Bernd Kowall

Wolfgang Rathmann
email: rathmann@ddz.uni-duesseldorf.de

Klaus Strassburger

Margit Heier

Rolf Holle

Barbara Thorand

Guido Giani

Annette Peters

Christine Meisinger

Á W Rathmann

Á K Strassburger

Á M Heier

Á G Giani

Á B Thorand

Á A Peters

Á C Meisinger

Association of passive and active smoking with incident type 2 diabetes mellitus in the elderly population: the KORA S4/F4 cohort study

Keywords: Diabetes mellitus, Prediabetes, Prospective studies, Smoking, Environmental tobacco smoke

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

Introduction

The effect of environmental tobacco smoke (ETS) on the risk of type 2 diabetes (T2DM) has so far been investigated in two prospective studies, and the results were not unambiguous [START_REF] Houston | Active and passive smoking and development of glucose intolerance among young adults in a prospective cohort: CARDIA study[END_REF][START_REF] Hayashino | A prospective study of passive smoking and risk of diabetes in a cohort of workers: the high-risk and population strategy for occupational health promotion (HIPOP-OHP) study[END_REF]: In the CARDIA study, no significantly increased risk of T2DM was found for never smokers exposed to ETS, whereas a significantly higher incidence of diabetes was observed in a Japanese study in a cohort of workers. Moreover, exposure to ETS was significantly associated with the incidence of glucose intolerance, a precursor of T2DM, in the CARDIA study [START_REF] Houston | Active and passive smoking and development of glucose intolerance among young adults in a prospective cohort: CARDIA study[END_REF].

Contrary to the sparse and unclear results concerning the association between ETS exposure and T2DM, active smoking has been shown to be an established risk factor for T2DM. Willi et al. recently pooled data of 25 cohort studies on the association of smoking and diabetes incidence, and found an adjusted relative risk (RR) of 1.44 (95% confidence interval (CI): 1.31, 1.58) [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF]. In 24 of these studies, a relative risk larger than one was found, and in 19, a significantly increased risk of diabetes was reported. Moreover, a dose-response relationship was shown in this meta-analysis: subjects smoking more than 20 cigarettes per day had a larger diabetes risk than current smokers smoking less (pooled RR = 1.61 (95%-CI: 1.43, 1.80), and pooled RR = 1.29 (95%-CI: 1.13, 1.48), respectively, compared to non-smokers). Three further studies from the 1980s were not considered in the review [START_REF] Keen | The ten-year follow-up of the Bedford Survey (1962-1972): glucose tolerance and diabetes[END_REF][START_REF] Wilson | Epidemiology of diabetes mellitus in the elderly. The Framingham study[END_REF][START_REF] Feskens | Cardiovascular risk factors and the 25-year incidence of diabetes mellitus in middle-aged men[END_REF]. In two of these studies with rather small numbers of diabetes cases, no association between active smoking and diabetes incidence was reported [START_REF] Keen | The ten-year follow-up of the Bedford Survey (1962-1972): glucose tolerance and diabetes[END_REF][START_REF] Wilson | Epidemiology of diabetes mellitus in the elderly. The Framingham study[END_REF]. Recently, results of four additional cohort studies with somewhat inconsistent findings were published [START_REF] Hur | Smoking cessation and risk of type 2 diabetes mellitus: Korea Medical Insurance Corporation Study[END_REF][START_REF] Park | The effects of smoking status upon occurrence of impaired fasting glucose or type 2 diabetes in Korean men (Korean)[END_REF][START_REF] Onat | Prospective epidemiologic evidence of a ''protective'' effect of smoking on metabolic syndrome and diabetes among Turkish women-without associated overall health benefit[END_REF][START_REF] Nagaya | Heavy smoking raises risk for type 2 diabetes mellitus in obese men; but, light smoking reduces the risk in lean men: a follow-up study in Japan[END_REF]. Nevertheless, effects of active smoking on the development of diabetes are hardly in doubt. As Willi et al. stated in their review, further studies should focus on the careful measurement and adjustment for potential confounders in order to clarify the mechanism of the smoking-diabetes-relationship [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF].

In this study, we examined the association of the risk of developing T2DM assessing active and passive smoking jointly in a population-based cohort of elderly subjects. We hypothesized that both passive and active smoking are predictors of diabetes incidence, and we investigated the relationship between smoke exposure and T2DM with adjustment for a large number of sociodemographic and lifestyle factors, as well as for the components of the metabolic syndrome.

Materials and methods

Study population

The KORA (Cooperative Health Research in the Region of Augsburg) S4 Survey is a population-based study conducted in Southern Germany using the same region and study methods as the previous WHO MONICA Augsburg project. 2,656 subjects in the age of 55-74 years were invited to participate in the baseline survey between 1999-2001, and 1,653 (62%) subjects were investigated. 131 subjects with known T2DM were excluded, and after exclusion of further drop-outs 1,353 non-diabetic subjects underwent an oral glucose tolerance test (OGTT) at baseline [START_REF] Rathmann | High prevalence of undiagnosed diabetes mellitus in Southern Germany: target populations for efficient screening. The KORA survey[END_REF].

The present study includes all subjects without known or newly diagnosed T2DM at baseline (n = 1,223). All subjects with completed OGTT at baseline were re-invited in 2006-2008 (F4 survey). Among these subjects, 98 had died before the time of the follow-up examination, and 887 (73%) subjects participated in the follow-up. Informed consent was obtained from the participants. The survey was approved by the ethics committee of the Bavarian Medical Association.

Ascertainment of diabetes and prediabetes in the follow-up

Subjects reporting a physician diagnosis of T2DM or use of anti-diabetic medications in the follow-up were classified as incident diabetes cases only, if their reports were validated by contacting the physicians who had treated them. For the remaining subjects, OGTTs with a 75 g oral load of anhydrous glucose were conducted to ascertain diabetes status. OGTTs were performed in the morning hours (7:00-11:00 h). Subjects were instructed to fast for 10 h overnight, to avoid heavy physical activity and not to smoke before or during the OGTT. Subjects with fever, infections or acute gastrointestinal diseases were excluded from the test. Impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and newly diagnosed T2DM were defined according to 1999 WHO criteria [START_REF] Alberti | Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation[END_REF]. Prediabetes comprised isolated IFG, isolated IGT, and combined IFG and IGT. We used the original IFG criteria (6.1-6.9 mmol/l) for the present analysis, as recommended by the European Diabetes Epidemiology Group [START_REF] Forouhi | The threshold for diagnosing impaired fasting glucose: a position statement by the European Diabetes Epidemiology Group[END_REF]. Newly diagnosed diabetes and validated physician diagnosis were considered as incident diabetes.

Assessment of smoking status

The category of current (or, synonymously, active) smokers comprised regular smokers (smoking daily) and occasional smokers (not smoking daily). The baseline questionnaire included the smoking status (regular/occasional/past/never smoker), the number of cigarettes smoked daily (for regular smokers only), the largest number of cigarettes ever smoked daily for a whole year (for current and past smokers), and the year of beginning and (in case of past smokers) of stopping smoking. ETS was characterized by questions whether, and if so, how much other persons smoked in the workplace or in the household of the participants at baseline (very much/much/hardly/not at all). At follow-up, the current smoking status was inquired again. Subjects were classified into five mutually exclusive categories at baseline: never smokers unexposed to ETS, never smokers exposed to ETS, past smokers unexposed to ETS, past smokers exposed to ETS, and current smokers. Subjects were regarded as passive smokers if any ETS exposure was reported. Assuming 20 cigarettes per pack, pack-years were calculated using the formula ''(cigarettes per day/20) 9 number of years smoked''.

Anthropometric and laboratory measurements have been described elsewhere [START_REF] Rathmann | High prevalence of undiagnosed diabetes mellitus in Southern Germany: target populations for efficient screening. The KORA survey[END_REF]. Information about sociodemographic variables, medical history, alcohol consumption and physical activity was gathered in a structured interview. Socioeconomic status (SES) was assessed as previously described, based on income, educational level and occupational status [START_REF] Rathmann | Sex differences in the associations of socioeconomic status with undiagnosed diabetes mellitus and impaired glucose tolerance in the elderly population: the KORA survey 2000[END_REF]. Dietary intake was assessed with a short 27 item qualitative food frequency list (FFL). The participants were asked to recall their ''average intake'' of each item in the following six frequency categories: almost daily, several times per week, about once a week, several times per month, once a month or less, never. Details on a very similar FFL have been described elsewhere [START_REF] Winkler | Validation of a short qualitative food frequency list used in several German large scale surveys[END_REF].

Statistical analyses

Continuous variables were calculated as mean ± standard deviation (SD) or geometric mean 9/7standard deviation factor (SDF). Baseline characteristics of the five smoke exposure categories were compared using F-tests in case of normally distributed variables; and for log-normal variables, F-tests were performed on a log-scale. Logistic regression was used to compare binomial proportions. For the total of all subjects, for subjects having prediabetes at baseline, as well as for subjects with normoglycemia (baseline), cumulative incidences of diabetes cases were calculated for each smoking category.

Multivariate logistic regression models were fitted with smoking status as main independent variable and incident diabetes as dependent variable. Four different models were fitted: two models adjusting for potential confounders (age, sex, parental diabetes, SES, alcohol consumption, physical activity, intake of meat and sausage, intake of salad and vegetables, intake of whole-grain bread, coffee consumption), and two models additionally adjusting for potential mediators (waist circumference, blood pressure, triglycerides, HDL-cholesterol, fasting insulin, serum adiponectin).

Age, waist circumference, and the log values of serum adiponectin and fasting insulin were included as continuous variables. The following covariables were dichotomized: high alcohol intake: C40 g/day in men, C20 g/day in women; high physical activity level: C1 h sports per week during leisure time in either summer or winter; large waist circumference C102 and C88 cm for men and women, respectively; hypertension: blood pressure of 140/ 90 mmHg or higher, or antihypertensive medication, given that the subjects were aware of being hypertensive; hypertriglyceridemia C 175 mg/dl; low HDL-cholesterol: B40 mg/dl in males, B50 mg/dl in women; intake of meat and sausage (intake of meat, sausage or ham almost daily or several times a week); intake of salad and vegetables (intake of salad, cooked or uncooked vegetables almost daily or several times a week); intake of whole-grain bread (intake of whole-grain bread, brown bread or crispbread almost daily or several times a week); coffee consumption (more than three cups of coffee a day).

Each of the four models was fitted for the total cohort, for subjects with baseline prediabetes and for subjects with normoglycemia. Some additional analyses were done. The ETS-diabetes-association was investigated in analyses confined to diabetes cases identified by OGTTs, and it was studied in a subgroup without passive smokers in the workplace. Moreover, in a multivariate linear regression model, the influence of the smoking status on the log HOMA-IR value was assessed. The insulin resistance score HOMA-IR was calculated as fasting plasma glucose (mmol/l) 9 fasting serum insulin (mU/l)/22.5.

Separately for regular and past smokers, the impact of lifetime pack-years until the baseline investigation on glucose values (fasting and 2 h glucose values at baseline and in the follow-up) as well as on incident diabetes was assessed in confounder-adjusted linear and logistic regression models, respectively.

To examine how duration of abstinence influenced the diabetes risk of ex-smokers, we fitted model 2 (Table 3) again, splitting the category of ex-smokers without ETS exposition into two categories, i. e., ex-smokers with abstinence times of less than 10 years, and ex-smokers with abstinence times of at least 10 years.

The level of significance was set at 5%. The analyses were carried out using SAS version 9.2 (SAS institute, Cary, NC, USA).

Results

Descriptive statistics

For two participants, information about smoking status was incomplete, so analyses were done with 885 subjects. 93 cases of T2DM were observed in the follow-up (cumulative incidence of diabetes: 10.5%) among whom 60 cases were identified by OGTTs and 33 by validated physician diagnoses.

Table 1 shows the baseline characteristics of the survey participants stratified by the five smoke exposure categories. Subjects who were neither current smokers nor exposed to ETS were more than 3 years older than subjects currently smoking or exposed to ETS. The proportion of men was lowest in unexposed never smokers (29% compared to 41% in never smokers exposed to ETS). In men, the BMI was lowest in current smokers; in women, past smokers exposed to ETS had an even lower BMI than current smokers. In both sexes, current smokers had lower waist circumferences than never smokers unexposed to ETS. The lowest fasting and 2-h glucose levels, as well as the lowest fasting insulin levels, were found in current smokers. High amounts of alcohol were more often consumed in past and current smokers than in never smokers.

Among the 88 never smokers exposed to ETS, 6 (7%) reported that they were very much exposed, 28 (32%) reported that they were much exposed, and 54 (61%) reported little exposure. For the 73 former smokers with ETS exposure, the corresponding figures were 14 (19%), 25 (34%) and 34 (47%), respectively.

Association of smoking status with risk of diabetes

The cumulative incidence of T2DM in the follow-up was lowest in never smokers not exposed to ETS (7.1%) (Table 2). The cumulative incidence ranged from 11.4 to 13.7% in the other smoke exposure categories. When the analysis was confined to prediabetic subjects, incident diabetes was observed in 16.5% of the never smokers unexposed to ETS. This incidence was about two times greater in never smokers exposed to ETS, and it was about three times greater in current smokers. In a subgroup analysis with normoglycemic subjects, there were little differences in the cumulative incidence of T2DM between the five smoke exposure categories.

Table 3 shows the results of multivariate logistic regression models, investigating the relationship of smoking status with diabetes risk (cumulative 7 year incidence) for the total sample and for the subgroups of prediabetic and normoglycemic subjects, respectively. In model 1 adjusting for sex, age, and parental diabetes, never smokers exposed to ETS displayed a significantly higher risk of T2DM compared to unexposed never smokers. Significantly increased diabetes risks were also found for past smokers with passive smoke exposure as well as for current smokers. Additional adjustment for SES and lifestyle factors including diet did not attenuate these odds ratios (model 2), and for current smokers, the association with diabetes incidence became even stronger. The smokingdiabetes relationships were slightly attenuated upon further adjustment for waist circumference (model 3). After additional adjustment for components of the metabolic syndrome including waist circumference, adiponectin, and fasting insulin, a threefold diabetes risk was found for current smokers, and a borderline significant risk was observed for never smokers with ETS exposure (model 4). Similar results were obtained when adjusting for BMI instead of waist circumference (data not shown). Model 2: adjusted for age, sex, parental diabetes, socioeconomic status, alcohol intake, physical activity, intake of meat and sausage, intake of salad and vegetables, intake of whole-grain bread, and coffee consumption Model 3: adjusted for age, sex, parental diabetes and waist circumference Model 4: adjusted for age, sex, parental diabetes, socioeconomic status, alcohol intake, physical activity, intake of meat and sausage, intake of salad and vegetables, intake of whole-grain bread, and coffee consumption, waist circumference, blood pressure, hypertriglyceridemia, HDLcholesterol, log insulin, log adiponectin

The KORA S4/F4 cohort study

Separate analysis of prediabetic and normoglycemic subjects

Overall, a stronger relationship between smoking and diabetes risk was found in prediabetic subjects than in the total sample (Table 3). Significantly higher risks of T2DM were found in all smoke exposure categories except for past smokers with ETS exposure after adjusting for age, sex, parental diabetes, SES and lifestyle factors (model 2).

In subgroup analyses for normoglycemic subjects, no significant effects of smoke exposure were observed. Interaction between prediabetes at baseline (yes/no) and smoking status was not statistically significant (P = 0.44 in model 1, P = 0.39 in model 2).

Association of pack-years with glucose values

In regular smokers, lifetime pack-years until the baseline investigation had a statistically significant effect on fasting glucose both at baseline and follow-up, as well as on baseline 2-h glucose (Table 4). In past smokers, a significant effect of pack-years on glucose levels was found for fasting glucose at baseline (Table 5). In regular smokers, pack-years had a statistically significant effect on incident diabetes after adjusting for sex, age, parental diabetes, SES, alcohol intake, physical activity and diet (OR = 1.05; 95%-CI: 1.01, 1.09). In past smokers, this association was not significant (OR = 1.00; 95%-CI: 0.99, 1.02).

Additional analyses

In additional analyses including only diabetes cases identified by OGTTs in the follow-up, a significantly increased risk of T2DM was again found for never smokers exposed to ETS in the total sample and in prediabetic subjects, respectively (adjustment for age, sex, parental diabetes, SES and lifestyle factors, data not shown).

In an analysis where never and past smokers exposed to ETS at the workplace were excluded, the diabetes risk of never smokers exposed to ETS at home compared to never smokers without ETS exposure was larger than in the whole sample (N = 804; OR = 3.3; 95%-CI: 1.3, 8.7 with adjustment for age, sex, parental diabetes, SES, alcohol intake, physical activity and diet, according to model 2 in Table 3). A corresponding analysis confined to never smokers exposed to ETS at the workplace was not done because there were only three diabetes cases in this category.

In a confounder-adjusted model with baseline data, it could be seen that in never smokers exposed to ETS and in current smokers log HOMA-IR values as indicator of insulin resistance were not significantly different from the log HOMA-IR values of unexposed never smokers (data not shown).

Models according to model 2 (table 3) were fitted again, splitting the category of ex-smokers without ETS expositions into subjects with abstinence times of less than 10 years, and abstinence times of at least 10 years, respectively. When normoglycemic and prediabetic subjects were included, ex-smokers with at least 10 years of abstinence showed a decline in the OR for the risk of diabetes (OR = 1.3, 95%-CI: 0.7-2.5), whereas ex-smokers with less than 10 years of abstinence showed an increase in diabetes risk (OR = 3.5, 95%-CI: 1.3-9.4) compared to the effect observed in the total group (OR = 1.6, 95%-CI: 0.9-3.0). For prediabetes subjects alone, the odds ratio of ex-smokers with at least 10 years of abstinence was 1.9 (95%-CI: 0.8-4.8), and the odds ratio of ex-smokers with less than 10 years of abstinence was 6.1 (95%-CI: 1.6-22.8).

Discussion

This 7-year prospective study showed that both passive and active smoking increased the risk of T2DM. After adjusting for potential confounders including age, sex, parental diabetes, SES, alcohol consumption, physical activity and diet, a significantly increased diabetes risk was observed in current smokers and in never smokers exposed to ETS. Further adjustment for variables of the metabolic syndrome, for adiponectin, and insulin levels did not attenuate the smoking-diabetes relationships. In subgroup analyses among subjects with baseline prediabetes, higher diabetes risks among current smokers and passive smokers were found than in the total sample. However, there was no indication of an interaction between prediabetes and smoking with regard to T2DM development. In normoglycemic subjects, in none of the smoke exposure categories a significantly increased diabetes risk was found.

Comparison with other studies

In the CARDIA study, a significantly increased risk of diabetes had not been observed for never smokers with ETS exposure compared to never smokers without ETS exposure (HR = 1.40, 95%-CI: 0.84-2.33). Two reasons might explain the different findings of the CARDIA study and the present study: First, in the CARDIA study, fasting blood glucose levels as well as self-reported history of diabetes but no OGTTs were used to define the outcome. In this study, however, OGTTs were used unless there was a self-reported history of T2DM validated by a physician diagnosis. In analyses confined to diabetes cases identified by OGTTs, never smokers exposed to ETS were still shown to be at a significantly increased risk of T2DM. Second, the two study populations were quite different: In the CARDIA study, the mean age at baseline was 25 years, but it was 63 in the KORA study. Moreover, in the CARDIA study, 50% of the participants were Afro-Americans.

The association between current smoking and T2DM found in this study after multivariable adjustment (OR = 2.8, 95%-CI: 1.3, 6.1) was stronger than the corresponding association calculated in a recent meta-analysis including 25 studies (RR = 1.44, 95%-CI: 1.31, 1.58) [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF]. For means of better comparison, our odds ratio was transformed into a relative risk (RR = 2.5, 95%-CI: 1.3, 4.5) using a method given by Zhang and Yu [START_REF] Zhang | What 0 s the relative risk? A method of correcting the odds ratio in cohort studies of common outcomes[END_REF]. However, two differences have to be considered. First, the category of reference in other studies consisted of never smokers, irrespective of exposure to ETS whereas in our study the reference category consisted of never smokers without ETS exposure [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF]. Second, contrary to most of the other studies included in the meta-analysis [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF], we separated models adjusting for potential confounders (Table 3, models 1-2) from models which additionally included potential mediators like components of the metabolic syndrome (models 3-4). Even after adjusting for the metabolic syndrome, for the adipocytokine adiponectin, and for fasting insulin as indicator of insulin resistance, we found an almost threefold elevated risk for current smoking. The lack of reduction of the smoking-diabetes association upon this additional adjustment suggests that the effect of active smoking on diabetes incidence can be explained neither by waist circumference nor by potential influences of smoking on factors of the metabolic syndrome.

Separate analysis for prediabetic and normoglycemic subjects

We did the multivariate analyses separately for prediabetic and normoglycemic subjects. For current smoking, a strongly significant effect was found for prediabetic subjects. For normoglycemic subjects, the risk of active smoking was even somewhat larger than the one found in the metaanalysis by Willi et al. [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF] but it was not statistically significant due to the small sample size. Among never smokers with ETS exposure, an effect was only seen in prediabetic subjects. In spite of the different effects of smoking in prediabetic and normoglycemic subjects, an interaction term of prediabetes (yes/no) and smoking status which was added to model 2 in the logistic regression analysis, was not statistically significant. This interaction term had four degrees of freedom, and we suppose that the sample was too small to show a statistically significant interaction. We looked at changes in the risk factor profile during the follow-up (like BMI, waist circumference, blood The KORA S4/F4 cohort study pressure), and we compared these changes between never smokers with ETS exposure and never smokers without ETS exposure in prediabetic subjects on the one side and normoglycemic subjects on the other side. However, changes in the risk factor profiles could not explain why in never smokers an effect of ETS exposure was only found in prediabetic subjects. Subgroup analyses for prediabetic and normoglycemic subjects were rarely done in other studies. Contrary to our results, Foy et al. found that the diabetes risk of current smokers was lower in prediabetic subjects and higher in normoglycemic subjects compared to the total sample [START_REF] Foy | Smoking and incidence of diabetes among US adults: findings from the Insulin Resistance Atherosclerosis Study[END_REF]. However, subjects were younger at baseline (40-69 years) than subjects in the KORA cohort.

Association of pack-years with glucose values in regular and past smokers

In this study, in regular smokers pack-years until the baseline investigation were significantly associated with fasting glucose at baseline and at follow-up, as well as with baseline 2-h glucose. In past smokers, a significant association was found between lifetime pack-years and fasting glucose at baseline. These findings demonstrate that there is a dose-relationship between the cumulative exposure to cigarette smoking and blood glucose. Dose-effect relationships between smoking and T2DM incidence have been found in other studies using either pack-years [START_REF] Manson | A prospective study of cigarette smoking and the incidence of diabetes mellitus among US male physicians[END_REF][START_REF] Rimm | Prospective study of cigarette smoking, alcohol use, and the risk of diabetes in men[END_REF][START_REF] Nakanishi | Cigarette smoking and risk for impaired fasting glucose and type 2 diabetes in middle-aged Japanese men[END_REF] or cigarettes per day [START_REF] Nakanishi | Cigarette smoking and risk for impaired fasting glucose and type 2 diabetes in middle-aged Japanese men[END_REF][START_REF] Rimm | Cigarette smoking and the risk of diabetes in women[END_REF][START_REF] Will | Cigarette smoking and diabetes mellitus: evidence of a positive association from a large prospective cohort study[END_REF] as measures of exposure. Other authors, however, did not find such a dose-effect relationship [START_REF] Kawakami | Effects of smoking on the incidence of non-insulin-dependent diabetes mellitus[END_REF][START_REF] Sairenchi | Cigarette smoking and risk of type 2 diabetes mellitus among middle-aged and elderly Japanese men and women[END_REF][START_REF] Wannamethee | Smoking as a modifiable risk factor for type 2 diabetes in middle-aged men[END_REF].

Causality of the association and possible pathways

For the relationship of active smoking and diabetes, it has been pointed out that several of the Bradford Hill criteria for causation are fulfilled [START_REF] Willi | Active smoking and the risk of type 2 diabetes. A systematic review and meta-analysis[END_REF]. For passive smoking, evidence for causation is less clear. First, in the present cohort study with a follow-up of 7 years, the exposure preceded the incidence of diabetes. Nevertheless, a temporal relationship cannot be taken for granted because most subjects with incident diabetes were prediabetic at baseline so that processes leading to later diabetes had possibly already started at baseline. Second, in never smokers, the association of exposure to ETS with incident diabetes could be considered as fairly strong because odds ratios were larger than two. However, this association was based on only ten new diabetes cases. Third, in never smokers, a dose-relation-ship could not be analyzed due to the small figure of incident cases. Fourth, there is still a lack of studies showing that the relationship of passive smoking and diabetes risk holds for different populations using different methods. Fifth, potential pathways leading from tobacco smoking to T2DM have so far mainly been suggested for active smoking. Possible mechanisms of the effects of secondhand smoke have hardly been investigated. However, as the concentration of many smoke ingredients is higher in sidestream smoke than in mainstream smoke [START_REF]The Health Consequences of Involuntary Exposure to Tobacco Smoke[END_REF][START_REF]Proposed Identification of Environmental Tobacco Smoke as a Toxic Air Contaminant[END_REF], secondhand smoke leads to large health risks although passive smokers have a lower overall smoke exposure than active smokers. So, the association between passive smoking and diabetes risk is in line with the present knowledge of pathological processes and can be considered as plausible.

Possible pathways leading from smoking to type 2 diabetes

In smokers, it has been shown that the insulin-mediated glucose-uptake is lower than in non-smokers in some studies [START_REF] Attvall | Smoking induces insulin resistance-A potential link with the insulin resistance syndrome[END_REF][START_REF] Facchini | Insulin resistance and cigarette smoking[END_REF][START_REF] Eliasson | The insulin resistance syndrome and postprandial lipid intolerance in smokers[END_REF]. However, other studies did not find impaired insulin sensitivity in smokers [START_REF] Nilsson | Increased level of hemoglobin A1c, but not impaired insulin sensitivity, found in hypertensive and normotensive smokers[END_REF][START_REF] Henkin | Cigarette smoking, environmental tobacco smoke exposure and insulin sensitivity[END_REF][START_REF] Sonmez | Adiponectin and insulin resistance in young and healthy smokers[END_REF]. Smoking leads to an adverse distribution of body fat with smaller body mass indices but larger waist-to-hip ratios in smokers [START_REF] Shimokata | Studies in the distribution of body fat. III. Effects of cigarette smoking[END_REF][START_REF] Albanes | Associations between smoking and body weight in the US population: analysis of NHANES II[END_REF][START_REF] Troisi | Cigarette smoking, dietary intake, and physical activity: effects on body fat distribution-The Normative Aging Study[END_REF]. Thus, abdominal obesity, which is a strong risk factor for T2DM [START_REF] Freemantle | How strong is the association between abdominal obesity and the incidence of type 2 diabetes?[END_REF], is more prevalent in smokers. Furthermore, tobacco ingredients are likely to have toxic effects on the pancreas [START_REF] Chowdhury | Pathophysiological effects of nicotine on the pancreas: an update[END_REF], a finding which is consistent with a higher risk of pancreatic cancer in smokers [START_REF] Gallicchio | Active cigarette smoking, household passive smoke exposure, and the risk of developing pancreatic cancer[END_REF]. Furthermore, elevated levels of C-reactive protein [START_REF] Bazzano | Relationship between cigarette smoking and novel risk factors for cardiovascular disease in the United States[END_REF][START_REF] Wannamethee | Associations between cigarette smoking, pipe/cigar smoking, and smoking cessation, and haemostatic and inflammatory markers for cardiovascular disease[END_REF] and plasma fibrinogen [START_REF] Eliasson | The insulin resistance syndrome and postprandial lipid intolerance in smokers[END_REF][START_REF] Bazzano | Relationship between cigarette smoking and novel risk factors for cardiovascular disease in the United States[END_REF][START_REF] Wannamethee | Associations between cigarette smoking, pipe/cigar smoking, and smoking cessation, and haemostatic and inflammatory markers for cardiovascular disease[END_REF] have been found in smokers, suggesting that tobacco smoke might activate inflammation leading to T2DM. Smoking causes oxidative stress, which has been implicated to be responsible for the observed systematic inflammatory responses and might also contribute to endothelial dysfunction [START_REF] Burke | Oxidative stress and smoking-induced vascular injury[END_REF]. Finally, smoking contributes to dyslipidaemia because smokers have higher levels of free fatty acids, triglycerides, and LDL cholesterol, and lower levels of HDL-cholesterol [START_REF] Eliasson | The insulin resistance syndrome and postprandial lipid intolerance in smokers[END_REF][START_REF] Berlin | Smoking-induced metabolic disorders: a review[END_REF], factors usually associated with T2DM. Thus, the smoking-diabetes relationship is plausible from a pathophysiological point of view. However, our study failed to show an impairment of insulin sensitivity in smokers as measured by the HOMA-IR index. Moreover, in this study, effects of smoking on diabetes incidence could be explained neither by altered fat distribution nor by factors of the metabolic syndrome.

Strengths and limitations of the study

Our study had several strengths. It was a population-based study, and contrary to many other studies, incident diabetes was assessed by OGTT in addition to validated self-reports. Smoke exposure status was thoroughly assessed by several questions, some of which were asked more than once in slightly different formulations. Associations between smoke exposure and T2DM were adjusted for a large number of factors, and potential confounders were separated from potential mediators.

There are also important limitations in our study. First, possible confounders like physical activity or alcohol consumption were only assessed once at baseline. However, insufficient adjustment for confounders could also lead to a downward bias, e.g. in case of moderate alcohol intake which is inversely related to diabetes incidence [START_REF] Rimm | Prospective study of cigarette smoking, alcohol use, and the risk of diabetes in men[END_REF][START_REF] Hays | Prevention and treatment of type 2 diabetes: current role of lifestyle, natural product, and pharmacological interventions[END_REF]. Passive smoking often occurs in the workplace, so professional activity could be a confounder of the relationship between ETS exposure of never smokers and diabetes risk. However, this relationship was not attenuated when analyses were confined to passive smokers at home. Second, we considered only current passive smoking, and, if subjects stated that other persons smoked in their workplace or in the household, this might not in any case mean an exposure to ETS (e.g., partners smoking on the balcony). However, classifying subjects not exposed to ETS as passive smokers would be non-differential misclassification and lead to an underestimate of the relationship between passive smoking and diabetes incidence. Third, as the number of cases among current smokers was small, we did not distinguish current smokers who had stopped smoking in the follow-up from the ones who continued smoking. Depending on the effects of smoking cessation this could have led to an under-or overestimate of the diabetes risk of active smoking. Fourth, non-participants turned out to be sicker than participants (data not shown). An elevation of baseline glucose levels in nonparticipants compared to participants was found in current smokers and in the reference category (i.e. never smokers without ETS exposure), and this elevation turned out to be larger in current smokers. This suggests that the healthyparticipant effect probably led to a downward bias of the smoking-diabetes relationship.

In conclusion, this study shows that both passive and active smoking are risk factors of T2DM in an elderly population. Thus, it gives support to efforts to ban smoking in public buildings, encourage the declaration of private homes as smoke-free, and encourage the delivery of smoking cessation interventions.

Table 1

 1 Baseline characteristics by smoke exposure status: the KORA S4/F4 cohort study (Augsburg, Southern Germany)

		Never smokers/no	Never smokers/yes	Past smokers/no	Past smokers/yes	Current	P
		ETS	ETS	ETS	ETS	smokers
	N	350	88	264	73	110
	Age	64.1 (5.3)	60.5 (4.8)	64.7 (5.3)	60.5 (4.7)	60.5 (4.8)	0.001**, #
	Sex (males) (%)	29.1	40.9	69.3	80.8	61.8	0.001**, ###
	BMI (males) (kg/m 2 )	27.6 (2.9)	27.6 (4.0)	28.3 (3.2)	29.1 (4.4)	27.2 (3.0)	0.01*, #
	BMI (females) (kg/m 2 )	28.2 (4.5)	29.5 (5.0)	28.1 (4.4)	25.3 (2.8)	27.4 (4.7)	0.03*, #
	Waist circumference (males) (cm) 99.1 (7.8)	97.8 (10.0)	100.0 (8.5)	102.9 (10.7)	98.2 (9.3)	0.02*, #
	Waist circumference (females) (cm) 89.4 (10.6)	91.2 (11.2)	88.3 (10.4)	82.9 (7.1)	88.5 (10.7)	0.12#
	Systolic blood pressure (mmHg)	132.2 (19.4)	133.9 (19.5)	137.0 (17.4)	131.7 (19.7)	126.3 (17.2) 0.001**, #
	Fasting glucose (mg/dl)	98.1 (9.3)	99.1 (8.8)	99.4 (9.0)	100.4 (9.5)	97.0 (9.7)	0.11#
	2-h glucose (mg/dl)	113.9 (29.8)	114.6 (33.0)	115.5 (30.4)	112.3 (31.8)	103.9 (29.1) 0.06#
	HOMA-IR	2.5 {1.9}	2.7 {2.2}	2.4 {1.8}	2.5 {1.9}	2.3 {2.1}	0.43##
	Triglycerides (mg/dl)	110.0 {1.6}	117.9 {1.7}	111.6 {1.6}	113.8 {1.6}	128.1 {1.6}	0.07##
	Fasting insulin (mU/l)	10.4 {1.8}	11.2 {2.2}	9.9 {1.7}	10.0 {1.8}	9.7 {2.0}	0.55##
	Alcohol intake (% above	14.9	14.8	25.8	27.4	25.5	0.09###
	critical value a )
	Physically active b (%)	48.6	42.1	52.7	31.5	42.6	0.01*, ###
	Low SES (%)	25.4	19.3	10.2	12.3	10.9	0.01*, ###

BMI body mass index; ETS environmental tobacco smoke; HOMA-IR homeostatis model assessment of insulin resistance; SES socioeconomic status Means (standard deviation) or geometric means {standard deviation factor} * P \ 0.05, ** P \ 0.01 a C40 g/day (males), C20 g/day (females) b C1 h sports/week # F-test; ## log F-test; ### logistic regression. All analyses adjusted for age and sex

Table 2

 2 Cumulative 7 year incidence of diabetes by smoke exposure status at baseline: the KORA S4/F4 cohort study (Augsburg, Southern Germany)

		Total sample (n = 885)	Subjects with prediabetes at baseline	Subjects with normoglycemia at baseline
		N	Cases (%)	N	Cases (%)	N	Cases (%)
	Never smokers/no ETS	350	25 (7.1%)	97	16 (16.5%)	253	9 (3.6%)
	Never smokers/yes ETS	88	10 (11.4%)	24	8 (33.3%)	64	2 (3.1%)
	Past smokers/no ETS	264	35 (13.3%)	75	27 (36.0%)	189	8 (4.2%)
	Past smokers/yes ETS	73	10 (13.7%)	23	8 (34.8%)	50	2 (4.0%)
	Current smokers	110	13 (11.8%)	19	9 (47.4%)	91	4 (4.4%)
	ETS environmental tobacco smoke

Table 3

 3 Association of smoke exposure status at baseline with T2DM risk in the total sample and in stratified analyses for prediabetic and normoglycemic participants, multivariate logistic regression models: the KORA S4/F4 cohort study (Augsburg, Southern Germany)

	Smoke exposure category	Model 1		Model 2		Model 3		Model 4
		OR	95% CI	OR	95% CI	OR	95% CI	OR	95% CI
	Total sample (prediabetes ? normal glucose tolerance)
	n	885		882		884		862
	Never/no ETS	1.0		1.0		1.0		1.0
	Never/yes ETS	2.4	1.1, 5.5*	2.5	1.1, 5.6*	2.3	1.02, 5.3*	2.4	0.996, 5.9
	Past/no ETS	1.5	0.8, 2.7	1.6	0.9, 3.0	1.5	0.8, 2.8	1.7	0.9, 3.3
	Past/yes ETS	2.4	1.01, 5.7*	2.8	1.1, 6.8*	2.0	0.8, 4.8	2.6	0.99, 6.8
	Current smokers	2.2	1.01, 4.7*	2.8	1.3, 6.1*	2.2	1.01, 4.8*	3.2	1.4, 7.5**
	Prediabetes (baseline)
	n	238		238		238		233
	Never/no ETS	1.0		1.0		1.0		1.0
	Never/yes ETS	4.0	1.4, 11.8*	4.4	1.5, 13.4**	4.5	1.5, 13.5**	5.0	1.5, 17.1*
	Past/no ETS	2.3	1.01, 5.1*	2.4	1.1, 5.6*	2.5	1.1, 5.8*	2.3	0.9, 5.9
	Past/yes ETS	2.8	0.9, 8.7	3.1	0.9, 10.3	2.4	0.7, 7.9	3.0	0.8, 11.3
	Current smokers	5.4	1.8, 16.5**	7.8	2.4, 25.7**	5.4	1.7, 17.0**	8.0	2.2, 28.6**
	Normal glucose tolerance (baseline)
	n	647		644		646		604
	Never/no ETS	1.0		1.0		1.0		1.0
	Never/yes ETS	1.2	0.2, 6.1	1.3	0.3, 6.8	1.1	0.2, 5.3	1.1	0.2, 6.0
	Past/no ETS	1.0	0.3, 2.9	1.0	0.3, 3.0	1.0	0.3, 2.8	1.0	0.3, 3.1
	Past/yes ETS	1.6	0.3, 8.5	1.9	0.3, 10.3	1.3	0.2, 7.1	1.7	0.3, 10.2
	Current smokers	1.7	0.5, 6.1	2.1	0.5, 7.9	1.6	0.4, 5.9	1.8	0.5, 7.4

OR odds ratio; CI confidence interval; ETS environmental tobacco smoke * P \ 0.05, ** P \ 0.01 Model 1: adjusted for age, sex and parental diabetes

Table 4

 4 Association of lifetime pack-years up to baseline investigation with glucose values (mg/dl, fasting and 2-h glucose, at baseline and in the follow-up) in regular smokers, multivariate linear regression models: the KORA S4/F4 cohort study (Augsburg, Southern Germany)

		Pack-years A a			Pack-years B b
		N	b	95%-CI	P	N	b	95%-CI	P
	Baseline fasting glucose	94	0.14	0.03, 0.25	0.01*	93	0.08	0.001, 0.16	0.03*
	Baseline 2-h glucose	94	0.51	0.19, 0.83	0.002**	93	0.32	0.11, 0.55	0.003**
	Fasting glucose (follow-up)	94	0.30	0.12, 0.47	0.001**	93	0.20	0.08, 0.32	0.001**
	2-h glucose (follow-up)	89	0.15	-0.46, 0.75	0.63	88	0.10	-0.31, 0.52	0.61
	Adjusted for: age, sex, parental diabetes, socioeconomic status, alcohol intake, physical activity, intake of meat and sausage, intake of salad and
	vegetables, intake of whole-grain bread, and coffee consumption
	b regression coefficient; CI confidence interval

* P \ 0.05, ** P \ 0.01 a Pack-years based on the average number of cigarettes per day b Pack-years based on the largest number of cigarettes per day ever smoked for a whole year

Table 5

 5 Association of lifetime pack-years up to baseline investigation with glucose values (mg/dl, fasting and 2-h glucose, at baseline and in the follow-up) in past smokers, multivariate linear regression models: the KORA S4/F4 cohort study (Augsburg, Southern Germany)

		n	b	95%-CI	P
	Baseline fasting glucose	317	0.05	0.01, 0.10	0.02*
	Baseline 2-h glucose	317	0.09 -0.06, 0.23	0.25
	Fasting glucose (follow-up) 317	0.06 -0.005, 0.12 0.07
	2-h glucose (follow-up)	304 -0.01 -0.21, 0.18	0.92
	Pack-years based on the largest number of cigarettes per day ever
	smoked for a whole year
	Adjusted for: age, sex, parental diabetes, socioeconomic status,
	alcohol intake, physical activity, intake of meat and sausage, intake of
	salad and vegetables, intake of whole-grain bread, and coffee
	consumption
	b regression coefficient; CI confidence interval
	* P \ 0.05


Libbe ; Kooistra

Professor Victor J Pop

High thyrotrophin levels at end term increase the risk of breech presentation

Keywords: Thyroxine < Hormones/related: < Thyroid, Thyrotropin thyrotrophin, breech presentation, neurodevelopment

Objective. To study the relationship between maternal TSH and breech presentation at term. Design. Combined data sets of two prospective studies to obtain adequate epidemiological power.

Patients: 1058 healthy pregnant women (58 breech, 1000 cephalic) and 131 women who presented in breech at an obstetrical outpatient clinic.

Measurements: Maternal thyroid parameters (TSH, FT4, TPO-Ab) and fetal presentation were assessed in both groups between 35-38 weeks gestation. Power calculations suggested that at least 148 breech cases were required.

Results. The characteristics of the women in breech in both samples were similar. Women in breech (n=58+131) had significantly higher TSH (but not FT4) than those (n=1000) with cephalic presentation (Mann-Whitney U, p = 0.003). Different cut-offs were used to define high TSH in the 916 TPO-Ab negative women with cephalic presentation: the 90 th , 95 th and 97.5 th percentiles were 2.4mIU/l (n=149), 2.7 mIU/l (n=77) and 3.2 mIU/l (n=37). The prevalence rates of breech presentation in these women were all higher compared to the prevalence of breech in women below these cut-offs (df=1, p < 0.01). The R.R. of the 149 women with a TSH > 90 th percentile (> 2.4 mIU/l) to present in breech was 1.82 (95% CI: 1.30 -2.56).

Conclusions. Women with high TSH at end term are at risk for breech presentation.

Substantial evidence for a relation between breech presentation and neurodevelopmental delay exists. Since high TSH during gestation has also been linked to poor neurodevelopment, the relation between breech presentation and poor neurodevelopment might be thyroid-related.

Introduction

Breech presentation occurs in 3-5 % of term women and is associated with neonatal as well as maternal morbidity and mortality. Breech is often an indication for elective caesarean section (CS) [START_REF] Rietberg | The effect of the Term Breech Trial on medical intervention behaviour and neonatal outcome in The Netherlands: an analysis of 35,453 term breech infants[END_REF] , which, in itself, constitutes a prominent risk factor for decreased reproductive health. [START_REF] Ecker | Cesarean delivery and the risk-benefit calculus[END_REF] According to a recent review, the currently known risk factors associated with breech presentation only account for up to 15 % of the variance. [START_REF] Nordtveit | Maternal and paternal contribution to intergenerational recurrence of breech delivery: population based cohort study[END_REF] We previously reported that women who presented in breech had higher TSH levels than those who presented in cephalic position. [START_REF] Kuppens | Maternal thyroid function during gestation is related to breech presentation at term[END_REF] This study, however, was limited by small sample size. To overcome this limitation, the current study expanded on our previous findings by combining the breech cases (n=58) from our initial study [START_REF] Kuppens | Maternal thyroid function during gestation is related to breech presentation at term[END_REF] with a newly obtained larger sample of breech cases (n=131). Moreover, we reviewed the literature on possible consequences of breech presentation and long term infant development.

Methods

Subjects

The current study combines two samples of pregnant women from the same area in the South-East of the Netherlands. The first sample comprises 1058 pregnant women who were recruited between 2002 -2004 for a follow-up study in which maternal thyroid status was assessed at 12, 24 and 36 weeks gestation. [START_REF] Kuppens | Maternal thyroid function during gestation is related to breech presentation at term[END_REF] Fetal position was assessed by ultrasound between 35 -38 weeks gestation. The second sample comprises 161 pregnant women who, between 2007-2009, presented in breech at an obstetric clinic for an External Cephalic Version (ECV) attempt. Of these women, 151 were eligible and ultimately 141 women consented to participate. Prior to the ECV attempt, obstetrical parameters were assessed and blood samples were taken. While term of gestation in this sample varied between 35 -39 weeks, for the purpose of the current study only women with a term between 35 -38 weeks (n = 131, 93%) were included.

In both samples, women with twin pregnancies, on thyroid hormone replacement therapy, those with known auto-immune disorders (e.g., diabetes-I), or those who became pregnant after hormonal stimulation, were excluded, as well as women with preterm birth (study 1). To avoid potential language problems only women of Dutch Caucasian background were included. This resulted in a total of 121 women who were excluded from the first sample and 20 women from the second sample.

The study was approved by the Medical ethical Committee of the Catharina Hospital Eindhoven.

Assessments

Fetal presentation was assessed by ultrasound in study one by one researcher (HW) who was blind to the thyroid hormone status, and in study two prior to the ECV attempt. Breech position was categorized into complete, incomplete and frank breech. In all women a careful obstetrical history was taken.

Thyroid parameters

Thyroid parameters, free thyroid hormone (FT4), thyrotrophin (TSH) and thyroid peroxidase antibody (TPO-Ab) were in the first sample assessed during the ultrasound visit at 35 -38 weeks gestation, and in the second sample during the visit at the obstetrical outpatient clinic just before the ECV attempt. Only the third trimester TSH/FT4 assessments at 35 -38 weeks in the first sample were taken into account and compared with the thyroid parameters of the women with a similar term seen in the ECV outpatient clinic. TSH was measured using a solid-phase, two site chemiluminescent enzyme immunometric assay (IMMULITE Third generation TSH, Diagnostic Products Corporation, Los Angeles USA). FT4 (concentration was also measured by means of a solid-phase immunometric assay (IMMULITE Free T4). The IMMULITE Anti-TPO Ab kit was used for the determination of antibodies against thyroid peroxidase (TPO). The anti-TPO assay was standardized in terms of the International Reference Preparation for anti-TPO MRC 66/387. TPO-Ab concentrations higher than 35 IU/ml were regarded as antibody positive.

Statistical analysis

Statistical analysis was performed using the Statistical Package of Social Science (SPSS 17.0). First, a power calculation was performed. The TSH numbers at 36 weeks gestation of study one were log transformed and the difference (delta) in

Results

The characteristics of breech women in sample one (n = 58) and those in sample two (n = 131) were compared (Table 1). As can be seen, there were no significant differences between the groups.

Insert table 1 about here

Table 2 shows the characteristics of the total group of breech women (58 + 131 = 189) versus the women of sample one who presented in cephalic position (n = 1000). In the latter group, TSH and FT4 reference ranges were calculated (between the 2.5 th and 97.5 th percentiles) at 36 weeks gestation for those women who were TPO-Ab negative at 12 weeks gestation (n = 916). These ranges were: 0.42 -3.2 mIU/l for TSH and 9.4 -17.1 pmol/l for FT4. As can be seen in Table 2, the women who presented in breech had significantly higher TSH levels compared to those who presented in cephalic position (Mann-Whitney-U, p = 0.003), while no between group differences were found for FT4 (Mann-Whitney-U, p = 0.62).

Insert table 2 about here

Significantly more primiparous women were found among the breech-presenting women versus the cephalic-presenting women. Groups did not differ in terms of the prevalence of women with elevated TPO-Ab. Subsequently, for the overall group (n = 1189) different TSH cut-off values were defined in the TPO-Ab negative women with cephalic presentation: the highest 90 th (> 2.4 mIU/l, n =149), 95 th (> 2.7 mIU/l, n = 77) and 97.5 th (> 3.2 mIU/l, n =37) percentiles. Comparing prevalence rates above and below these cut-offs of TSH categories, breech-presenting women were significantly more prevalent than cephalic-presenting women (25% versus 14%, 27% versus 15% and 35% versus 15%, p < 0.01, figure 1). The number of women with elevated TPO-Ab bodies in the group with TSH > 90 th , the 95 th and the 97.5 th percentile was significantly higher compared to the number of women below these cut-offs: 14 versus 4.5%, 17% versus 5% and 18 versus 5.2%. The mean FT4 values between groups did not differ: 13.2 pmol/l (in TSH > 3.2 mIU/l) versus 13.3 pmol/l (TSH <3.2mIU/l) or 13.4 pmol/l in women with a TSH < 50 th percentile.

Insert figure 1 about here When high TSH (> 97.5 th percentile) and parity were entered into a logistic regression analysis, both were found to be significantly related to breech presentation (dependent variable): O.R.: 2.3, 95% CI: 1.2 -4.9 and O.R.: 5.1, 95% CI: 3.4 -7.5, respectively. The 37 women with a TSH > upper reference limit (> 3.2 mIU/l) had a median TSH of 3.7 mIU/L with a range between 3.3 -10 mIU/L, and 13 presented in breech. Finally, women with a TSH > 90 th percentile (>2.4 mIU/l) already had a 1.82 increased risk of breech presentation (R.R.:

1.82, 95% CI: 1.30 -2.56).

Discussion

The current study combined the data sets of two studies in which the relationship between maternal thyroid status and fetal breech position was investigated. The present combined results obtained in 189 breech versus 1000 cephalic cases were clear in showing that high maternal TSH during pregnancy is related to increased rates of breech position. It is unlikely that this relationship has arisen by chance, given the 189 breech cases in this study which was substantially higher than the minimum group size of 148 suggested by the power calculations. In line with the literature, primiparity was significantly more prevalent in breech cases compared to cephalic cases [START_REF] Nordtveit | Maternal and paternal contribution to intergenerational recurrence of breech delivery: population based cohort study[END_REF] .

In a recent large retrospective study elevated maternal TSH levels in both the first and second trimesters were not related to adverse obstetrical outcome. [START_REF] Cleary-Goldman | Maternal thyroid hypofunction and pregnancy outcome[END_REF] In contrast, another large retrospective study did report a relation between increased TSH and poor obstetrical outcome (without detailed reports on fetal position), with TSH being assessed between 12 and 24 weeks gestation. [START_REF] Casey | Subclinical hypothyroidism and pregnancy outcomes[END_REF] How can the relation between high maternal TSH and breech position be explained? As discussed in a previous paper, one might speculate that TSH directly acts to modulate uterine contractility. [START_REF] Kuppens | Maternal thyroid function during gestation is related to breech presentation at term[END_REF] Another (more indirect) explanation might be that high maternal TSH reflects suboptimal maternal thyroid function, which, in turn, would affect maternal-fetal T4 transfer, with impaired fetal neuromotor outcome as a final consequence. The finding that women with elevated TPO-Ab levels are especially at risk for high TSH levels does suggest that high maternal TSH in these women is pathologic rather than physiologic (the latter, for example, to compensate for high T4 turnover by deiodinase activity of the placenta).

In view of this latter explanation, several examples of delayed fetal neural maturation in breech pregnancies are worth addressing. First, the fetus, from 24-35 weeks onwards changes position through sudden leg extensions and active whole body movements (kicking). [START_REF] Suzuki | Fetal movement and fetal presentation[END_REF] These changes are caused through excitation of antigravity muscles, i.e. extensors of the body axis. [START_REF] Sekulic | Possible explanation of cephalic and non-cephalic presentation during pregnancy: a theoretical approach[END_REF] This excitation is a result of the interplay between inhibitory and stimulatory influences generated in the reticular nuclei, cerebellum and motor cortex. One may speculate that suboptimal maternal thyroid function (reflected by high TSH) might affect the neural processes involved in changes of fetal position. Secondly, neonates born in breech show different sensory experiences compared to those presenting in cephalic position. [START_REF] Van Der Meulen | Fetal sensory-elicited body movements differ in breech compared to cephalic position[END_REF] Although this was explained in terms of more amniotic fluid surrounding the head in breech, also this effect might have been due to suboptimal maternal-fetal thyroid hormone supply. In another study the development of lateralized head-position preference was found to be less pronounced in breech fetuses than in cephalic fetuses. [START_REF] Fong | Does intra-uterine environment influence fetal head-position preference? A comparison between breech and cephalic presentation[END_REF] Although this may have been related to a smaller difference in stimulation between the left and right otoliths, inadequate thyroid hormone supply for development of the cochlear system might have been involved. With regard to neonatal maturation, breech babies tend to be smaller [START_REF] Luterkort | The role of asphyxia and slow intrauterine growth in morbidity among breech delivered infants[END_REF] , have lower scores on neurological tests [START_REF] Kean | A comparison of fetal behavior in breech and cephalic presentations at term[END_REF][START_REF] Molkenboer | Birth weight and neurodevelopmental outcome of children at 2 years of age after planned vaginal delivery for breech presentation at term[END_REF][START_REF] Sekulic | Decreased expression of the righting reflex and locomotor movements in breech-presenting newborns in the first days of life[END_REF] and are balance-impaired until the age of 12-18 months. [START_REF] Sival | The effect of intra-uterine breech position on postnatal motor functions of the lower limbs[END_REF] Again, for all these findings inadequate maternal-fetal T4 supply may have been an underlying etiological factor with regard to optimal neurodevelopment. A large cohort study reported a significant IQ delay in 20 years old men born in breech compared to men born in cephalic presentation [START_REF] Sørensen | Long-term follow-up of cognitive outcome after breech presentation at birth[END_REF] . A similar IQ delay has been reported in children of women with high TSH during gestation 18 . Finally, autism spectrum disorder has been found to occur more frequently in children born in breech position 19 . Autism has also been linked to poor maternal thyroid function during gestation 20,21. A limitation of the current study was that two different studies were combined. These studies, however, were performed in the same region, in women with similar characteristics, using identical thyroid assessments tests. Combining studies, however, also constituted a key strength, as it heightened the statistical power, thereby allowing a more accurate estimation of the relationship between high TSH and breech position. Other study strengths were its prospective design, and the fact that obstetric staff was blind to the thyroid status of the women.

In conclusion, this study strongly suggests a relationship between high maternal TSH and fetal position at end term. It also suggests that neurodevelopmental delay, which has been related to breech presentation at term, might in fact reflect sub-optimal maternal thyroid function during gestation. Because breech presentation is associated with neonatal as well as maternal morbidity and mortality, future prospective studies are needed to further elucidate the relation between high maternal TSH and fetal presentation at term.

  women presenting in breech versus those presenting in cephalic position was calculated. With an alpha of 0.05 and a power of 0.8, it was calculated that the size of the breech group in the current study needed to be at least 148 women.Kolmogorov-Smirnov tests revealed a non-normal distribution of FT4 and TSH in the current sample. Therefore, group comparisons on FT4 and TSH used Mann-Whitney U tests. T-tests (two-tailed) and chi-square tests were used for other group comparisons. Subsequently, we looked at different cut-offs for maternal TSH with regard to breech position, and performed chi squares to test associations.

11

 11

  JE, Palomaki GE, Allan WC et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999;341(8):549-55 19. Bilder D, Pinborough-Zimmerman J, Miller J, McMahon W. Prenatal, perinatal, and neonatal factors associated with autism spectrum disorders. Pediatrics 2009;123:1293-1300 20. Sadamatsu, M., Kanai, H., Xu, X., et al. Review of animal models for autism: implication of thyroid hormone. Congenital Anomalies, 2006: 46:1-9. 21. Roman GC. Autism: transient in utero hypothyroxinemia related to maternal flavonoid ingestion during pregnancy and to other environmental antithyroid agents. Journal of Neurol sciences 2007;262:15-26.

  TSH percentile cut-off's * : p < 0.01, chi-square, df=1.1

Figure 1 :

 1 Figure 1: Comparison of prevalence rate of breech presentation at 35 -38 weeks between women above and below different percentile cut-off's of TSH. (n total group =1189, of which 189 breech and 1000 cephalic presentations)

Table 1

 1 Characteristics (relevant to thyroid function) of a sample of 58 breech women of a general population sample of 1000 women, compared to 131 women in breech who visited an outpatient clinic for ECV.

		Term in both groups was between 35 -38 weeks gestation.
	_______________________________________________________________________________________________
				Breech women	Breech women
			of general population sample	of outpatient ECV clinic
				N = 58	N = 131
			Mn (SD)	n (%) median (range)	Mn (SD) n (%)	median (range)	P
	Age		31.2 (3.7)		31.6 (4.1)	0.6
	Life style habits
	smoking (yes/no)		6 (11)	8 (6)	0.3
	any alcohol use		10 (18)	12 (9)	0.1
	Obstetrical features
	Parity
	Primiparity		33 (57)	87 (66)
	Multiparity		25 (43)	44 (34)	0.2
	one child		21 (37)	42 (32)
	two children		5 (9)	8 (6)
	>two children		1 (2)	4 (3)	0.7
	Miscarriage earlier in life		11 (19)	31 (24)	0.5
	Fetal characteristics
	Boy			34 (59)	62 (48)
	Girl			24 (41)	68 (52)	0.2
	Birth weight (grams)	3190 (464)		3329 (438)	0.1
	Placental weight (grams)	583 (128)		607 (132)	0.2
	Thyroid function parameters
	Family history of thyroid dysfunction	5 (8)	13 (10)	0.8
	TSH	median (range)		1.60 (0.58 -5.4)		1.59 (0.51 -9.7) 0.6
	FT4	median (range)		13.3 (9.8 -17.5)		13.2 (9.6 -18.2)	0.7
	TPO-Ab > 35		5 (8)	10 (7.6)	0.8
	________________________________________________________________________________________________

Table 2

 2 Characteristics of a sample of 189 breech women and 1000 women who present in cephalic position.

	Term in both groups was between 35 -38 weeks gestation.
	____________________________________________________________________________________________________
		Breech women	Cephalic women
		N = 189	n = 1000
		Mn (SD) n (%) median (range)	Mn (SD) n (%) median (range)
					P
					t-test	χ2 M-W-U
	Age	31.7 (3.9)		32.3 (3.6)
	Life style habits
	smoking (yes/no)		13 (7)	120 (12)
	alcohol use		23 (12)	130 (13)
	Obstetrical features
	Parity
	Primiparity	120 (64)	480 (48)
	Multiparity		69 (36)	520 (52)	0.003
	Miscarriage earlier in life		51 (27)	190 (19)
	Fetal characteristics
	Boy		96 (51)	520 (52)
	Girl		93 (49)	480 (48)
	Birth weight (grams)	3299 (440)	3530 (485)	< 0.001
	Placental weight (grams)	612 (109)	623 (113)
	Thyroid function parameters
	Family history thyroid dysfunction	23 (12)	170 (17)
	TSH		1.61 (0.48 -9.7)	1.30 (0.05 -5.1)	0.003
	FT4		13.3 (9.6 -18.2)	13.2 (7.2 -24.6)	0.58
	TPO-Ab > 35		15 (8)	60 (6)
	____________________________________________________________________________________________________
	M-W-U: Mann-Whitney-U test


Dr Hakan Cangul
email: h.cangul@bham.ac.uk

Neil V Morgan

Julia R Forman

Halil Saglam

Zehra Aycan

Tahsin Yakut

Tuna Gulten

Omer Tarim

Ece Bober

Yasar Cesur

Gail A Kirby

Shanaz Pasha

Mutlu Karkucak

Erdal Eren

Semra Cetinkaya

Veysel Bas

Korcan Demir

Sevil A Yuca

Esther Meyer

Michaela Kendall

Wolfgang Hogler

Timothy G Barrett

Eamonn R Maher

Novel TSHR mutations in consanguineous families with congenital non-goitrous hypothyroidism

Keywords: congenital hypothyroidism, consanguineous, mutation, TSHR, gene mapping TSHR, congenital hypothyroidism, consanguineous, gene mapping, mutation

Objective: Non-syndromic autosomal recessively inherited non-goitrous congenital hypothyroidism (CHNG) can be caused by mutations in TSHR, PAX8, TSHB, and NKX2-5. We aimed to investigate mutational frequencies of these genes and genotype/phenotype correlations in consanguineous families with CHNG.

Design: Since consanguinity in individuals with a presumptive genetic condition is often an indicator of an autosomal recessive inheritance and allows firmer correlations to be established between genotype and phenotype, we planned to execute our study in consanguineous families.

Patients: 139 children with CHNG phenotype born to consanguineous families.

Measurements: First we investigated cases for evidence of linkage to the four known-CHNG genes by microsatellite marker analysis. Mutation analysis by direct sequencing was then performed in those cases in whom linkage to the relevant candidate gene could not be excluded. In addition in silico analysis of the predicted structural effects of TSHR mutations was performed and related to the mutation specific disease phenotype.

Results: Homozygous germline TSHR mutations were detected in 6 families (5%), but no mutations were detected in PAX8, TSHB, and NKX2-5. Four of TSHR mutations had not previously been described. Genotype-phenotype correlations were established and found to be related to the predicted structural effects of the mutations. Conclusions: Known-causative genes account for the development of CHNG only in a minority of cases and our cohort should provide a powerful resource to identify novel causative genes and to delineate the extent of locus heterogeneity in autosomal recessively inherited CHNG.

INTRODUCTION

Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder with an incidence of 1 in 3000 to 4000 live births and, if untreated, results in severe neurodevelopmental impairment and infertility. Worldwide the most frequent cause of hypothyroidism, including congenital forms, is iodine deficiency. However, particularly in areas where dietary iodine is adequate, developmental defects of the hypothalamicpituitary-thyroid axis are the main important cause of CH. Thus CH is most often caused by defects in thyroid development leading to thyroid dysgenesis (85%) [which can be subdivided into thyroid agenesis (40% of all cases), failure of the gland to descend normally during embryological development with or without ectopy (40%), or hypoplasia of a eutopic gland] and the remaining cases are associated with either goitre or a normal thyroid gland. Rarely, central (secondary/tertiary) hypothyroidism may be caused by pituitary or hypothalamic disease leading to deficiency of thyrotropin (TSH) or thyrotropin releasing hormone (TRH), respectively [START_REF] Park | Genetics of congenital hypothyroidism[END_REF][START_REF] Kopp | Genetic defects in the etiology of congenital hypothyroidism[END_REF] .

In this study we focused on primary non-goitrous congenital hypothyroidism (CHNG) likely to be caused by single gene defects. Therefore we executed our study in consanguineous families in whom recessive gene defects are the most likely cause for the development of the disease. In OMIM database four clinical entries with known genetic causes, CHNG 1, 2, 4, and 5, have been described to date (MIM numbers 275200, 218700, 275100, and 225250 respectively). The causative genes for these are respectively TSHR, PAX8, TSHB, and NKX2-5. There is one additional locus on The presence of parental consanguinity in individuals with a presumptive genetic condition is often an indicator of an autosomal recessive inheritance. In consanguineous families with autosomal recessively inherited disorders there is a high likelihood of mutation homozygosity (both parents having inherited the same mutation from a common ancestor) and so autozygosity mapping can provide a powerful strategy to map regions containing a disease gene. Conversely, candidate genes that are not contained within an autozygous segment are unlikely to cause the relevant disorder. We have ascertained a large cohort of consanguineous families (n=120) with non-goitrous congenital hypothyroidism (CHNG) and have undertaken genetic mapping and mutation analysis studies for four candidate genes (TSHR, PAX8, TSHB, and NKX2-5). We identified four novel and two previously reported TSHR mutations but most cases were not linked to any known CHNG genes.

MATERIALS AND METHODS

Subjects

We collected blood samples from a total of 139 cases from 120 consanguineous families with primary non-syndromic CH without evidence of goitre or auto-immune thyroid disease. Families in which autosomal dominant inheritance was suspected, with at least one parent affected, were excluded from the study. Cases with clear evidence of central hypothyroidism were also excluded and almost all cases had elevated TSH levels at the time of diagnosis (range between 33 and 1578 mIU/L), levels for some cases not being available. Tissue scans showed a spectrum of thyroid morphologies ranging from agenesis to normal sized gland but none of the cases had enlarged gland at the time of diagnosis. 40 consanguineous families were recruited through the West Midlands

Regional Clinical Genetics Service in Birmingham, UK. Most of these families were originated from the Mirpur region of Pakistan in whom CH occurs at an unusually high rate (1:770 live births). In addition 80 consanguineous Turkish families with CHNG were ascertained through four large clinical centres in Turkey where consanguinity rates are high. All families gave informed consent and the study was approved by local ethics committees. This study was conducted according to the principles stated in the Declaration of Helsinki. DNA was extracted by using standard methods and stored at -20 °C until analysed.

Microsatellite marker analysis

Initially cases were genotyped by using microsatellite markers (short tandem repeats-STRs) that closely flank the existing loci for CHNG (see

RT-PCR

In one family a homozygous germline TSHR exon 2 deletion was suspected, and we proceeded to investigate the status of the TSHR transcript between exons 1 and 3. Thus we extracted RNA from all family members, synthesized their cDNA, and performed PCR and sequencing reactions with primers covering the region between the end of exon 1 and the beginning of exon 3. Total RNA was extracted from lymphocytes using the RNeasy Mini Kit (Qiagen), and then cDNA was synthesized using random primers and AMV reverse transcriptase using the Promega reverse transcription system A3500 according to manufacturers' instructions (Promega). The primers used for RT-PCR and sequencing reactions were: TSHR_1_FOR (5'-GAGAAATAGCCCCGAGTCCC-3') and TSHR_3_REV (5'-GAGTCACTTTACTCAAATTG -3').

Structural prediction model for missense mutations

To analyse the likely structural effects of the missense mutations identified in this work, we considered their evolutionary conservation, as illustrated by a multiple alignment of the top 50 Blast hits (performed using the Uniprot server). We also examined potential intermolecular interactions, to determine if they would be disrupted upon mutation.

TSHR is a G-coupled receptor with 764 amino acids, comprised of a large, extracellular leucine-rich repeat domain which binds the hormone, and a transmembrane domain with seven helices. Sanders et al. [START_REF] Sanders | Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody[END_REF] solved the crystal structure of the extracellular domain, residues 1-213, in complex with a thyroid-stimulating antibody (PDB code 3G04). The transmembrane domain structure is not known, however the bovine rhodopsin structure (e.g. PDB code 1GZM 4 ) provides a suitable template for analysis or homology modelling (based on GRIS [START_REF] Van Durme | GRIS: glycoprotein-hormone receptor information system[END_REF] and swissmodel.expasy.org [START_REF] Arnold | The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[END_REF] ). In addition, given the sequence similarity between TSHR and FSHR, the structure of the FSH/FSHR complex (PDB code 1XWD 7 )

suggests the most likely binding site of TSH with TSHR. Also, there is evidence that TSHR functions as a homodimer. To consider the structural effects on dimerisation, we analysed the dimer contacts of a published model of dimeric bovine rhodopsin (1N3M 8 ).

Finally, individual missense mutations may destabilise a protein domain, even to the extent of preventing folding. To consider this effect, we predicted quantitative stability changes (∆∆G D-N ) with the software tool SDM [START_REF] Topham | Prediction of the stability of protein mutants based on structural environment-dependent amino acid substitution and propensity tables[END_REF]

RESULTS

Genotyping with 16 microsatellite markers associated with the TSHR, TSHB, PAX8 and NKX2-5 genes (see Table 1) was performed in 139 individuals with CHNG (44 of Pakistani origin and 95 of Turkish origin). Heterozygosity of the disease genes was detected in 65 cases (13 of Pakistani origin and 52 of Turkish origin) from a total of 37 kindreds. Details of the number of affected individuals and genes for which linkage could not be excluded are shown in Table 2.

Direct sequencing of the TSHR gene was performed in 24 individuals from 16 kindreds in whom linkage to TSHR could not be excluded. Germline TSHR mutations were characterised in 6 kindreds (see Table 3). A homozygous G to A transition at codon 553 of the TSHR gene, causing substitution of an alanine residue by a threonine (A553T) was detected in two affected siblings from a family of Pakistani origin (B1). The parents and an unaffected sibling were heterozygous for this missense mutation confirming the segregation of the disease allele. Both affected children showed absent I 133 uptake in thyroid scintigraphy but had severely hypoplastic glands on ultrasonography at the time of diagnosis. Our studies showed that this mutation was significantly destabilising (∆∆G D-N -2.7) and that the Ala553 residue was conserved for 49 of the 50 sequences in our alignment.

A homozygous splice site mutation c.317+1G>A was identified in a Turkish family with two affected children (T1) and it segregated with disease status (both parents and an In family T5 exon 2 of the TSHR gene failed to amplify in the two affected siblings.

F
Analysis of the TSHR transcript by RT-PCR revealed two differently sized fragments in the parents. In cDNA derived from blood of both affected children only the smaller fragment (268 bp) was detected whereas both parents and an unaffected sibling also expressed a larger (340 bp) fragment (which corresponded to the expected size).

Sequencing confirmed that the 340 bp fragment contained exon 1, 2 and 3 sequence whereas the 268 bp fragment did not include exon 2 (Figure 1). These findings were consistent with a homozygous exon 2 deletion in the two affected children.

Phenotypically, the older sibling had no activity in thyroid scintigraphy and a hypoplastic gland on US scan but hormone levels at diagnosis were not available. The younger sibling had a TSH level of 1578 mIU/L and a FT4 level of 0,16 ng/dL at the time of diagnosis, and his US scan detected a very hypoplastic thyroid tissue.

No mutations were detected in the TSHB, PAX8 and NKX2-5 in the patients in whom linkage to one or more of these genes could not be excluded. 4. Mutation Pro162Ala is predicted to be only mildly destabilising (∆∆G D-N -0.7). However, it is conserved in all of the TSHRs in our alignment. To consider its intermolecular interactions, we examined the interactions of the equivalent residue, Ile155, in the FSH/FSHR complex (PDB code 1XWD 7 , Figure 2). There are no known direct contacts, however it is in the local environment of the binding site so we predict it could slightly disrupt binding. The remaining missense mutations were found in three sites in the transmembrane domain. As the structure was not available, the stability predictions were made for the mutations at equivalent positions in bovine rhodopsin (PDB code 1GZM 4 ; see Table 4 and Figure 3; equivalent positions were found using an alignment made using Uniprot [START_REF]The Universal Protein Resource (UniProt) in 2010[END_REF][START_REF] Jain | Infrastructure for the life sciences: design and implementation of the UniProt website[END_REF] and the alignments could be seen in Supplements 1 and 2)

Residue Arg531 was conserved in all of the TSHRs in our alignment. Mutation Arg531Trp (Phe146 in bovine rhodopsin) is predicted to be slightly stabilising (∆∆G D-N 1.3), while Arg531Glu is predicted to be significantly destabilising (∆∆G D-N -2.1). The equivalent site in bovine rhodopsin, Phe146, is at the dimer interface, so these mutations may act by disrupting dimerisation. Residues Ala553 and Pro556 (Ala168 and Pro171 in bovine rhodopsin, respectively) are both buried, and therefore do not make any intermolecular contacts, however the bovine rhodopsin equivalent residues are found in one of the helices at the dimer interface (as modelled in 1N3M 8 ). Ala553Thr (Ala168Thr in bovine rhodopsin) is predicted to be significantly destabilising (∆∆G D-N -2.7). In Therefore early and accurate diagnosis of the disease is of crucial importance for timely therapeutic intervention. In this study we performed linkage and mutation analysis of four known causative genes for CHNG (TSHR, PAX8, TSHB, and NKX2-5) in a large cohort of patients with CHNG phenotype from consanguineous families.

Individuals with autosomal recessively inherited disorders born into consanguineous families are most likely to be homozygous for a mutation inherited from a common parental ancestor. This observation is the basis for using an autozygosity mapping strategy to identify disease genes for recessively inherited disorders. Conversely the finding of heterozygosity at microsatellite markers flanking a disease gene makes it unlikely that the gene contains a mutation. In order to prioritise patients for gene sequencing we initially typed affected individuals for 16 microsatellite markers linked to the four disease genes. Whilst this might have excluded some patients who (despite being from consanguineous families) were compound heterozygotes or who harboured a homozygous mutation within a small region of autozygosity, such patients are likely to be infrequent. Mutation analysis demonstrated that 15% of the families with thyroid dysgenesis carried a TSHR mutation but no mutation was detected for the other three genes tested in our cohort. To our knowledge, we report the first large (exonic) deletion of TSHR. The exon 2 deletion is predicted to cause an in frame deletion (IFD) and skipping of exon 2 in the transcript. However this mutation would not have been detected

if the probands had been heterozygous and our finding suggests that techniques that can As expected, all six consanguineous kindreds had homozygous TSHR mutations. The finding of homozygosity for a mutation facilitates the elucidation of genotype-phenotype correlations. The p.Ala553Thr missense mutation identified in family B1 was originally described in a consanguineous Moroccan family with two affected siblings in whom initial physical examination and 99m Tc scintigraphy failed to demonstrate the presence of thyroid tissue (leading to an incorrect diagnosis of athyreosis) but subsequent ultrasonography (US) revealed the presence of a very hypoplastic thyroid tissue in normal position in both cases [START_REF] Abramowicz | Familial congenital hypothyroidism due to inactivating mutation of the thyrotropin receptor causing profound hypoplasia of the thyroid gland[END_REF] . Similarly in our study this mutation was associated with a severe phenotype and our results indicated highly destabilising nature of this alteration and high degree of conservation of the altered residue. These results combined with the in vitro observations showing that the expression of the mutant protein in transfected cell lines 12- 17 is associated with very low expression of the mutant protein at the cell surface, are consistent with the severe phenotype associated with this mutation. The novel c.317+1G>A splice site mutation was also associated with a very severe phenotype, indicating a deleterious effect of this alteration on the resulting receptor.

The missense mutation p.Pro556Arg detected in family T2 was associated with a severe phenotype, suggesting a critical role for this residue in the structure of the molecule. Even though this change was not previously reported, another missense mutation at the same site (p.Pro556Leu) was characterised in severely hypothyroidic hyt/hyt mice and 17 functional investigations showed that this substitution totally abolished TSH binding and receptor function [START_REF] Gu | The thyrotropin receptor transmembrane domain mutation (Pro 556 Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but dfective TSH binding[END_REF] . In vitro studies also revealed that p.Pro556Leu eliminated G proteincoupled cAMP production and cAMP-responsive reporter gene expression. Overall these data support the association of this mutation with the severe phenotype observed in our patient.

The missense mutation p.Pro162Ala associated with a mild phenotype in family T3

was previously described as a loss-of-function mutation and heterozygous carriers of this mutation had mild resistance to TSH manifesting as euthyroidism with slight hyperthyrotropinemia [START_REF] Sunthornthepvarakul | Resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene[END_REF][START_REF] Tonacchera | Identification of TSH receptor mutations in three families with resistance to TSH[END_REF][START_REF] Refetoff | Resistance to thyrotropin[END_REF] . There have been only a few previous reports of homozygous individuals for this mutation and all four cases coming from three different consanguineous families had moderately elevated TSH levels (< 100 µU/mL) with normal thyroid radioiodine scan and FT 4 levels at normal range [START_REF] De Roux | Four families with loss of function mutations of the thyrotropin receptor[END_REF][START_REF] Tonacchera | Low prevalence of thyrotropin receptor mutations in a large series of subjects with sporadic and familial nonautoimmune subclinical hypothyroidism[END_REF][START_REF] Costagliola | Structure-function relationships of two loss-of-function mutations of the thyrotropin receptor gene[END_REF] . Similar phenotypic features of our case provide further evidence for the association of this mutation with a moderate TSH resistance. The other mild missense mutation detected in family T4, p.Arg531Trp, is the first naturally occurring homozygous mutation reported to date at this site. An alternative missense substitution at the same residue was investigated previously in an in vitro functional study 25 Overall there is further evidence from our results that TSHR loss-of-function mutations cause a wide spectrum of phenotypic consequences, ranging from a slight TSH resistance to very severe hypothyroidism. Similarly while some deleterious TSHR mutations may result in severe hypoplasia rendering the gland undetectable by initial scintigraphy, some milder mutations may be associated with normal-sized or even slightly enlarged thyroid glands, suspecting defective hormone synthesis.

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  Novel TSHR mutations in consanguineous families with congenital non-goitrous hypothyroidism

  3 causing CHNG 3 for which the causative gene is yet to be identified.

13 Predicted

 13 structural consequences of missense mutations detected SDM scores (for predicted effect on protein stability) for each of the missense mutations detected in this study are summarised in Table

15 DISCUSSION

 15 alanine is conserved for 49 of the 50 sequences in our alignment (the only exception is Danio rerio TSHR, with glycine in the equivalent position). Pro556Arg and Pro556Leu (at the Pro171 site in bovine rhodopsin) are predicted by SDM to be neutral and disease-associated mutations, respectively (∆∆G D-N 0.7 and 3.45). However, proline is conserved at this site in all of the sequences in our alignment, and is found at the end of a helix, suggesting it is structurally required at this site.Congenital hypothyroidism is the most common preventable cause of mental retardation.

  exonic deletions (e.g. MLPA) should have a role in TSHR mutation testing.

  and Feng et al. identified a motif in the second intracellular loop (IL2) of the TSHR molecule involving R531 as a potential G s -coupling motif. They found that substitution of arginine residue with a glutamine (p.Arg531Glu) resulted in a significant decrease in the basal activity of the receptor but had very little or no effect on the hormone-stimulated cAMP production, and further studies showed that this mutation was structurally conservative. The clinical features observed in our patient mild TSH resistance and might be attributed to a slight structural change and/or reduced basal activity of the receptor brought about with the mutation.The severe phenotype associated with exon 2 deletion in family T5 might be explained by the fact that this exon encodes an amino terminal extracellular ligand binding domain of the receptor molecule. Our literature review suggested that homozygous LOF point mutations at the sites encoding the extracellular domain (ECD) of the molecule generally caused mild phenotypes whereas homozygous LOF point mutations in the transmembrane domain were rather associated with severe phenotypes. However our results implicate that gross alterations in the ECD might also severely affect the function of the receptor and thus cause severe phenotypes as observed in our patients.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. A) Pedigree (upper panel) and TSHR exon 1-3 RT-PCR results (lower panel) in family T5 with two affected cases showing two different-sized fragments, 268-bp and 340-bp. (Note the homozygous state of the bands in affected cases versus heterozygous state in carrier family members). B) Sequence traces of TSHR exon 1-3 cDNA from an affected case (upper panel) and one of her parents (lower panel) representing homozygous and heterozygous states of the deletion, respectively.

Table

excluded the cases with clear evidence of central hypothyroidism, TSHB locus was still included in the analyses since hormone levels were not available for some cases, and also since some TSHB mutations can completely abolish the bioactivity but resulting mutant molecule can still form heterodimer with the alpha-subunit and preserve immunoreactivity in some TSH measurement methods. Therefore variable TSH levels could be detected in patients with TSHB mutations depending on the method used. In

	families with two or more affected siblings, microsatellite marker
	F o r analyses of all family members were carried out to observe the segregation of conserved F o haplotypes. Standard polymorphic microsatellite markers (UniSTS; r
	P http://www.ncbi.nlm.nih.gov) were used for each of the causative loci investigated. P
	e e r R e v i e w Overall genotypes for 24 markers were determined for each patient, including eight markers for the CHNG3 locus. Table 1 shows the details of the causative genes examined e e r and the markers used for each locus. One oligonucleotide of each primer pair was fluorescently labelled (5` HEX or TET or FAM) and genomic DNA samples at a concentration of 20ng/µl were amplified by using these primers in a Tetrad2 PCR machine (Bio-Rad, USA). Fluorescently-labelled PCR products then were loaded onto ABI 3730 DNA capillary electrophoresis machine in a 96-well plate format and subsequent data was analysed with the use of ABI Prism GeneMapper™ v3.0 software R e v i e w
	suite. The haplotypes, i.e. set of closely linked alleles inherited as a unit, were deduced
	for all patients. When an affected individual was homozygous for the haplotypes closely
	surrounding a region where a disease gene is located, i.e. both haplotypes derived from
	the two homologous chromosomes are identical, this was taken as suggestive of linkage
	whereas heterozygosity was considered as the proof of no-linkage.
	7

). Even though we

8 Mutation analysis of the known-causative genes in cases with positive linkage

	In families with potential linkage to a known-CHNG gene (TSHR, PAX8, TSHB, and
	NKX2-5) we then proceeded to perform mutation analysis of the relevant gene by direct
	sequencing. Each gene was investigated by sequencing of all exons and intron-exon
	boundaries on an ABI Prism 3730 DNA Analyzer, using the ABI BigDye Terminator
	v3.1 Cycle Sequencing kits. Primer pairs were designed for exon-specific PCR

amplification of all exons of four causative genes. At the purification stage, amplified products were either treated with microCLEAN (Microzone Ltd) or gel-extracted (Qiagen). DNA sequences were viewed and analysed using Chromas software.

Table 3 )

 3 . None of these 3 mutations were detected in 340 ethnically matched control chromosomes and each segregated with disease status. Family T2 with a homozygous p.Pro556Arg mutation presented when the proband was 3 months old. At the time of diagnosis a TSH level of >100 µU/mL (normal < 20 µU/mL) and a FT 4 level of 0,4 ng/dL (normal 0,8-1,9 ng/dL) were detected, and thyroid gland was not detected on scintigraphy or US scan. Even though treatment immediately started at the age of 3 months, the 11 year-old case now shows moderate mental retardation. At age 11 years a US scan showed an apparently absent right thyroid lobe and a very hypoplastic left lobe. We note that the proline556 residue is conserved in all of the 50 sequences in our alignment and located at the end of a transmembrane helix. ), and a thyroid gland of normal size and location in US scan at the time of diagnosis. Our SDM predictions suggested that p.Arg531Trp mutation might slightly disrupt the dimerisation of the receptor molecule.

	unaffected sib were heterozygous). Both cases carrying this novel mutation had apparent
	thyroid aplasia on US scan.
	Three different homozygous missense mutations were detected in 3 Turkish families with
	o r P e e r R e v i a single affected individual (see Two of the mutations characterised in this study (p.Pro162Ala and p.Arg531Trp) were e associated with a milder phenotype. The case from family T3 had the missense mutation w p.Pro162Ala and was diagnosed with a moderately high TSH level (48 µU/mL) in
	neonatal screening and had normal thyroid scintigraphy and US scan with euthyroid
	hormone levels. Our structural predictions suggested that p.Pro162Ala would only cause
	a mild destabilisation (∆∆G D-N -0.7) in the receptor molecule and slightly disrupt ligand
	binding. The other mild missense mutation p.Arg531Trp was detected in family T4 and
	the case had a mildly elevated level of TSH (33,3 mIU/L), a normal level of FT4 (1.3
	11

Table 1 .

 1 Features of known-causative genes for congenital non-goitrous hypothyroidism (CHNG)

	Gene	Chromosomal	CHNG locus	Microsatellite markers 2	Inheritance	Thyroid	Biocemical
		location 1				morphology	spectrum
	TSHR	14q31	CHNG1	D14S1433, D14S1008, D14S606,	AR	Agenesis-	High TSH
				D14S610		Normal	Low TH to
							euthyroid
	PAX8	2q12-q14	CHNG2	D2S2269, D2S160, D2S410,	AR, AD with	Agenesis,	High TSH
				D2S1893	variable	hemiagenesis,	Low to normal
					expressivity	hypoplasia,	TH
						ectopy
	TSHB	1p13	CHNG4	D1S2756, D1S2881, D1S2852,	AR	Hypoplasia	Variable TSH
				D1S189			Low TH
	NKX2-5	5q34	CHNG5	D5S400, D5S2075, D5S211,	AD?	Hypoplasia	High TSH
				ATA52D02			Low TH

[START_REF] Park | Genetics of congenital hypothyroidism[END_REF]

Location according to USCS Genome Bioinformatics (http://zeon.well.ox.ac.uk/HumanGenome) 2 Sequences available from Genome Database at http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists AR, autosomal recessive; AD, autosomal dominant; TH, thyroid hormones

Table 2 .

 2 Number of patients at each locus in whom linkage could not be excluded.

				CHNG loci
	Population	TSHR	TSHB	PAX	NKX2-5 CHNG3
	All (n=139)	24	17	15	9	9
	Pakistani Cases (n=44)	10	8	5	6	2
	Turkish Cases (n=95)	14	9	10	3	7

Table 4 .

 4 Structural prediction scores calculated by the software tool SDM 9 PDB code for the extracellular domain of TSHR molecule based on crystal structure as resolved by Sanders et al. 3 b PDB code for bovine rhodopsin molecule. Equivalent residues corresponding to the mutation sites in TSHR molecule are indicated and prediction scores are based on alterations in these residues.

	3G04 a			SDM prediction
	Pro162Ala		-0.72	neutral
	TSHR	1GZM b		SDM predictions
	Arg531Trp	F146W	1.25	neutral
	Arg531Glu	F146Q	-2.08	disease-associated
	Ala553Thr	A168T	-2.65	disease-associated
	Pro556Arg	P171R	0.69	neutral
	Pro556Leu	P171L	3.45	disease-associated

a

Clinical Endocrinology

Acknowledgments

We thank NewLife, WellChild and the Wellcome Trust for financial support. HC was partly supported by European Union under its Framework 7 programme, FP7-PEOPLE-2009-Marie Curie IEF. JRF was sponsored in part by a grant from the Pasteur Foundation of New York. We also thank families for their help with this research. Authors declared no conflicting interests. Word count : 3,457

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