END_REF]. For validation purposes, we first recorded lung sounds of 40 selected patients with exacerbated chronic lung diseases and compared the measurements with those received by simultaneous auscultation through a trained physician. The appearance of the key respiratory symptoms wheezing and coughing were noted by the physician and compared with the detection of lung sounds by our device. Analysis of lung sounds revealed a sensitivity and specificity of 100% for both symptoms, respectively.

The Lung-Sound-Monitoring device system is displayed in Figure 1. Four air-coupled microphones are used to capture lung sounds. Two microphones are positioned on the basis of each lung, a further one is fixed over the trachea and the remaining microphone detects environmental sounds. Signals are filtered and subsequently amplified. Analogue filtering applied to the captured sound signal varies from 60 Hz (high-pass filter) to 2.500 Hz (low-pass filter). Analogue-to-digital converters are used with 16 bits for digitalisation, followed by storage on a computer. The analysis device allows the audio-visual interpretation of the recorded lung sounds. Two independent and specifically trained investigators analysed the recorded lung sounds and marked coughing and wheezing events temporally exact (Figure 2). Wheezing was empirically defined as clinically relevant when exceeding a period of 2 minutes.

PH Monitoring and measurement of Duodenal-Gastro-Oesophageal Reflux (DGER) by Bilitec2000:

Oesophageal pH monitoring was performed using an antimony pH electrode with a separate skin reference electrode (Medtronic, Germany) in an outpatient setting. All data were stored on a portable digital recorder (Digitrapper Mk III). Prior to each measurement, the pH probe was calibrated in standard buffer solutions of pH 7 and

1. An episode of acid reflux was defined as a decrease of the oesophageal pH value below 4 for at least 10s. pH values below 4 for longer than 4% of the total 24h period were classified as pathological.

The fiber-optic spectrophotometer Bilitec2000 was used to quantify DGER. The system consists of a miniaturised probe, which carries light signals into the oesophagus and back trough a plastic fiber-optic bundle. Two light emitting diodes (470 and 565nm) represent the source for the measurement of bilirubin. The portable photodiode system converts the light into electrical signal. After amplification, the signals are processed by an integrated microcomputer, which calculates the differences of the absorbence at 470 and 565nm. This value is directly proportional to the bilirubin concentration in the sample under study. Bilirubin absorbance is an established marker for DGER [START_REF] Bechi | Long-term ambulatory enterogastric reflux monitoring. Validation of a new fiberoptic technique[END_REF]. Prior to each measurement, the probe was calibrated in water. A significant episode of DGER was defined as an increase in oesophageal bilirubin absorbance> 0.14 for at least 10s.

Bilirubin absorbance above 0.14 for longer than 4,6% of the total 24h period was classified as pathological. A specialised diet was prescribed to exclude an

Analysis of data

Our analysis software displays the results of the reflux measurements (pH-Monitoring and Bilitec 2000) and the detected respiratory symptoms in temporal association.

Reflux events (acidic, biliary or combined) were automatically detected within a period 2 minutes before and after the occurrence of a respiratory symptom (Figure 3).

Coughing and wheezing events with temporal association to reflux episodes were divided into those preceding or following reflux. To calculate statistical significance of the temporal association a Symptom Association Probability (SAP) analysis was done [START_REF] Wunderlich | Temporal correlation between chronic cough and gastroesophageal reflux disease[END_REF]. To assure the exact temporal synchronisation of lung sound and reflux measurement a synchronisation was routinely done at the beginning and the end of each measurement.

Statistics:

Data are presented as mean ± S.D. unless stated otherwise. Two-sided Wilcoxon-Mann-Whitney-Test was performed to compare the frequency of coughing and wheezing events in healthy subjects and patients. Fisher's exact test was used to calculate SAP. Statistical significance was accepted when p was <0.05 (SAP>95%).

Comparison between healthy controls and patients

Lung-Sound Monitoring was performed in 20 healthy volunteers (14 male, 6 female, average of age 24.1 years +-2.1) to prove the feasibility of the technique and to determine the normal frequency of nocturnal coughing and wheezing. Coughing was detected in 6 out of the 20 healthy volunteers (11 events; average per night 0.55±0.9; median 0; Q 75 1.5; Q 25 0; max. 2; min. 0). Wheezing occurred in 2 out of the 20 healthy subjects (2 events; average per night 0.1±0.3; median 0; Q 75 0; Q 25 0; max. 1; min. 0). In the patient group (N=25; 13 male; 12 female; average of age 43 years ±19) coughing occurred in 19 (116 events; average per night 4.64±4.7; median 3; Q 75 8; Q 25 1; max. 13; min. 0) and wheezing in 11 out of the 25 patients (39 events; average per night 1.56±2.8; median 0; Q 75 2; Q 25 0; max. 9; min. 0). The statistical analysis of healthy volunteers (n=20) compared to the patients (n=25) resulted in a significant difference of both, coughing and wheezing (p<0.05). These results are displayed in Figure 4.

To evaluate whether the presence of an oesophageal tube has irritating effects and causes and increase in the frequency of respiratory symptoms Lung-Sound isolated biliary reflux). The average fraction of time with a pH smaller than 4 within the 24 hours of measurement was 5.3% (±3.5). The average time with a bilirubin absorbance greater than 0.14 was 14% (±12.6). Overall 653 nocturnal reflux events were detected (Average 26.1 events per night, ±26.9) consisting of 108 combined, 230 acidic and 315 biliary reflux events.

Coughing

Our mobile device detected nocturnal coughs in 19 out of the 25 patients. A total number of 116 nocturnal coughs were identified. A temporal association between coughing and reflux was assumed if a reflux event occurred in a 2 minutes interval before or after each coughing event. 59 of the total number of coughing events (51%) were considered independent of reflux, whereas 57 of 116 coughing events (49%) showed a temporal association with reflux events. These 57 coughing events were associated with different types of reflux events: I. biliary reflux (N=37; 64%), II. acidic reflux (N=10; 18%) and III. combined reflux (N=10; 18%). For a more precise description of the exact temporal association of coughing and reflux we separated the resulting 57 coughing events with temporal association to reflux events into I. a reflux-coughing succession (RC-S) and II. a coughing-reflux succession (CR-S). The majority of coughing events showed a RC-S (51/57; 89%). In 32 (63%) of these coughing events the reflux event persisted until coughing started.

In the remaining 19 events (37%) reflux occurred in the 2 minutes interval before the cough but was already normalised when the patient started to cough. Only a small percentage of the 57 coughing events with a temporal correlation to reflux showed a coughing-reflux succession (6/57; 11%).

To determine if the temporal correlation between coughing and reflux events is significant a symptom association probability (SAP) analysis was performed. In 9 out of the 19 patients with nocturnal coughing (47%) a positive SAP analysis indicated a statistically significant temporal correlation between the respiratory symptom coughing and reflux events.

Notably, all patients showing a positive association in the SAP-analysis had a RC-S.

Furthermore we analysed if the type of reflux had an influence on the results of the SAP-analysis. SAP-positive patients displayed biliary (N=6), acidic (N=1) and combined (N=2) reflux events (Figure 5), thus indicating a predominance of biliary reflux.

Wheezing

In 11 out of 25 patients nocturnal wheezing was detected. A temporal association between wheezing and reflux was assumed if a reflux event occurred in a 2 minutes interval before or after a wheezing event. A total number of 39 nocturnal wheezing episodes were identified of which 23 (59%) were considered to occur independent of reflux, whereas 16 (41%) showed a temporal association with reflux events. These In analogy to the evaluation of coughing the detected wheezing episodes with temporal association to reflux events (N=16) were separated into a reflux-wheezing succession (RW-S) and a wheezing-reflux succession (WR-S). All wheezing episodes with temporal association to reflux showed a RW-S (16/16; 100%). In 10 cases (63%) the reflux event persisted until the occurrence of wheezing. In the remaining 6 cases (37%) reflux occurred in the 2 minutes interval before the wheezing event but had already stopped before wheezing started. Interestingly, reflux in the 2 minutes period after wheezing was not observed.

To determine if the correlation between wheezing and reflux events is significant or only by chance a symptom association probability (SAP) analysis was performed. In 5 out of the 11 patients (45%) with nocturnal wheezing, SAP analysis was positive (greater than 95%) for wheezing episodes. All these patients had a RW-S and showed biliary (N=1), acidic (N=3) and combined (N=1) reflux events (Figure 6), thus indicating a predominance of acidic reflux. An increased prevalence of respiratory symptoms has been commonly observed in reflux patients lending support to the plausible assumption that GERD might contribute to the occurrence of pulmonary disorders in certain patients. Unfortunately, however, methodological limitations of current available approaches to detect respiratory symptoms in GERD patients have hampered the design of clinical studies to address this issue and to demonstrate a clear temporal correlation between refluxevents and the presence of respiratory symptoms such as coughing and wheezing.

Here, we present a novel and unique diagnostic tool that combines the simultaneous measurement of acidic and biliary reflux episodes with lung sounds recording in GERD patients. This novel device operates via air-coupled microphones that accurately detect lung sounds in a similar way as a doctor's stethoscope. By use of analogue filters and analogue-to-digital converters the device provides an audiovisual graphical display of the recorded lung sounds [START_REF] Gross | Mobile nocturnal long-term monitoring of wheezing and cough[END_REF]. Subsequent computer analysis then allows to determine a putative temporal association of both events, reflux and respiratory symptoms.

In this pilot study, we exclusively focused on the detection of nocturnal lung sounds since (i) there is a growing body of epidemiological data suggesting that nocturnal reflux plays the major role in inducing respiratory symptoms [START_REF] Gislason | Respiratory symptoms and nocturnal gastroesophageal reflux: a population-based study of young adults in three European countries[END_REF], (ii) physiological alterations including slower gastric emptying, decreased saliva production and swallowing frequency as well as a diminished pressure in the upper oesophageal sphincter during night-time may facilitate aspiration of gastric contents into the upper airways [START_REF] Fass | Review article: supraoesophageal manifestations of gastro-oesophageal reflux disease and the role of night-time gastro-oesophageal reflux[END_REF] and (iii) multiple other stimuli such as exercise, temperature or air pollution may trigger respiratory symptoms during day time.

First, we aimed to evaluate the feasibility of the diagnostic approach to monitor lung sounds in parallel to acidic and biliary reflux in healthy subjects. As expected, Overall the healthy control group showed a significantly lower number of coughing and wheezing events as compared to GERD patients. To exclude that the presence of pH-monitoring and Bilitec catheters in the oesophagus alone is sufficient to induce respiratory symptoms, we recorded the nocturnal lung sounds in a subgroup of healthy controls in the presence and in the absence of the pH-monitoring/Bilitec catheters. This analysis revealed no significant differences between the frequency of respiratory symptoms in presence or absence of the oesophageal catheters.

Nocturnal coughing was detected in 19 out 25 GERD patients and almost half of the coughing events (49%) coincided with reflux events. Interestingly, 89% of these coughing events were preceded by reflux whereas reflux was rarely detected after a cough. In contrast, nocturnal wheezing was only observed in 11 out of 25 patients and 41% of these wheezing events coincided with reflux events. Notably, 100% of these wheezing events were preceded by reflux episodes. At a first glance, it may appear surprising that the most frequently observed temporal association was that reflux events were followed by respiratory symptoms. This may be due to the selection of patients enrolled in our study. In contrast to previous studies primarily investigating patients with chronic coughing or asthma [30;31;37-39], we exclusively enrolled patients with typical symptoms of GERD and respiratory symptoms during the last 6 months. Therefore, the probability to detect patients in which coughing or wheezing provokes reflux was naturally rather low. Symptom-associated probability (SAP) analysis was first introduced by Weusten et al. in 1994 [40] as a tool to estimate, if there is a real association between two observed events and thus to exclude that the association is caused only by chance.

Using this SAP analysis on our data set, we identified 9 patients with values indicating a positive association for the reflux-coughing succession whereas for none of the patients with coughing-reflux succession a positive association could be shown. Furthermore the SAP analysis yielded a statistically significant correlation between reflux and wheezing for 5 out of 11 patients with wheezing events, which, as mentioned above, were all preceded by the reflux event. Although our pilot study does not have the power to draw general conclusions on the type of reflux inducing the corresponding respiratory symptoms, it is interesting to note that the majority of patients showing a positive association between coughing and reflux had predominantly biliary reflux events, whereas wheezing was more often preceded by acidic reflux. These observations are in line with previous data further supporting the hypothesis that non acidic components of the refluxate (such as biliary acids) may, at least in part, be responsible for reflux induced coughing, whereas obstructive respiratory symptoms such as wheezing may be primarily caused by acidic reflux [38;41;42].

To conclude, we have developed a novel technical approach for simultaneously measuring lung sounds and acidic and biliary reflux episodes. The novelty of the presented device is the long time detection of all nocturnal respiratory symptoms with the quality of an auscultation with a stethoscope. Thus, this approach is superior, more reliable and objective than previously used techniques to detect respiratory symptoms. Using the mobile Lund-Sound-Monitoring device tool we could identify subgroups of patients with a clear temporal correlation between reflux and respiratory symptoms such as coughing and wheezing. These results support the hypothesis of a causative role of reflux episodes in triggering respiratory symptoms in GERD patients. Our study was primarily aimed at showing the feasibility of this new approach which can now be used for prospective studies to further investigate the mechanisms of reflux associated pulmonary diseases and to control for success under therapy. Follow up investigations of patients off and on proton pump inhibitors Taken together this novel technology might be a useful tool for further scientific approaches but also capable to routinely identify a subgroup of patients with a causal relationship of GERD and pulmonary symptoms in the future.

  temporal association between reflux episodes and respiratory

6 nocturnal

 6 Lung-Sound-Monitoring. Nocturnal Lung-Sound-Monitoring was performed at the patients´ home and supervised by trained medical staff. Oesophageal manometry and gastroscopy were routinely performed in all patients to exclude oesophageal motility disorders or malignancy. If the patient was on acid suppression therapy this was withdrawn at least 7 days prior to the diagnostic procedures. The study protocols were approved by the local ethics committee of the University of Marburg.

  measurement. Both, the pH-and the Bilitec-catheter were positioned 5cm above the lower oesophageal sphincter. The exact position of the lower oesophageal sphincter was determined in all patients using oesophageal manometry.

From

  January 2008 to January 2010 20 healthy controls and 30 patients with suspected GERD and respiratory symptoms as wheezing, nocturnal coughing or hoarseness were enrolled into this pilot study. In the patients group, 5 out of 30 patients had to be excluded due to incomplete Lung-Sound Monitoring (N=2), incomplete reflux measurement (N=1) and occlusion of the Bilitec sensor by a bolus (N=2). The resulting 25 patients were completely analysed according to the study protocol.

10 Monitoring

 10 was repeated in the following night with a pH/Bilitec catheter in place in 8 out of the 20 healthy volunteers. The statistical analysis revealed no significant difference between the measurement with and without the oesophageal tube, providing evidence that the oesophageal tube itself did not affect the frequency of nocturnal coughing and wheezing.Reflux measurementIn all of the remaining 25 patients a combined 24 hour pH-and Bilitec-Measurement was performed in parallel to Lung-Sound Monitoring. Over the 24 hour period 21 out of the 25 patients exhibited increased reflux (8 combined, 6 isolated acidic and 7

  were associated with different types of reflux events including biliary reflux (N=5; 31%), acidic reflux (N=9; 56%) and combined reflux (N=2; 13%).

  in an outpatient setting was well tolerated by all participants.

  initiation of a sufficient antireflux therapy has an impact on the objective frequency of respiratory symptoms.

20 Figure 1 :

 201 Figure 1: Configuration of the novel Lung-Sound Monitoring device. 4 air-coupled

Figure 2 :

 2 Figure 2: Audio-visual analysis of lung sounds. The typical pattern of normal lung

Figure 3 :

 3 Figure 3: Computerized analysis of the simultaneous measurements of pH, biliary reflux in correlation to coughing events. As an example a coughing event (blue arbour) is displayed in temporal association with a combined reflux event (pH<4 and bilirubin absoption > 0.14).

Figure 4 :

 4 Figure 4: Number of coughing and wheezing events per night. HV (healthy

Figure 5 :

 5 Figure 5: Flow diagram for detection of nocturnal coughing in the study population.

Figure 6 :Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :

 6123456 Figure 6: Flow diagram for the detection of nocturnal wheezing in the study

This study was supported by a Research Grant of the University Medical Center Giessen and Marburg (UKGM)