PhD Anna-Maija Tolppanen
email: am.tolppanen@bristol.ac.uk

Dylan Williams Msc

PhD John Henderson

PhD Debbie A Lawlor

Serum 25-hydroxy-vitamin D and ionised calcium in relation to lung function and allergen skin tests

Background: Evidence suggests that higher levels of vitamin D and calcium are associated with greater lung function and that vitamin D is inversely associated with atopic sensitisation. It is unknown whether the associations of vitamin D and calcium with lung function are independent of each other or mediated by atopic sensitisation. Objective: To study the associations of 25-hydroxyvitamin D [25(OH)D] and ionised calcium levels with lung function and specific allergen sensitisation in adolescents (12-19 years) and adults (20-59 years) and to assess whether the associations with lung function are due to altered atopic sensitisation. Methods: Cross-sectional analysis of the data from the third National Health and Nutrition Examination Survey. Results: 25(OH)D levels were positively associated with forced vital capacity in adolescents [0.035(95 %CI: 0.007-0.064) standard deviations;SD in model adjusted for multiple confounders]

. This association and the previously reported association between higher serum levels of 25(OH)D and better lung function in adults were independent of serum calcium levels, which were not associated with lung function. In adults, calcium was associated with sensitisation to grass allergens [OR per SD,1.17(1.03-1.32), 1.15(1.01-1.31) and 1.18 (1.06-1.32) for white oak, Bermuda grass and short ragweed, respectively] and peanut OR 1.21 (95%CI 1.02-1.43) after adjusting for age, gender and race/ethnicity, but these associations attenuated towards the null after adjusting for additional confounders. The associations were independent of 25(OH)D levels, which were not associated with allergen sensitisation.

Conclusions:

Circulating levels of 25(OH)D are positively associated with lung function and this does not appear to be driven by allergen sensitisation or influenced by calcium levels.

INTRODUCTION

Atopy is characterised by a tendency to mount immunoglobulin E (IgE)-mediated, type I hypersensitivity reactions to allergens. It is strongly associated with allergic diseases, including asthma. In many Western countries 10-20 percent of the population are affected by atopy and the prevalence of atopic diseases in children and adults has increased in these countries from the 1970s [START_REF] Asher | Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys[END_REF][START_REF] Upton | Intergenerational 20 year trends in the prevalence of asthma and hay fever in adults: the Midspan family study surveys of parents and offspring[END_REF]. Although there is evidence of genetic heritability [START_REF] Swarr | Unraveling the complex genetic underpinnings of asthma and allergic disorders[END_REF], little is known about modifiable risk factors for atopy that might be used in prevention.

Vitamin D is a key regulator of calcium homeostasis, and biological evidence suggests that vitamin D and calcium might be implicated in atopic diseases and respiratory health, [START_REF] Hughes | Vitamin D and respiratory health[END_REF][START_REF] Litonjua | Childhood asthma may be a consequence of vitamin D deficiency[END_REF][START_REF] Hirota | Ionic mechanisms and Ca2+ handling in airway smooth muscle[END_REF][START_REF] Chhabra | Increased intracellular calcium and decreased activities of leucocyte Na+,K+-ATPase and Ca2+-ATPase in asthma[END_REF] though epidemiological evidence is limited and inconsistent [START_REF] Devereux | Maternal vitamin D intake during pregnancy and early childhood wheezing[END_REF][START_REF] Laaksi | An association of serum vitamin D concentrations < 40 nmol/L with acute respiratory tract infection in young Finnish men[END_REF][START_REF] Ginde | Association between serum 25hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey[END_REF][START_REF] Erkkola | Maternal vitamin D intake during pregnancy is inversely associated with asthma and allergic rhinitis in 5-year-old children[END_REF][START_REF] Kunisaki | Vitamin D Status and Longitudinal Lung Function Decline in the Lung Health Study[END_REF][START_REF] Hypponen | Infant vitamin d supplementation and allergic conditions in adulthood: northern Finland birth cohort[END_REF][START_REF] Hypponen | Serum 25-hydroxyvitamin D and IgE -a significant but nonlinear relationship[END_REF][START_REF] Searing | Decreased serum vitamin D levels in children with asthma are associated with increased corticosteroid use[END_REF][START_REF] Miyake | Dairy food, calcium and vitamin D intake in pregnancy, and wheeze and eczema in infants[END_REF]. Differences in how vitamin D exposure and outcomes were assessed or differences in the confounders that were taken into account in different studies may have contributed to differences between them.

Wheezing and associated reduced lung function are amongst the wide range of symptoms associated with atopy and there is some evidence of positive association between vitamin D (measured as serum 25-hydroxyvitamin D [25(OH)D] and lung function [START_REF] Devereux | Maternal vitamin D intake during pregnancy and early childhood wheezing[END_REF][START_REF] Searing | Decreased serum vitamin D levels in children with asthma are associated with increased corticosteroid use[END_REF][START_REF] Brehm | Serum vitamin D levels and markers of severity of childhood asthma in Costa Rica[END_REF][START_REF] Black | Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey[END_REF][START_REF] Sutherland | Vitamin D Levels, Lung Function and Steroid Response in Adult Asthma[END_REF], although a null association has also been reported [START_REF] Devereux | A case-control study of vitamin D status and asthma in adults[END_REF].

Previous studies in the US third National Health and Nutrition Examination Survey (NHANES III) have shown positive association between serum calcium and forced expiratory volume in one second (FEV 1) [START_REF] Mckeever | A multivariate analysis of serum nutrient levels and lung function[END_REF] but not with atopy [START_REF] Mckeever | Serum nutrient markers and skin prick testing using data from the Third National Health and Nutrition Examination Survey[END_REF]. In addition, higher serum 25(OH)D levels were associated with better lung function in the same study [START_REF] Black | Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey[END_REF], but it is unknown whether the associations of 25(OH)D were independent of calcium and vice versa.

Our study adds to these previous papers by: (i) examining whether the associations of 25(OH)D and calcium are independent of each other: (ii) examining the association of 25(OH)D and calcium with specific allergen sensitisation: (iii) studying the association between serum calcium and FVC and (iv) comparing whether the magnitudes and/or directions of these associations differ between adolescents and adults. The previous lung function studies have included participants older than 17 years only [START_REF] Mckeever | A multivariate analysis of serum nutrient levels and lung function[END_REF] or older than 20 years [START_REF] Black | Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey[END_REF]. The association between calcium and atopy was studied in 6-16 year olds and 17-59 year olds [START_REF] Mckeever | Serum nutrient markers and skin prick testing using data from the Third National Health and Nutrition Examination Survey[END_REF].

METHODS

Sample

Outcomes

Lung function and allergy tests were conducted at mobile examination centres (MECs). Lung function [FEV 1 and forced vital capacity (FVC)] was assessed with a Ohio 827 rolling seal spirometer (Ohio Medical Instrument Company, Cincinnati, OH USA) according to American Thoracic Society criteria (1987) (American Thoracic Society, 1987).

The allergen panel consisted of 10 common allergens (Dermatophagoides farinae, cat, German cockroach, short ragweed, perennial rye, Alternaria alternata, Bermuda grass, Russian thistle, white oak and peanut) and a negative (glycerinated diluent) control.

Hypersensitivity reactions were evaluated 15 minutes after administering the allergens on separate sites of examinee's forearms with prick-puncture technique. A positive test response was defined as maximum weal diameter≥3mm for allergen and <3mm for negative control.

Atopy was defined as a positive response to one or more allergens. Individuals with severe eczema or signs of skin infection on both arms, those with previous severe response to allergen testing or participants with signs of breathing difficulties were excluded from the allergy tests. We appreciate that these might include individuals with most severe atopy, but due to small numbers (39 adolescents; 111 adults) it was not possible to examine this as an outcome by itself in both adolescents and adults.

Potential confounders

The following were considered potential confounding factors due to their known or plausible associations with exposures and outcomes: age, gender, race/ethnicity, height, health status, socioeconomic position, outdoor activity and smoking.

Height and weight were measured using standard research procedures at the MECs and were used to calculate body mass index (BMI). Physicians' impressions of health status (Poor/Fair/Good/Very good/Excellent) at the MECs were used as an indicator of general health. Data on self-reported race/ethnicity, smoking, pets, socioeconomic background, outdoor physical activity and history of respiratory/allergic diseases (emphysema, chronic bronchitis, asthma and hay fever) were obtained during interview. Family poverty-income ratio (below/equal to or above 1) was used as indicator of socioeconomic position. Outdoor activity during past month for adults was derived from the interview data according to method by [START_REF] Scragg | Frequency of leisure-time physical activity and serum 25-hydroxyvitamin D levels in the US population: results from the Third National Health and Nutrition Examination Survey[END_REF]. These data were not collected from those aged 18 or younger. In adolescents, we adjusted for pubertal stage, which was assessed by Tanner scales [START_REF] Tanner | Growth at adolescence[END_REF]. Among adults, smoking status was defined as never, former (has smoked ≥100 cigarettes but not currently smoking), and current smoker. Pack-years of smoking were calculated using answers to 12 tobacco questions and included assessment of current number of cigarettes smoked, maximum number of cigarettes smoked, and periods during which a current smoker did not smoke. Due to the skewed distribution, a categorical variable for pack-years was derived (0, 0.1-4.9, 5-9.9, 10-19.9 and ≥20 pack-years). Because of missing data on pack-years (22% of the adults), in our main analyses we adjusted for smoking status only and in sensitivity analyses we examined the effect of adjusting for pack-years in those with these data. In adolescents, smoking status was classified as current smoker or non-smoker currently due to low frequency of ex-smokers (n=67, 6.3% of those with smoking data available). Because 61.7% of adolescents did not answer the smoking questionnaire, we did not include smoking as a covariable in the main analyses in adolescents but conducted a sensitivity analysis in those with smoking data available to see if adjusting for smoking affected the associations.

Statistical analyses

Statistical analyses were conducted with Stata 11.0 (Stata Corp LP, College Station, TX USA), using the svy procedure to account for cluster effects and sampling weights from MECs (lung function) and allergy subsamples (allergen sensitisation). A more detailed description of recommended statistical methodology is available in [START_REF] Landis | A statistical methodology for analyzing data from a complex survey: the first National Health and Nutrition Examination Survey[END_REF]. The associations of 25(OH)D and calcium levels with lung function were analyzed with multivariable linear regression. The association with atopy and sensitisation to allergens were assessed with multivariable logistic regression. Serum calcium, 25(OH)D and spirometry measurements were analyzed as z-scores.

RESULTS

The weighted mean (standard error) 25(OH)D and ionised serum calcium levels among adolescents were 80.3 (1.80) nmol/l and 1.25 (0.003) mmol/l. Among adults, the weighted mean 25(OH)D was 75.0 (1.00) nmol/l and weighted mean calcium level was 1.24 (0.003) mmol/l. The correlation coefficient r for serum 25(OH)D and ionised calcium was 0.02 (P=0.34) in adolescents and 0.04 (P=0.0009) in adults. There was no statistical evidence that any associations differed between males and females (P for interaction ≥0.13). Therefore, results are presented for males and females combined.

Supplementary Tables 1234 The proportion of men increased linearly across the quartiles of ionised serum calcium in adults. Mean age and number of pack-years smoked decreased across the calcium quartiles in adults. Among the 12-19 year olds, mean BMI increased across the quartiles. In the adult group, mean serum 25(OH)D increased across the quartiles, while the proportion of Mexican-American and Other ethnicities decreased across the quartiles.

With respect to outcomes, in these age-and gender-adjusted models 25(OH)D was associated positively with FEV 1 and FVC and inversely with responsiveness to German cockroach allergen in both age groups. In adults, 25(OH)D was inversely associated with the prevalence of atopy, and in adolescents, decreased responsiveness to ragweed, Bermuda grass and perennial rye. Serum ionised calcium was positively associated with sensitisation to short ragweed, Bermuda grass, white oak and peanut allergens in adults only. Ionised calcium was not associated with any outcomes in adolescents, or with other outcomes in adults. Where associations occurred they appeared linear across the quartiles.

Tables 1 and2 show multivariable associations of 25(OH)D and calcium with lung function in adolescents and adults respectively. In adolescents (Table 1), 25(OH)D was positively associated with FVC in the confounder adjusted model, but not with FEV 1 . Additional adjustment for pubertal stage (n=1566) did not alter the associations presented (results available from authors). In adults (Table 2) serum 25(OH)D levels were positively associated with both FEV 1 and FVC after adjusting for all potential confounders (Model 2). Ionised serum calcium was not associated lung function in adolescents or adults (Tables 12). With additional mutual adjustment (associations of 25(OH)D adjusted for ionised calcium and vice versa) the associations remained unchanged (Model 3 in Tables 12).

Tables 3 and4 summarise the multivariable associations of 25(OH)D and calcium levels with previously diagnosed respiratory or allergic diseases and atopic outcomes in adolescents and adults. No associations were observed in adolescents (Table 3) and serum 25(OH)D levels were not associated with allergic diseases or allergen sensitisation in adults, apart from slightly decreased sensitisation to German cockroach allergen (Table4), Higher calcium levels were associated with increased sensitisation to short ragweed, Bermuda grass, white oak and peanut allergens, when adjusted for age, gender, race/ethnicity and height (model 1).

With adjustment for additional confounders (Model 2) and 25(OH)D (Model3) the associations with sensitisation to short ragweed and Bermuda grass allergen remained, but other associations were attenuated to the null.

Sensitivity analyses are shown in supplementary Tables 5-12. In the subgroups of adolescents with smoking data and adults with data on pack-years associations that were the equivalent of Models 1-3 for the whole eligible sample (Tables 1234) were markedly different in inconsistent ways, suggesting that analyses in these subgroups may be affected by selection bias. Adjusting for smoking in adolescents and pack-years in adults did not markedly alter associations in these subgroups.

DISCUSSION

We showed that the previously reported positive association of 25(OH)D with FVC and FEV 1 in adults [START_REF] Black | Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey[END_REF] is independent of serum calcium levels, and not due decreased risk of allergen sensitisation or respiratory diseases, as serum 25(OH)D levels were not associated with these outcomes. In addition, the association between 25(OH)D levels and FVC, but not with FEV 1 was observed in adolescents, whereas a previous publication using NHANES III data had only explored this association in adults [START_REF] Black | Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey[END_REF]. As with adults, this association was independent of calcium and allergen sensitisation. We did not find an association between ionised serum calcium and lung function in either adolescents or adults, although a previous study reported a positive association between ionised calcium and FEV 1 among NHANES III participants age≥17 years [START_REF] Mckeever | A multivariate analysis of serum nutrient levels and lung function[END_REF]. That previous study did not address the association between FVC and calcium. The difference between our results and those of that previous NHANES study are likely to be due to differences in age ranges and numbers of participants included in the analyses of the two papers. We were interested in exploring whether associations differed between adolescents (defined as those aged 12-19) and adults, who had data on allergen sensitisation ( 20

Vitamin D and lung function

Several mechanisms may link higher vitamin D levels to better lung function. The positive association of 25(OH)D with lung function might be the result of vitamin D protecting against atopic asthma, but we found no evidence that higher levels of 25(OH)D were associated with decreased allergic responsiveness to common allergens. FEV 1 is primarily used as a measure of intrathoracic airway obstruction, as seen in asthma, in clinical and epidemiological settings, although changes in total lung capacity (TLC) will affect both FEV 1 and FVC. FVC is a dynamic representation of the difference between TLC and residual volume and, in healthy subjects, it is likely to vary with TLC and reflect lung growth. It is therefore possible that the positive association of 25(OH)D with FVC in adolescents reflects the established role of vitamin D in skeletal growth [START_REF] Holick | The influence of vitamin D on bone health across the life cycle[END_REF], i.e. higher 25(OH)D is related to greater height via beneficial effects on skeletal development and greater height is associated with greater lung volume. However, all associations were adjusted for height and so this is unlikely to fully explain the association. In adults 25(OH)D was positively associated with both FEV 1 and FVC, supporting a role beyond the relation to skeletal development.

High consumption of antioxidants have been associated with lung function,(e.g [START_REF] Gilliland | Children's lung function and antioxidant vitamin, fruit, juice, and vegetable intake[END_REF]) so it is possible that the association of vitamin D is mediated via its antioxidative properties. Alternative mechanisms might be the regulation of immune response, cellular differentiation and/or proliferation by vitamin D [START_REF] Bikle | Nonclassic actions of vitamin D[END_REF][START_REF] Van Etten | Regulation of vitamin D homeostasis: implications for the immune system[END_REF].

Vitamin D receptor is present in human bronchial smooth muscle cells, epithelial cells lining the respiratory tract, activated lymphocytes and antigen-presenting cells [START_REF] Van Etten | Regulation of vitamin D homeostasis: implications for the immune system[END_REF][START_REF] Bosse | 1alpha,25-dihydroxy-vitamin D3 stimulation of bronchial smooth muscle cells induces autocrine, contractility, and remodeling processes[END_REF], which support the local functions of vitamin D in the lungs and in the immune system. Therefore, it could be that vitamin D regulates the growth of bronchial smooth muscle cells or protects from the lung inflammation by promoting the expression of antimicrobial peptides and through anti-inflammatory mechanisms [START_REF] Bikle | Nonclassic actions of vitamin D[END_REF][START_REF] Van Etten | Regulation of vitamin D homeostasis: implications for the immune system[END_REF]. Alterations in calcium homeostasis are known to occur in asthma sufferers [START_REF] Chhabra | Increased intracellular calcium and decreased activities of leucocyte Na+,K+-ATPase and Ca2+-ATPase in asthma[END_REF], so it would be tempting to speculate this as a possible mechanism, although in our study circulating levels of ionised calcium were not associated with lung function, However, despite the lack of association between serum calcium levels and lung function, local calcaemic effects such as alterations in intracellular calcium signalling could still relate to the mechanism.

Ionised calcium and atopy

Our results suggest that variation in ionised serum calcium is more robustly associated with allergic response to common allergens in adults than is variation in vitamin D. In adults positive associations were found with sensitisation to grass allergens and peanut, although apart from short ragweed and Bermuda grass, these associations were attenuated towards the null after adjusting for confounders. Experimental studies suggest that calcium is involved in allergic response: bronchial smooth muscle contraction, mast cells granulation and histamine release from mast cells are calcium-dependent [START_REF] Hirota | Ionic mechanisms and Ca2+ handling in airway smooth muscle[END_REF][START_REF] Alm | Histamine liberators and the mechanisms of mediator release[END_REF] and effects of vitamin D on the immune system depend on adequate calcium levels [START_REF] Cantorna | Dietary calcium is a major factor in 1,25-dihydroxycholecalciferol suppression of experimental autoimmune encephalomyelitis in mice[END_REF].

Both intra-and extracellular calcium levels have been shown to regulate the histamine release [START_REF] Alm | Histamine liberators and the mechanisms of mediator release[END_REF], so one would perhaps expect to see a disruption of calcium homeostasis in atopy, although intracellular calcium levels are strictly controlled and thus unlikely to be affected by normal variation in serum calcium levels. In addition, the calcium-binding motifs in allergens, especially of grass origin, are necessary for IgE binding and the chelation of calcium ions from sera of allergic patients lead to decreased allergen-IgE binding or prevented this occurring [START_REF] Suphioglu | Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen[END_REF][START_REF] Ledesma | Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens? Olive pollen allergens as model of study[END_REF]. To our knowledge only one previous study has examined the association of circulating calcium with allergic response and that cross-sectional study found no association [START_REF] Mckeever | Serum nutrient markers and skin prick testing using data from the Third National Health and Nutrition Examination Survey[END_REF]. The positive associations we have found here in adults require further exploration.

Study strengths and limitations

The main strengths of this study are its sample size, examination of associations with serum measurements of 25(OH)D and calcium rather than dietary reports, examination with objectively assessed outcomes of lung function and allergic response to common allergens, comparison of associations in adolescents and adults and the ability to adjust for a wide range of potential confounding factors. In adults we were only able to adjust for smoking status (current/ex/never) for our main analyses and, whilst sensitivity analyses suggested that adjustment for pack-years in the subgroup with these data did not result in greater attenuation compared with smoking status, there was some evidence that selection bias may have influenced this subgroup analysis. Similarly, in adolescents we were unable to adjust for smoking in the main analysis and those with smoking data appeared to be a selected subgroup.

Because this study is cross-sectional, we cannot rule out reverse causality as an explanation for the observed associations. Further exploration in prospective studies are warranted, though we are unaware of any studies that currently have measurements of serum 25(OH)D and calcium with later objectively measured lung function and response to common allergens. NHANES III has single measurements of calcium and 25(OH)D, which may be inadequate to reflect long-term status [START_REF] Schram | Serum calcium and cognitive function in old age[END_REF], although a single measurement may be a useful biomarker of season-specific vitamin D status over a longer time [START_REF] Hofmann | Long-term variation in serum 25-hydroxyvitamin D concentration among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial[END_REF] and serum calcium levels are normally maintained at relatively narrow limits within individuals [START_REF] Parfitt | Bone and plasma calcium homeostasis[END_REF]. Other studies, including those finding associations with bone phenotypes [START_REF] Jesudason | Relationship between serum 25-hydroxyvitamin D and bone resorption markers in vitamin D insufficiency[END_REF], have used single measurements of 25(OH)D. A further limitation is the lack of data on IgE and parathyroid hormone levels.

Conclusions

Our findings contribute to the emerging literature on a possible role of vitamin D and calcium with lung function and atopy. We showed that the positive cross-sectional association between serum 25(OH)D levels and lung function is independent of variation in ionised calcium levels and does not appear to be driven by increased allergen sensitisation. By contrast we report a novel positive association between serum levels of ionised calcium and The views expressed in this paper are those of the authors and not necessarily those of any funding body or others whose support is acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

COMPETING INTERESTS

None

  Data from the third US NHANES III were used.NHANES III, conducted between 1988 and 1994, is a survey of the civilian non-institutionalized population of the United States. Data and documentation are available at http://www.cdc.gov/nchs/nhanes/nh3rrm.htm. Serum 25(OH)D and calcium measurements were available for participants aged ≥12 years and skin prick allergen responses for participants up to age 59 years (with a random 50 percent of 20-59 year olds having these tests). Thus, we included participants aged 12-59 years, examining adolescents (12-19 years) and adults (20-59 years) separately. Figure1shows the flow of participants who attended mobile examination clinics and final analyses samples for the studies in adolescents and adults. We only included individuals with complete data on exposures, outcome and all covariables in any analyses meaning that the final sample sizes were 2446 and 2315 adolescents for associations with lung function and allergy tests, respectively and 8049 and 3656 adults for associations with lung function and allergy tests, respectively. The NHANES III study was approved by NHANES Institutional Review Board and documented consent was obtained from participants. Exposures Serum levels of 25(OH)D and calcium were measured on venous samples. 25(OH)D was assayed with INCSTAR 25-OH-D I 25 radioimmunoassay (Diasorin, Stillwater, MN, USA) and serum calcium with NOVA 7 analyzer (Nova Biomedical, Waltham, MA, USA). Ionised calcium (adjusted for serum pH) was used in these analyses.

  summarise the age-and gender-adjusted characteristics of adolescents and adults across quartiles of serum 25(OH)D and ionised calcium. The proportion of females, non-Hispanic blacks and persons from households with poverty/income ratio below 1 decreased across the quartiles of 25(OH)D in adolescents, while the proportion of non-Hispanic whites increased across the quartiles. Adolescents in the higher quartiles had lower Tanner scores. Among adults, mean age and BMI decreased across quartiles of 25(OH)D, and mean serum level of ionised calcium and height increased across the quartiles. The proportion of men, non-Hispanic whites, those reporting outdoor physical activity during past month, and those who had a pet increased across the quartiles of 25(OH)D. The proportion of non-Hispanic blacks, individuals with fair health status and individuals from households with poverty-income ratio below 1 decreased across the quartiles of 25(OH)D in adults.

  -59 year old). By contrast McKeever et al. were exploring associations in adults only (defined as anyone aged 17 years or older), consequently they had larger numbers. McKeever et al. also categorised calcium in a different way to the way we have used this variable. When we repeat our analyses using calcium in the same way as that used by McKeever et al. we still find no strong statistical evidence of an association in either of the age groups we examined, by contrast we can perfectly replicate McKeevers results in those aged 17 years and above.

FUNDING

  Work on this study is funded by an UK Medical Research Council (MRC) Grant (G0701603), which also pays AMT's salary. The MRC and the University of Bristol provide core funding for the MRC Centre of Causal Analyses in Translational Epidemiology (G0600705). DW is funded by a Wellcome Trust 4-year PhD studentship in Molecular, genetic and lifecourse epidemiology [WT083431MA].

  ABBREVIATIONS

Figure legends Figure 1 .

 legends1 Figure legends

Table 1 .

 1 Association of circulating 25(OH)D and ionised calcium (n2446) with lung function in 12-19 year olds.

	Mean difference in respiratory function (SD) per 1 SD change	Mean difference in respiratory function (SD) per 1 SD
	in 25(OH)D (95%CI)			change in ionised serum calcium (95%CI)
	SD	Model 1 1	Model2 2	Model3 3	Model 1 1	Model2 2	Model3 3
	FEV 1 753	0.009	0.009	0.008	0.010	0.010	0.010
		(-0.028 to 0.046)	(-0.028 to 0.046)	(-0.028 to 0.045)	(-0.024 to 0.045)	(-0.024 to 0.045)	(-0.024 to 0.044)
	FVC 901	0.036	0.036	0.035	0.007	0.007	0.005
		(0.007 to 0.065)	(0.007 to 0.065)	(0.007 to 0.064)	(-0.024 to 0.038)	(-0.024 to 0.038)	(-0.025 to 0.034)
	FEV 1 forced expiratory volume in 1 second; FVC forced vital capacity
	1 Model 1 is adjusted for age, ethnicity/race, gender and height
	2 Model 2 as Model 1, plus additional adjustment for BMI, poverty index, household pets (cat/dog/bird), general health and diagnosed
	asthma/hay fever/chronic bronchitis
	3 Model 3 as Model 2, plus additional adjustment for calcium in 25(OH)D analysis and 25(OH)D in calcium analysis

Table 2 .

 2 Association of circulating 25(OH)D and ionised calcium(n=8049) with lung function in 20-59 year olds.

	Mean difference in respiratory function(SD) per 1 SD change in	Mean difference in respiratory function(SD) per 1 SD
	25(OH)D(95%CI)			change in ionised serum calcium(95%CI)
	SD	Model 1 1	Model2 2	Model3 3	Model 1 1	Model2 2	Model3 3
		0.034	0.023	0.023	0.006	0.002	0.000
	FEV 1 821
		(0.016 to 0.052)	(0.004 to 0.041)	(0.005 to 0.041)	(-0.012 to 0.025)	(-0.018 to 0.021)	(-0.019 to 0.019)
		0.043	0.032	0.032	0.004	-0.001	-0.003
	FVC 1006
		(0.024 to 0.061)	(0.015 to 0.049)	(0.015 to 0.049)	(-0.018 to 0.025)	(-0.022 to 0.020)	(-0.024 to 0.018)
	FEV 1 forced expiratory volume in 1 second; FVC forced vital capacity
	1 Model 1 is adjusted for age, ethnicity/race, gender and height
	2 Model 2 as Model 1, plus additional adjustment for BMI, poverty index, household pets(cat/dog/bird), general health, outdoor activity during
	last month, smoking and diagnosed asthma/emphysema/hay fever/chronic bronchitis
	3 Model 3 as Model 2, plus additional adjustment for calcium in 25(OH)D analysis and 25(OH)D in calcium analysis

Table 3 .

 3 Association of circulating 25(OH)D and ionised calcium levels with previously diagnosed respiratory/allergic diseases (asthma, chronic bronchitis or hay fever) (n=2446) and allergy test results (n=2317) in 12-19 year olds Odds ratio for each allergen outcome per 1SD change in 25(OH)D

						Odds ratio for each allergen outcome per 1SD change in
		(95%CI)				ionised serum calcium (95%CI)
		Prev
			Model 1 1	Model2 2	Model3 3	Model 1 1	Model2 2	Model3 3
		(%)
	Respiratory
		16.3	0.92 (0.76-1.13) 0.92 (0.76-1.13)	0.92 (0.75-1.12) 1.09 (0.91-1.29) 1.09 (0.91-1.29) 1.09 (0.92-1.30)
	disease
	Atopy	60.6	1.05 (0.89-1.24) 1.06 (0.90-1.26)	1.06 (0.90-1.26) 1.03 (0.89-1.19) 1.01 (0.88-1.17) 1.01 (0.87-1.16)
	D.farinae	31.1	0.96 (0.79-1.18) 0.97 (0.80-1.17)	0.97 (0.80-1.18) 0.98 (0.83-1.15) 0.96 (0.81-1.14) 0.96 (0.81-1.14)
	Cat	18.4	0.93 (0.78-1.10) 0.93 (0.78-1.11)	0.94 (0.78-1.12) 0.94 (0.76-1.18) 0.93 (0.73-1.18) 0.93 (0.73-1.18)
	German
		32.9	0.90 (0.77-1.05) 0.92 (0.79-1.08)	0.92 (0.79-1.08) 1.03 (0.85-1.26) 1.03 (0.83-1.27) 1.03 (0.84-1.27)
	cockroach
	Short ragweed	31.6	0.92 (0.79-1.06) 0.94 (0.81-1.08)	0.93 (0.81-1.08) 1.06 (0.91-1.23) 1.04 (0.90-1.22) 1.05 (0.90-1.22)

Table 4 .

 4 Association of circulating 25(OH)D and ionised calcium levels with previously diagnosed respiratory/allergic diseases(asthma, chronic bronchitis or hay fever, n=8049) and allergy test results (n=3656) in 20-59 year olds Odds ratio for each allergen outcome per 1SD change in 25(OH)D

						Odds ratio for each allergen outcome per 1SD change
		(95%CI)				in ionised serum calcium (95%CI)
		Prev (%)	Model 1 1	Model2 2	Model3 3	Model 1 1	Model2 2	Model3 3
	Respiratory
		16.1	0.96 (0.85-1.09) 0.96 (0.85-1.09) 0.96 (0.84-1.09) 1.04 (0.95-1.15) 1.04 (0.94-1.15) 1.04 (0.94-1.16)
	disease
	Atopy	57.8	0.97 (0.85-1.11) 0.98 (0.86-1.12) 0.98 (0.85-1.12) 1.06 (0.97-1.15) 1.03 (0.95-1.13) 1.03 (0.95-1.13)
	D.farinae	29.0	1.01 (0.88-1.16) 1.02 (0.89-1.18) 1.02 (0.89-1.17) 1.04 (0.93-1.17) 1.03 (0.91-1.16) 1.03 (0.92-1.15)
	Cat	18.1	1.07 (0.91-1.27) 1.08 (0.91-1.27) 1.07 (0.91-1.27) 1.09 (0.98-1.22) 1.04 (0.92-1.16) 1.03 (0.92-1.16)
	German
		32.3	0.89 (0.79-1.01) 0.89 (0.79-1.01) 0.89 (0.78-1.01) 1.05 (0.95-1.15) 1.04 (0.94-1.15) 1.05 (0.95-1.16)
	cockroach
	Short ragweed	29.2	1.04 (0.91-1.19) 1.05 (0.92-1.21) 1.05 (0.91-1.20) 1.15 (1.01-1.31) 1.14 (1.01-1.29) 1.15 (1.02-1.30)
	Bermuda grass	20.8	0.96 (0.83-1.10) 0.95 (0.82-1.10) 0.94 (0.81-1.09) 1.18 (1.06-1.32) 1.14 (1.01-1.29) 1.15 (1.02-1.30)

grass allergen sensitisation in adults, which is independent of a wide-range of potential confounding factors and of vitamin D.

Supplementary information is available at The European Journal of Clinical Nutrition's website.

Prev: Prevalence


Dr Ute Grasshoff
email: ute.grasshoff@med.uni-tuebingen.de

Michael Bonin

Ina Goehring

Arif Ekici

Andreas Dufke

Kirsten Cremer

Nicholas Wagner

Eva Rossier

Anna Jauch

Michael Walter

Claudia Bauer

Peter Bauer

Karl Horber

Stefanie Beck-Woedl

Dagmar Wieczorek

De novo MECP2 duplication in two females with random X-inactivation and moderate mental retardation

Keywords: Xq28 duplication, MECP2, X-inactivation, Mental Retardation in Females

Xq28 duplications including MECP2 are a well known cause of severe mental retardation in males with seizures, muscular hypotonia, progressive spasticity, poor speech and recurrent infections that often lead to early death. Female carriers usually show a normal intellectual performance due to skewed X-inactivation. We report on two female patients with a de novo MECP2 duplication associated with moderate mental retardation. In both patients, the de novo duplication occurred on the paternal allele, and both patients show a random X-inactivation, which can be assumed as the triggering factor for the phenotype. Furthermore, we describe the phenotype, which might be restricted to unspecific mild to moderate mental retardation with neurological features in early adulthood.

Introduction

The X-linked Rett syndrome (OMIM #312750) has been known for a long time as a mental retardation syndrome, with its first clinical description in 1966/1977, and the identification of its underlying cause with loss-of-function mutations of the MECP2 gene in 1999 1,2,3 . The Rett syndrome is almost always sporadic due to de novo mutations and is seen almost exclusively in girls. Typical findings of classic Rett syndrome are a regression of motor skills, communicative abilities and especially a loss of purposeful hand movements, accompanied by secondary microcephaly after a period of (near to) normal development. The few affected males with deleterious MECP2 mutations usually present with clinical features consisting of fatal seizures and severe mental retardation including microcephaly that often leads to death during the first years. The genetically related Lubs syndrome (OMIM #300260) was first described in 1999, and etiologically resolved in 2005 as the MECP2 duplication syndrome 4,5,6 . Xq28 duplications of different sizes spanning the MECP2 gene have been identified to date as a common cause of mental retardation in males. In familial cases with X-linked pedigrees, the asymptomatic female carriers show a significant skewing of Xinactivation 7,8,[START_REF] Vandewalle | Dosage-dependent severity of the phenotype in patients with mental retardation due to a recurrent copy-number gain at Xq28 mediated by an unusual recombination[END_REF] . Besides mental retardation, the main features of the affected boys are muscular hypotonia, progressive spasticity, seizures, poor speech and recurrent infections. The reported duplications vary from 0.1 to 2.6 Mb with the minimal critical region containing the IRAK and MECP2 genes 7 . In familial cases, carrier females might suffer from endocrinological abnormalities, autoimmune diseases as well as psychiatric disorders [START_REF] Ramocki | Autism and other neuropsychiatric symptoms are prevalent in individuals with MeCP2 duplication syndrome[END_REF] . A phenotype with mental retardation has been reported only two times [START_REF] Reardon | Progressive cerebellar degenerative changes in the severe mental retardation syndrome caused by duplication of MECP2 and adjacent loci on Xq28[END_REF][START_REF] Makrythanasis | De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features[END_REF] .

We report on two unrelated female patients with a de novo MECP2 duplication and a random X-inactivation revealing a moderate but unspecific mental retardation in childhood and the development of neurological features in the second decade of life.

Materials and Methods

Patients

Both patients were examined for diagnostic evaluation because of mental retardation.

Karyotype analysis was performed for each patient on metaphase chromosomes from cultured blood lymphocytes using Giemsa banding at 550 bands resolution with normal results. The parents of patient 1 also showed normal karyotypes. Informed consent for the investigation was obtained from both families.

SNP Microarray

17.11.2010

Genomic DNA was isolated from peripheral blood leucocytes using routine procedures.

In both samples, we used the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA) with an average distance of 1.3 kb between neighboring probes. Samples were processed according to the manufacturer's instructions. Genotypes were called with Affymetrix Genotyping Console Software v3(GTC) using the Birdseed algorithm with the default calling threshold of 0.5 and a prior size of 10000 bases in a simultaneous analysis of the patient-parents trio.

Interpretation was based on Human Genome Build 36.

Multiplex Ligation-Dependent Probe Amplification (MLPA)

In patient 2, MECP2 duplication was confirmed by MLPA with the test kit P015D2 Human Probe Mix by MRC Holland, Amsterdam, The Netherlands (www.mlpa.com) according to the manufacturer's instructions. MLPA profiles were obtained using normal and pathological DNA samples as controls.

Quantitative PCR (qPCR)

In patient 2, the relative copy number of selected genomic regions was determined by quantitative PCR as described previously (data not shown).

X-Inactivation (XCI)

The highly polymorphic (CAG)n motif within the human androgen receptor gene (HUMARA) was used to distinguish and compare the methylation activity of the X chromosomes in both patients as described previously [START_REF] Allen | Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation[END_REF] .

Fluorescence in situ hybridization (FISH)

To investigate the localization of the duplicated segment, FISH analyses were performed on metaphase spreads and interphase nuclei of both patients and the parents of patient 2. Using standard two-color FISH protocols, a BAC clone from the duplicated region Xq28 (RP11-218L14, kindly provided by Joris Vermeesch, Center for Human Genetics, Leuven, Belgium) was hybridized together with a whole or partial chromosome painting probe for chromosome X (wcpX or pcpXq) [START_REF] Lichter | Chromosome analysis by non-isotopic in situ hybridization[END_REF] . Microscopic evaluation of 20 metaphase spreads and 100 nuclei from each patient and the parents of patient 2 was performed using a DM RXA epifluorescence microscope (Leica Microsystems, Bensheim, Germany) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ, USA).

Microsatellite analysis

The determination of the parental origin of the duplication was performed by microsatellite analysis, and the determination of the signal intensities for specific probes was performed on the 6.0 array. Microsatellite analysis was performed with four dinucleoteid sequences within the region of interest. Motifs were selected using the UCSC genome browser and repeat-spanning primers were designed with a 5'-fluorescence tag for the forward primer. PCR amplicons for trios were analyzed using capillary electrophoresis (Beckman Coulter CEQ8000) and amplicon lengths were determined.

Besides genotyping, we measured peak heights for all duplication carriers compared to normal controls in order to identify duplicated and non-duplicated alleles as described earlier [START_REF] Nguyen | Mosaic Trisomy 21/Monosomy 21 in a living female infant[END_REF] .

The SNP-microarray data were evaluated considering the genotype and the corresponding probe signals within the duplicated region, thus providing a correlation of a specific genotype and the according signal height. The origin of the duplication was identified by an allele-specific elevation of signal intensity in the patient in comparison to the parental genotypes.

Results

All position coordinates given below are based on Human Genome Build 36.

Case reports Patient 1

The girl was born to healthy, non-consanguineous parents of Turkish origin at 39 weeks gestation via forceps delivery with a birth weight of 2900 g (-1.0 SD), a length of 48 cm (-1.3 SD) and a head circumference of 34 cm (-0.3 SD). She had constipation from birth on, which had to be treated with a laxative. During her first years of life, she presented with a high frequency of respiratory infections. She learned to walk and spoke her first words at the age of 19 months. Her further psychomotor development was delayed. The parents noted autistic behavioural features in her daughter, for example she mostly plays alone.

We examined patient 1 for diagnostic evaluation at the age of 6 2 / 12 years and again at 7 6 / 12 years. At second examination, her body measurements were still normal: her height was 128 cm (0.3 SD), her weight was 28.5 kg (0.9 SD) and her OFC was 52.5 cm (0.7 SD). She visited a school for the mentally retarded, and was not able to write or count.

Her non-verbal IQ was 58. Her constipation improved, but sometimes a drug therapy was necessary. Adenoids were removed at the age of 5 years, but overall there was no history of recurrent or severe infectious diseases. She still displayed autistic behaviour, spending most of her time engaged in role-playing games with her dolls. Her psychomotor development was slow, but the parents did not report regression, and no epilepsy or stereotypies were reported. She had no facial dysmorphism, but facial similarity to her father, who also presented with synophrys. Bilateral simian creases as well as a sacral hemangioma were noted. No other abnormalities were present.

Patient 2

Patient 2 was born at 40 weeks of gestation by spontaneous vaginal delivery as the 3 rd of 4 children of non-consanguineous Turkish parents following an uncomplicated pregnancy. Birth measurements were normal with a weight of 3650 g (0.4 SD), a length of 52 cm (mean) and an OFC of 35.5 cm (0.5 SD). Her three brothers were healthy. She achieved normal motor milestones. Speech development was delayed and started at 3 years. At the age of 5 years, the parents reported a stagnation of development, but no regression. She mostly played alone and revealed some autistic features. After a first year at regular school, she changed to a special school. She never learnt to read or write.

At the age of 10 years, standardized non-verbal testing (SON) showed a developmental age of 6 years and a cognitive score of 55. On physical examination at the age of 10 years, her weight was 36.8 kg (BMI: 17.6, 50 th -70 th centile), her height was 145 cm (0.9 SD) and her OFC was 54 cm (1.2SD). She showed a primary enuresis until the age of 12 years, and furthermore a vesicouretral reflux II° was diagnosed. The EEG at that age was irregular with alpha-rhythm and abnormal beta waves. The brain MRI was normal.

At the age of 18 years a spastic hypertonia of the legs appeared, which led to gait disturbances. EEG at the age of 20 was physiological, MRI of the myelon revealed no pathological aspects. With 20 years, the patient's weight was 67 kg (BMI: 26.2, 90 th -97 th centile), her height was 160 cm (-1 SD) and her OFC was 54 cm (mean). Patient 2 did not present with any dysmorphic features. Her course of puberty was described as normal and no endocrinological problems were reported.

Detection of MECP2 duplications

De novo duplication of Xq28 material, including the MECP2 gene, was identified in both patients using array analyses. In patient 1, a 266 kb duplication (chrX:152 892 781-153 158 642, 266 kb, 71 array markers) containing 7 reference sequence genes was detected (RefSeq, according to the Genome browser of the University of California in Santa Cruz, UCSC). The proximal breakpoint was located within the TMEM187 gene, centromeric to the IRAK gene, and the telomeric breakpoint was located within the TEX28 gene (Fig. 1). In addition, patient 1 showed a second de novo duplication of In patient 2, we found a duplication of chromosome Xq28 material (chrX: 152 925 421-153 403 154, 478 kb, 215 array markers) containing 22 RefSeq-genes (UCSC), with the proximal breakpoint centromeric to the IRAK gene and the distal breakpoint telomeric to the FAM3A gene (Fig. 1). The nomenclature according to ISCN for patient 2 is arr Xq28(152,925,421-153,403,154)x3 dn. knowledge, only two mentally retarded females with an assured duplication have been reported [START_REF] Reardon | Progressive cerebellar degenerative changes in the severe mental retardation syndrome caused by duplication of MECP2 and adjacent loci on Xq28[END_REF][START_REF] Makrythanasis | De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features[END_REF] . This underrepresentation of girls may have different reasons. An investigation bias favouring affected boys to be investigated for MECP2 duplications may have occurred, while girls were simply not investigated for this disorder. The fact that all females carrying the duplication were described as intellectually normal could have added to that effect. There is a recent report that considers certain clinical findings in those "carrier mothers" as an affinity to endocrinological abnormalities (e.g. hypothyroidism, menstrual irregularity and diabetes), autoimmune diseases as well as psychiatric disorders (especially depression, anxiety and compulsion) [START_REF] Ramocki | Autism and other neuropsychiatric symptoms are prevalent in individuals with MeCP2 duplication syndrome[END_REF] . Nevertheless, the intellectual performance of all investigated females in that study was in the normal range (full scale IQ 81-123). During the last years, large cohorts of mentally retarded patients of both sexes were investigated with high resolution arrays for submicroscopic genomic imbalances through different array technologies. These should have shown MECP2 duplications in females, and yet no further affected girls have been reported [START_REF] Mcmullan | Molecular Karyotyping of patients with unexplained mental retardation by SNP Arrays: A multicenter Study[END_REF] .

This contradicts the investigation bias as an explanation for the rarity of those cases. We conclude that MECP2 duplications are a comparatively rare cause of mental retardation in females. This is confirmed by the identification of only 2 cases out of our cohort of 1000 unselected patients with mental retardation (incidence of 0,002).

In this study, we demonstrate two more female patients with an MECP2 duplication, and further delineate the aspect that, as Reardon et al first suggested, the random Xinactivation (XCI) may be causative for the phenotype [START_REF] Reardon | Progressive cerebellar degenerative changes in the severe mental retardation syndrome caused by duplication of MECP2 and adjacent loci on Xq28[END_REF] . Most of the published carrier women who were informative for the XCI test presented a highly skewed X-inactivation with the duplication-bearing X chromosome being preferentially inactivated [START_REF] Ramocki | Autism and other neuropsychiatric symptoms are prevalent in individuals with MeCP2 duplication syndrome[END_REF] . One can reasonably assume that the random X-inactivation is the crucial point leading to the mental retardation phenotype in these patients, while a highly skewed XCI with a preferential inactivation of the duplication carrying X-chromosome is a protective factor. The impact of the XCI in our patients is further emphasised by the fact that a translocation of the duplicated segment to an autosome was excluded by FISH investigation. Another remarkable aspect of our patients, besides their retardation and the random XCI, is that the MECP2 duplication occurred de novo in both cases. We propose that de novo occurrence of the MECP2 duplication, as in our cases, might have an effect on the XCI by preventing a protective "mutation induced" skewed Xinactivation by a yet unrecognized mechanism. This would mean that there could be a different trigger for the methylation process from an inherited MECP2 duplication than from one that occurred de novo. Furthermore, the analysis of the microsatellites and the signal intensities of the microarrays for our patients showed the de novo MECP2 duplication had been derived from the paternal X chromosome, which is a novelty in the literature.

For all but one female cases reported so far, a duplication on the maternal Xchromosome can be assumed due to an X-linked pedigree. In this case the duplication occurred de novo on an autosome (chromosome 10), thus completely escaping X inactivation [START_REF] Makrythanasis | De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features[END_REF] .

A postzygotic mosaic occurrence of the duplication is a feasible explanation for a random XCI, and mosaicism can not be excluded by any means. However, both patients showed no clinical signs of mosaicism, such as pigmental abnormalities of the skin or asymmetry, and the SNP-Arrays gave no evidence for a mosaic state of the MECP2 duplication. This contradicts a high-level postzygotic mosaicism as a cause for the random XCI in our cases.

It has to be mentioned that in patient 1 a second de novo duplication was detected spanning 112 kb of chromosome 2q37 material with the RefSeq-genes PASK, PPP1R7, ANO7, HDLBP (UCSC: NCBI36/hg18). Only weak PASK (OMIM *607505) expression is found in human thymus and testis. PPP1R7 (OMIM *602877) is expressed in all human tissues including the brain. ANO7 (OMIM *605096) is expressed only in the prostate. Haploinsufficiency of two of the genes within this region has been connected with neuropsychiatric disorders (HDLBP and PASK [START_REF] Felder | FARP2, HDLBP and PASK are downregulated in a patient with autism and 2q37.3 deletion syndrome[END_REF] ), but to our knowledge no phenotype has been demonstrated for a duplication of these genes. Thus, the significance of the 2q37 duplication remains unclear. An effect of the 2q37 duplication on the phenotype of the patient seems to be unlikely, but cannot be completely excluded. Regarding that the phenotypic effect of a MECP2 duplication is already well documented, and patient 1 presents no clinical signs of a neuropsychiatric disorder exceeding the phenotype of patient 2, we feel safe to assume that the phenotype is caused by the MECP2 duplication in both patients.

The typical symptoms of affected boys, such as seizures, poor speech development and recurrent severe infections, are not present in the females. The clinical signs in female patients with MECP2 duplication and random X-inactivation consist of unspecific mild to moderate mental retardation, with autistic features, recurrent infections in early childhood, constipation and late onset neurological features as variable symptoms.

Facial dysmorphism was absent in all females (Tab. 1) [START_REF] Clayton-Smith | Xq28 duplication presenting with intestinal and bladder dysfunction and a distinctive facial appearance[END_REF] . This unspecific phenotype does not allow the diagnosis of females with a MECP2 duplication and random Xinactivation on clinical grounds alone. Nevertheless the stagnation of development at the age of 5 years in patient 2 as well as the onset of spasticity in this patient at the age of 18 years seem remarkable, especially regarding the progressive spasticity in males. This underlines the necessity to consider late-onset neurological features as a possible aspect in affected females.

In conclusion, we report on two female patients with a de novo MECP2 duplication and a mental retardation. Both patients show a random X-inactivation, which can be postulated as the triggering factor for the phenotype. Furthermore, these are the first patients with a duplication derived from the paternal X chromosome and this report gives a description of the phenotype of affected females.

  2q37.3 material (chr2:241 714 171-241 825 938, 112 kb, 71 array markers), including the genes ANO7, HDLBP, PASK, PPP1R7. The genotype for patient 1 according to the International System for Human Cytogenetic Nomenclature 2009 (ISCN) is arr 2q37.3(241,714,171-241,825,938)x3 dn,Xq28(152,892,781-153,158,642)x3 dn.

1Figure 1 :

 1 Figure 1: Illustration of the genomic aberration of the MECP2 region with the 6.0

Figure 2 :

 2 Figure 2: Metaphase and interphase FISH results

Friez MJ, Jones JR, Clarkson K et al: Recurrent infections, hypotonia, and mental retardation caused by duplication of MECP2 and adjacent region in Xq28. Pediatrics.

Acknowledgements

This study would not have been possible without the invaluable assistance of the patients and their families. We wish to thank the cooperating physicians for referral of the patients to our departments, especially G. Dissemond, L. Kämmerling and V.

Horber. We also thank Bernhard Horsthemke, André Reis and Olaf Riess for continuous support as well as Juliane Hoyer and Markus Zweier for their essential help.

The Institutes for Human Genetics, Essen, Erlangen, Heidelberg and Tübingen, Germany, are members of the 'German Mental Retardation Network' (MRNET), which is funded by the German Federal Ministry of Education and Research (BMBF) as a part of the National Genome Research Network (NGFNplus/www.ngfn.de/englisch/15.htm, project reference numbers 01GS08164, 01GS0816, 01GS08168, 01GS08162).

Confirmation and delineation of MECP2 duplications

In patients 1 and 2, the duplication in Xq28 was confirmed by interphase FISH based on the presentation of three hybridization signals for the BAC clone RP11-218L14 (Fig. 2) A localization of the duplicated region on an autosome was excluded for both patients in metaphase spreads. In the parents of patient 2, FISH analyses for the Xq28 BAC clone revealed only normal signals on the X chromosomes. Furthermore, duplications of the genes IRAK, MECP2 and GDI1 were confirmed in patient 2 according to the genespecific probes from the MLPA kit, whereas a normal signal was obtained for the probe against the proximal flanking IRAK gene. In patient 2, the duplication of MECP2 was confirmed by quantitative PCR (data not shown).

X inactivation studies showed a random X-inactivation in both patients with a status of 61:39 and 71:29, respectively (Fig. 3). Microsatellite analysis revealed two alleles with different repeat lengths for the polymorphic marker in position chrX:153 050 087-153 050 120 (17xTG) in the father's and in the mother's DNA of patient 2. Semiquantitative analysis indicates a paternal origin of the MECP2 duplication in patient 2 (data not shown). This marker as well as all other tested markers were not informative for patient 1. The analysis of the SNP-microarray data regarding the correlation of genotype and signal height within the duplicated region indicates a paternal origin in both patients.

Discussion

There were a considerable number of reports on mentally retarded boys with MECP2duplications during the years following the first publication 6 . However, to best of our

Conflict of interest statement:

The authors declare no conflict of interest.

patient 2 presenting only one signal on the X chromosome and f) a normal signal in the interphase nuclei with the Xq28 BAC clone. g) Metaphase spread of the mother of patient 2 showing a specific hybridization signal on both X chromosomes and h) two normal signals in the interphase nuclei.


Jana Herholz

Alessandra Meloni

Mara Marongiu

Francesca Chiappe

Manila Deiana

Carmen Roche Herrero

Giuseppe Zampino

Hanan Hamamy

Yusra Zalloum

Per Erik Waaler

Giangiorgio Crisponi

Dr Laura Crisponi
email: laura.crisponi@inn.cnr.it

Frank Rutsch

Herholz et al. Phenotypic severity of CRLF1 associated disorders 1 Differential secretion of the mutated protein is a major component affecting phenotypic severity in CRLF1-associated disorders

Keywords: Crisponi syndrome, cold-induced sweating, hyperthermia, CRLF1

Crisponi syndrome (CS) and cold-induced sweating syndrome type 1 (CISS1) are disorders caused by mutations in CRLF1. The two syndromes share clinical characteristics such as dysmorphic features, muscle contractions, scoliosis and cold-induced sweating, with CS patients showing a severe clinical course in infancy involving hyperthermia, associated with death in most cases in the first years of life.

To evaluate a potential genotype/phenotype correlation and whether CS and CISS1 represent two allelic diseases or manifestations at different ages of the same disorder, we performed a detailed clinical analysis of 19 patients carrying mutations in CRLF1. We studied the functional significance of the mutations found in CRLF1, providing evidence that phenotypic severity of the two disorders mainly depends on altered kinetics of secretion of the mutated CRLF1 protein. Based on these findings we believe that the two syndromes, CS and CISS1, represent manifestations of the same disorder, with different degrees of severity. We suggest to rename the two genetic entities CS and CISS1 with the broader term of Sohar-Crisponi syndrome.

INTRODUCTION

Mutations in CRLF1 (cytokine receptor-like factor 1) account for both Crisponi syndrome (CS; MIM#601378) and cold-induced sweating syndrome type 1 (CISS1; MIM#272430).

Crisponi syndrome was initially described in 1996, in 17 patients from 12 different families in southern Sardinia. [START_REF] Crisponi | Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome?[END_REF] Further patients have been reported later. [START_REF] Dagoneau | Mutations in cytokine receptorlike factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes[END_REF][START_REF] Crisponi | Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1[END_REF][START_REF] Accorsi | Crisponi syndrome: report of a further patient[END_REF][START_REF] Nannenberg | Neonatal paroxysmal trismus and camptodactyly: the Crisponi syndrome[END_REF][START_REF] Marca | Central apneas in a case of Crisponi syndrome[END_REF][START_REF] Okur | Crisponi syndrome: a new case with additional features and new mutation in CRLF1[END_REF][START_REF] Thomas | Crisponi syndrome in an Indian patient: a rare differential diagnosis for neonatal tetanus[END_REF] The syndrome usually manifests at birth, when patients present with hyperthermia and abnormal paroxysmal contractions of the facial and oropharyngeal muscles, as well as feeding and respiratory difficulties often requiring the use of nasogastric feeding. Physical dysmorphisms such as a large face, broad nose and camptodactyly have been described in most of the patients. [START_REF] Crisponi | Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1[END_REF][START_REF] Accorsi | Crisponi syndrome: report of a further patient[END_REF][START_REF] Nannenberg | Neonatal paroxysmal trismus and camptodactyly: the Crisponi syndrome[END_REF][START_REF] Okur | Crisponi syndrome: a new case with additional features and new mutation in CRLF1[END_REF][START_REF] Thomas | Crisponi syndrome in an Indian patient: a rare differential diagnosis for neonatal tetanus[END_REF] Hyperthermia is frequently associated with death within the first months of life. Feeding difficulties and hyperthermia often resolve after infancy in the rare surviving patients, who then develop scoliosis and sometimes psychomotor retardation. In pre-adolescent patients, evidence of cold-induced sweating was reported. [START_REF] Crisponi | Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1[END_REF] Cold-induced sweating syndrome type 1 (CISS1) was first reported in two Israeli sisters in 1978. [START_REF] Sohar | Coldinduced profuse sweating on back and chest. A new genetic entity[END_REF] It involves paradoxical sweating at cold ambient temperatures on the upper part of the body, along with progressive scoliosis.

In both sisters, dysmorphic features including a high arched palate, nasal voice and joint contractures have been observed. Two Norwegian brothers and a Canadian woman with a similar phenotype have been described more recently. [START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF][START_REF] Knappskog | Cold-induced sweating syndrome is caused by mutations in the CRLF1 gene[END_REF] CS and CISS1 belong to a group of genetic disorders with similar phenotypes associated with mutations of genes in the ciliary neurotrophic factor receptor (CNTFR) pathway, which is known to be important for development and maintenance of the nervous system and muscles. [START_REF] Dechiara | Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth[END_REF] This group includes cold-induced sweating syndrome type 2 (CISS2; MIM#610313), caused by mutations in CLCF1, [START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF][START_REF] Rousseau | Inactivation of cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factor receptor, leads to cold-induced sweating syndrome in a patient[END_REF] and Stüve-Wiedemann syndrome (SWS; MIM#601559), caused by mutations in LIFR. [START_REF] Dagoneau | Null leukemia inhibitory factor receptor (LIFR) mutations in Stuve-Wiedemann/Schwartz-Jampel type 2 syndrome[END_REF] Mutations in CLCF1 lead to a phenotype similar to mutations in CLRF1, with cold-induced sweating, scoliosis and cubitus valgus. [START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF][START_REF] Rousseau | Inactivation of cardiotrophin-like cytokine, a second ligand for ciliary neurotrophic factor receptor, leads to cold-induced sweating syndrome in a patient[END_REF] Clinical features of SWS also include camptodactyly, feeding difficulties, scoliosis and temperature instability, also present in the other syndromes, but the characteristic bowing of the long bones is not present in CS, CISS1 and CISS2. [START_REF] Dagoneau | Null leukemia inhibitory factor receptor (LIFR) mutations in Stuve-Wiedemann/Schwartz-Jampel type 2 syndrome[END_REF][START_REF] Al-Gazali | Stuve-Wiedemann syndrome in children surviving infancy: clinical and radiological features[END_REF][START_REF] Chabrol | Stuve-Wiedemann syndrome and defects of the mitochondrial respiratory chain[END_REF][START_REF] Cormier-Daire | Clinical homogeneity of the Stuve-Wiedemann syndrome and overlap with the Schwartz-Jampel syndrome type 2[END_REF][START_REF] Kozlowski | Stuve-Wiedemann dysplasia in a 3 1/2year-old boy[END_REF][START_REF] Wiedemann | Stuve-Wiedemann syndrome: update and historical footnote[END_REF] Clinical similarities between Crisponi syndrome and coldinduced sweating syndrome type 1, along with the involvement of the same gene (CRLF1), led to the question whether these two syndromes represent two allelic diseases or, in fact, manifestations of the same disorder, reported at different ages of affected patients. [START_REF] Crisponi | Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome?[END_REF][START_REF] Dagoneau | Mutations in cytokine receptorlike factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes[END_REF] In an effort to delineate the specific clinical features attributed to CS and CISS1, we investigated the clinical history, physical characteristics and experimental data of 19 patients with mutations in CRLF1, 14 of them classified as CS (9 reported and 5 unreported cases) and 5 as CISS1 (all reported), [START_REF] Sohar | Coldinduced profuse sweating on back and chest. A new genetic entity[END_REF][START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF][START_REF] Knappskog | Cold-induced sweating syndrome is caused by mutations in the CRLF1 gene[END_REF] by means of a standardized questionnaire.

Because a genotype/phenotype correlation has not been found in former studies [START_REF] Dagoneau | Mutations in cytokine receptorlike factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes[END_REF][START_REF] Crisponi | Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1[END_REF] , we attempted to carry out a detailed analysis of the clinical phenotype and compare it with the genetic and functional data from the investigation of the CRLF1 gene and protein for all the probands analyzed.

We found that phenotypic severity of CRLF1-associated disorders depends on altered kinetics in the secretion of the mutated CRLF1 protein, suggesting that CS and CISS1 are manifestations of the same disease.

MATERIALS AND METHODS

Patients and clinical data

Inclusion in the study was based on the diagnosis of CS or CISS1 with proven mutations on both alleles of the CRLF1 gene. Diagnosis of either syndrome was established based on compilation of symptoms: 14 patients diagnosed with CS and 5 patients diagnosed with CISS1 were included in the study. The 14 patients classified as CS (9 females, 5 males) originated from 11 families from Italy, Turkey, Spain and Libya (Table 1). The 5 patients (3 females, 2 males) classified as CISS1 originated from 3 families from Norway, Canada and Israel and showed a heterogenic phenotype (Table 1). The study protocol was approved by the Münster University Hospital Ethical Committee in Germany and all subjects involved in this study gave informed written consent.

Clinical data and patient history were gathered through a standardized questionnaire, which was sent to the responsible clinicians and through review of the clinical data presented in the literature. Data on facial and skull height, as well as nasal and philtrum length were obtained from a subset of probands, measurements were plotted according to the Handbook of Normal Physical Measurements, 1989. [START_REF] Hall | Handbook of Normal Physical Measurements[END_REF]

DNA extraction and mutation analysis

Genomic DNA was isolated from peripheral blood by a salting-out protocol.

All the nine coding exons and surrounding intronic regions of CRLF1 (GenBank accession number NM_004750U) were amplified by PCR using specific primers [START_REF] Crisponi | Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1[END_REF] , and the amplicons subsequently analyzed by direct sequencing (ABI3130XL, Applied Biosystems).

DNA cloning

The clone pCMV6-XL5CRLF1 (Origene cat.TC126412 NM_004750.2) was used to subclone the cDNA encoding the human CRLF1 in the PEF5HA vector (provided by Doctor Nunzio Bottini, La Jolla Institute for Allergy and Immunology, CA) using EcoRI and XbaI restriction enzymes. The clone pCMV6-XL5CLCF1 (Origene cat.TC122789 NM_013246.2) was used to be co-transfected with PEF5HA-CRLF1, wild type and mutated, in COS-7 cells.

CRLF1 Site-directed Mutagenesis

The PEF5HA-CRLF1 clone was subjected to site-directed mutagenesis using the QuickChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primer sequences are available on request.

Cell culture

COS-7 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-Invitrogen CA, USA) supplemented with 10% fetal bovine serum, 100U/ml penicillin, 100µg/ml streptomycin and 2mM glutamine (Gibco Invitrogen). IMR32 neuroblastoma cell lines (DSMZ ACC165) were grown in RPMI 1640 (Gibco Invitrogen) supplemented with 10% fetal bovine serum, 100U/ml penicillin, 100µg/ml streptomycin and 2mM glutamine, 1% essential amino acids, 1% sodium pyruvate (Gibco Invitrogen).

DNA transfection and cell lysis

COS-7 cells were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions and after 24 h, 36 h, 48 h and 72 h were lysed with Lysis Buffer, (150mM NaCl, 50mM TrisHCl pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 4mM pefablock ) containing Complete Protease Inhibitor Cocktail (Roche).

Western blotting

Protein concentrations were determined by spectrophotometer, using Bio-Rad Protein Assay (BIO-RAD). Then 50µg of lysates and 30µl of supernatants were submitted to SDS-PAGE with 9% polyacrylamide gels and transferred to PVDF membranes Hybond-P (Amersham) by electroblotting. The membranes were blocked in PBS with 1% Tween 20 (PBS-T) and with 5% of milk for 1 h at room temperature and then incubated with the anti-CRLF1 (mouse monoclonal Abcam ab26125) or anti-CLCF1 (rabbit polyclonal Abcam ab56500) antibodies overnight at 4°C, with a dilution 1:500 and 1:1000 respectively. The membranes were washed 3 times for 10 minutes in PBS-T and incubated in the appropriate secondary antibody for 1 h at room temperature. After washing, the blots were developed by chemiluminescence using ECL plus reagent (Amersham Biosciences), according to the manufacturer's instructions. To perform densitometric analysis to evaluate CRLF1 secretion level, we used Image J software (http://rsbweb.nih.gov/ij/). We defined a rectangular shaped area surrounding each band, and keeping this area constant we measured the corresponding mean of intensities. We then subtracted the values of background area and calculated the percentage rate related to the mean of the intensities between supernatants and lysates. The level of wild type CRLF1 secretion in the supernatants was considered as 100%. The result is representative of at least three independent experiments.

Tyrosine Phosphorylation Analysis

After a 24 h serum starvation, IMR32 cells were stimulated for 10 min with the medium recovered from COS-7 cells co-transfected with wilde type or mutant CRLF1 and CLCF1, with serum free medium as negative control and with 100pM human recombinant Ciliary Neurotrophic Factor, expressed in E. coli (C3710 #086k1965-SIGMA) in serum free medium as positive control. The cells were then lysed with Lysis Buffer, (150mM NaCl, 50mM TrisHCl pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 4mM pefablock) containing Complete Protease Inhibitor Cocktail (Roche) and HaltPhosphataseInhibitor Cocktail (PIERCE). Then 50 µg of lysates were subjected to SDS PAGE analysis with 9% polyacrylamide gel and transferred to PVDF membrane Hybond-P (Amersham) by electroblotting. The filter was immunoblotted with an antibody specific for all of the phospho-STAT3 isoforms (Cell Signaling--#9131s dil. 1:1000). The membrane was then stripped in 0.1N NaOH, and reblotted with an antibody specific for STAT3 (Cell Signaling-#9132 dil. 1:1000).

RESULTS

Clinical features of patients classified with Crisponi syndrome and CISS1 syndrome

In our study, patients classified as CS, presented with typical symptoms in infancy, inlcuding severe hyperthermia, contraction of the facial muscles, feeding difficulties with hypersalivation, which often required nasogastric feeding, and dysmorphic features, e.g., camptodactyly, foot anomalies, high arched palate and chubby cheaks (Figure 1, Table 1 and Supplementary Table 1). The severe manifestations led to death in most of the historically described cases.

However, these classical symptoms of CS could be observed also in some patients with the established diagnosis of CISS1 (Figure 2, Table 1 and Supplementary Table 1). The Norwegian brothers described initially by Knappskog et al. [START_REF] Knappskog | Cold-induced sweating syndrome is caused by mutations in the CRLF1 gene[END_REF] feature a phenotype that shows considerable resemblance to Crisponi patients. Feeding difficulties and the need of nasogastric tube during infancy were described in the paper, while in one of the two brothers episodes of hyperthermia in the first period of life was reported only later during a reconsideration of the cases for this study. Also the eldest brother showed contractions of the facial muscles in infancy (Figure 2b). Feeding difficulties in infancy were also described in the Canadian woman. [START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF] Physical measurements performed in 14 patients with CRLF1 mutations did not reveal a significant difference of the facial attributes in comparison to the general population (data not shown). These observations show, in contrast to previous studies [START_REF] Crisponi | Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome?[END_REF] , that the attribute of a long and large face, long philtrum and broad nose cannot be included in the obligatory features of patients with CRLF1 mutations.

All surviving patients older than 2 years of our study cohort developed scoliosis and those older than 6 years developed paradoxical sweating in response to low ambient temperatures. Some patients had been treated with Clonidine to reduce the sweating. Patients 15 and 18 showed long lasting positive effects, whereas patient 16 showed no significant improvement and preferred physical training as a method against the sweating. 10 Patients 17 and 19 showed positive but decreasing effects over time. [START_REF] Hahn | Coldinduced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity[END_REF] Also, patients 7 and 8, originally classified as CS patients, responded to alpha-adrenergic agents with regard to paradoxical sweating. [START_REF] Herholz | Successful treatment of cold-induced sweating in Crisponi syndrome and its possible mechanism of action[END_REF] Diagnostic studies such as electroencephalogram, cranial computed tomography, muscle biopsy and nerve conduction velocity were performed in several cases mostly showing normal results (Supplementary Table 2).

Most of the initial symptoms resolved after the first 1 or 2 years of life. Some patients still showed hypersalivation or hyperthermia in adulthood (Table 1 and Supplementary Table 1).

In our cohort, there is a subgroup of patients with a mild phenotype in early childhood presenting no hyperthermia or feeding difficulties after birth (patients 17-19, Table 1). In pre-adolescence, these patients developed scoliosis and cold-induced sweating (Table 1).

CRLF1 mutational analysis

The CRLF1 gene (GenBank accession number NM_004750) is localized on human chromosome 19p13.11 and is composed of 9 coding exons (Figure 3a). The encoded protein (GenBank accession number NP_004741) comprises 422 amino acids. By direct sequencing of all the 9 coding exons as well as exonic/intronic boundaries of the CRLF1 gene, different types of mutations have been identified in all the patients included in this study (Figure 3, Table 1, Table 2). Six patients (Table 1) were compound heterozygotes for different mutations, the remaining were homozygotes for one type of mutation. No obvious correlation between the type/localization of mutation and the severity of the clinical course was detected (Table 1).

CRLF1 functional analysis

CRLF1 protein is known to be a member of the ciliary neurotrophic factor receptor (CNTFR) pathway by forming a complex with CLCF1. The stable secreted complex of CRLF1 and CLCF1 forms a ligand for CNTFRα, which, along with gp130 and LIFR, compose the CNTF-receptor complex. [START_REF] Elson | CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex[END_REF] CRLF1/CLCF1 binding to CNTFRα leads to dimerization of gp130/LIFR, which then induces downstream signalling events, including activation of the JAK1/STAT3 pathway. [START_REF] Elson | CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex[END_REF] To investigate the biological significance and to assess the secretion capacities of mutated CRLF1, mutations found in the 19 patients (Table 1) were introduced into the wild-type CRLF1 cDNA and co-transfected with wild type CLCF1 cDNA in COS-7 cells. Nontransfected COS-7 cells were used as control.

After a 48 h culture period, strong expression of wild type CRLF1 and mutants was detected in cell lysates, whereas only wild type CRLF1, mutants K368X (M4), W284C (M10), R81H (M12) and L374R (M13) were strongly detected in the supernatant of transfected cells. CRLF1 mutants W76G (M1), T226NfsX104 (M2), P238RfsX6 (M3), and V282GfsX47 (M11) were only partially secreted into the medium, while Y75D (M5), P239AfsX92 (M6), D181GfsX5 (M7), [N113I;L114P] (M8) and Q180X 12 (M9) were not secreted or detected at all (Supplementary Figure 1a, Table 2).

Previous studies reported that CLCF1 must interact with CRLF1 in the 'producer' cell to be released. [START_REF] Plun-Favreau | The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine[END_REF] To evaluate the ability of mutated CRLF1 to stimulate CLCF1 secretion, we co-transfected wild type and mutant forms of CRLF1 together with wild type CLCF1. After a 48 h culture period, we found CLCF1 in culture supernatants when transfected alone, as well as when co-transfected with the wild type and mutants CRLF1, although with different signal intensities (Supplementary Figure 1b). To better understand these findings, we performed a time course experiment with different culture periods (24 h, 36 h, 48 h and 72 h) for both wild type CRLF1 and CLCF1 transfected alone or together in COS-7 cells. Non-transfected COS-7 cells were used as control (Supplementary Figure 2a,b). The results indicate that in the cell lysate, CLCF1 alone is always expressed from 24 h to 72 h, whereas in the supernatants it is detected at 48 h and 72 h. When cotransfected with CRLF1, CLCF1 was only faintly detected in the supernatants at 24 h, while it was strongly detected at 36 h, 48 h and 72 h. Thus, it seems that co-expression with CRLF1 is not necessary for CLCF1 secretion, but is required to accelerate the kinetics of secretion into the extracellular medium.

Analysis of induction of STAT3 tyrosine phosphorylation by the CRLF1/CLCF1 complex

It has been previously reported that the secreted heterodimer CRLF1/CLCF1 activates the CNTF receptor with the induction of downstream signaling events including activation of the JAK1/STAT3 pathway. [START_REF] Elson | CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex[END_REF][START_REF] Perret | Two different contact sites are recruited by cardiotrophin-like cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines[END_REF] We therefore assessed the ability of CRLF1 mutants to induce STAT3 signal transduction in the IMR32 neuroblastoma cell line, expressing the CNTF receptor complex (gp130, LIFRβ and CNTFRα). [START_REF] Elson | CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex[END_REF] The supernatants of COS-7 cells transfected with wild type CLCF1 or wild type CRLF1 alone, and with wild type CLCF1 and wild type or mutant forms of CRLF1, were added to an IMR32 cell line after 24 h serum starvation. CNTF was used as a control at a concentration of 100pM. After a 10 min incubation, cells were lysed and analyzed by western blotting with anti STAT3 and anti phosphoSTAT3 antibodies. We found that CLCF1 alone can induce STAT3 phosphorylation in an IMR32 cell line, but CRLF1 alone cannot (data not shown). However, the complexes of wild type CLCF1 and wild type or mutated forms of CRLF1 were able to elicit STAT3 signaling.

DISCUSSION

Clinical and diagnostic considerations

In this study, we collected and analyzed in detail clinical data from 19 patients diagnosed with CS or CISS1. Patients with mutations in the CRLF1 gene until now have been classified as CS or CISS1 based on the severity of their clinical symptoms and the age at diagnosis. However, a detailed analysis of the clinical phenotypes in this study showed that there is a clinical course of varying severity in both groups of patients. The Israeli sisters described in 1978 by Sohar et al. [START_REF] Sohar | Coldinduced profuse sweating on back and chest. A new genetic entity[END_REF] , exhibited a less severe phenotype and have formed the entity of CISS1. Further patients like the Norwegian brothers, have been put in this group. However, our retrospective study showed that they presented symptoms in early childhood including feeding difficulties and hyperthermia, which have been reported only for CS. On the other hand, CS patients who have survived the critical first months of life, develop typical symptoms of CISS1 as they grew older. This makes it very likely that the initial CS and CISS1 patients reported in the paper by Crisponi, 1996 [START_REF] Crisponi | Autosomal recessive disorder with muscle contractions resembling neonatal tetanus, characteristic face, camptodactyly, hyperthermia, and sudden death: a new syndrome?[END_REF] and by Sohar et al., 1978 9 have been considered different entities only because the patients were reported at different ages. This is in line with a most recent study reporting a CISS1 patient with neonatal features of Crisponi syndrome supporting the notion that CISS1 and CS may be a single clinical entity. [START_REF] Yamazaki | Cold-induced sweating syndrome with neonatal features of Crisponi syndrome: longitudinal observation of a patient homozygous for a CRLF1 mutation[END_REF] The overlapping phenotypes (Table 1 and Supplementary Table 1), make it difficult today to classify new patients. In order to elucidate a potential genotype/phenotype correlation, we performed a detailed analysis in relation to the mutations found in the CRLF1 gene.

Classification according to the type of mutation

The different types of mutations were analyzed for all patients included into this study. Missense, nonsense, insertions, and frameshift mutations were found. Strikingly, patients who have suffered a severe clinical course with hyperthermia and feeding difficulties during childhood display all different kinds of mutations. The deceased patients do not differ from other patients with regard to the type of mutation. Also, the same class of mutation is responsible for different clinical courses, as exemplified by patient 5, who displays an extremely severe phenotype until now, and patients 18 and 19, who are known to have shown a very mild clinical course. As missense mutations were detected in all 3 of them, a strict correlation between the type of mutation and the phenotype seems very unlikely. The variety of mutations leading to the same phenotype and the variety of phenotypes caused by the same type of mutation lead to the conclusion that there is no strict correlation between phenotype and genotype based on the type/localization of CRLF1 mutation.

Classification according to the biochemical characteristics of CRLF1

We also performed a functional analysis of the mutated forms, showing that different mutations can be grouped according to their biochemical quality.

In the first group, absent or weak secretion (less than 40% compared to wild type) of mutant CRLF1 occurred; in the second group, the mutated protein was strongly secreted (more than 40% compared to wild type, Table 1, Table 2).

Patients 1-15 are carriers of mutations belonging to the first group. The clinical phenotype was severe for all patients in this group (Table 1). All these patients showed hyperthermia, contraction of the facial muscles, hypersalivation and feeding difficulties in early childhood and required nasogastric feeding. All of these patients older than two years developed a scoliosis, those older than six years also cold-induced sweating.

Patients 18 and 19 carry mutations associated with a strong CRLF1 secretion. These patients described by Sohar et al. [START_REF] Sohar | Coldinduced profuse sweating on back and chest. A new genetic entity[END_REF] exhibit a mild phenotype without severe manifestations in infancy such as hyperthermia, but with cold-induced sweating, scoliosis and some dysmorphic features. Based on these observations, we conclude that phenotypic severity of CRLF1 associated disorders mainly depends on altered secretion of the CRLF1 protein. Accordingly, patient 17, who is a compound heterozygous carrier of two mutations, associated with absent and strong CRLF1 secretion respectively, presents an "intermediate" phenotype with feeding difficulties in infancy, scoliosis and cold-induced sweating in adolescence. However, it is likely that in some cases, the severity of the phenotype could also be affected by other factors. For example, in patient 16, genetic factors may account for the milder phenotype compared to his brother (patient 15), who carries the same mutation.

Our analysis supports the conclusion that absent or weak CRLF1 secretion is associated with a severe clinical phenotype, whereas strong secretion is associated with a milder clinical phenotype. Based on the fact that our retrospective clinical study was limited either by the small number of patients in each CRLF1-secretion -based group and by clinical information not exhaustive, we suggest that defective secretion is a major component affecting phenotypic severity of CRLF1-associated disorders.

Analysis of the CRLF1/CLCF1 pathway

CRLF1 protein is a member of the ciliary neurotrophic factor -receptor (CNTFR) pathway by forming a complex with CLCF1, which binds to the CNTF-receptor. In contrast to the observations previously reported, where CLCF1 can be actively secreted from cells by forming a complex with CRLF1 [START_REF] Elson | CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex[END_REF][START_REF] Perret | Two different contact sites are recruited by cardiotrophin-like cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines[END_REF] , we found that CLCF1 was detected in culture supernatants when transfected alone, as well as when co-transfected with the wild type and mutant CRLF1, although with different signal intensities (Supplementary Figure 1b).

It is noteworthy that an alteration in the kinetics of CLCF1 secretion is associated with the co-expression with some mutant forms of CRLF1, which may act to inhibit its secretion into the medium. Further studies aiming to identify the CRLF1 interaction sites with CLCF1 will help to better understand the heterodimer complex formation as well as the role of the CRLF1 mutations analyzed.

It has been previously reported that the secreted heterodimer CRLF1/CLCF1 activates the CNTF receptor with the induction of downstream signaling events including activation of the JAK1/STAT3 pathway. Dagoneau et al. [START_REF] Dagoneau | Mutations in cytokine receptorlike factor 1 (CRLF1) account for both Crisponi and cold-induced sweating syndromes[END_REF] , already reported that STAT3 phosphorylation was normally triggered in fibroblasts of patients with Crisponi syndrome by LIF. We found the same result in our experiments (data not shown).

Although CRLF1 is not needed to activate the CNTF receptor, early secretion of the full CRLF1 protein seems to be necessary for adequate CLCF1 secretion and proper activation of the CNTF receptor pathway.

However, the results from the cell culture experiments cannot be directly transferred to the mechanisms in humans, so that it cannot be fully estimated how a delay of 48 hours in a cell culture would affect function in organ systems. Alternatively, another receptor may be involved, and the alteration of its pathway may cause the phenotype. This hypothesis gains some evidence from the recent detection of a previously unknown receptor for CRLF1. [START_REF] Crabé | The IL-27 p28 Subunit Binds Cytokine-Like Factor 1 to Form a Cytokine Regulating NK and T Cell Activities Requiring IL-6R for Signaling[END_REF] Our findings lead to the old matter about "lumpers" and "splitters" [START_REF] Mckusick | On lumpers and splitters, or the nosology of genetic disease[END_REF] and thus to the critical question how to classify and name these genetic entities.

In the presence of bowing of the long bones, the pediatric manifestation would lead to Stüve Wiedemann syndrome. [START_REF] Jung | Stüve-Wiedemann syndrome: longterm follow-up and genetic heterogeneity[END_REF] In the absence of bowing of the long bones, based on our findings, we believe that the two syndromes, CS and CISS1, represent manifestations of one disorder, with different degrees of severity. The rare cases of CISS1 most likely correspond to CS survivors. Genetic heterogeneity could be assumed with CISS2, which shows the same phenotype as CISS1, but is due to mutations in CLCF1.

However, since there has been only one case described in literature, this assumption might be too early at the moment.

Use of one facet of the syndrome, such as cold-induced sweating as a name for the whole, has some limitations since this feature is not the only one, may occur as an isolated anomaly or may be a feature of other syndromes. Therefore, we suggest to rename the two genetic entities CS and CISS1 with the broader term of Sohar-Crisponi syndrome. Furthermore, we conclude that defective secretion of the CRLF1 protein is a major factor contributing to the phenotypic severity of this syndrome.

Table 1. Patients included in the study with type of mutation, biochemical features of the mutated protein and main clinical features

Abbreviations: Mis, missense; Nons, nonsense; Fs, frameshift; n.a., not available. *CRLF1 secretion level of mutant forms compared to wild type.

Figure 1 .

 1 Figure 1. Clinical characteristics of patients classified as Crisponi

Figure 2 .

 2 Figure 2. Clinical characteristics of two Norwegian patients classified as

Figure 3 .

 3 Figure 3. Structure of the human CRLF1 gene and protein. a: Relative

ACKNOWLEDGEMENTS

We are grateful to all the patients and their family members, who with their enthusiastic, continuous and generous participation made this study possible. This work was supported by the Associazione Sindrome di Crisponi e Malattie Rare ONLUS (grant to L.C.) by the Regione Autonoma della Sardegna (grant to L.C.) and the by Interdisciplinary Center for Clinical Research, Münster, Germany (grant to F.R.). We thank Prof.

Antonio Cao, Prof. Francesco Cucca and Dr. David Schlessinger for the critical reading of the manuscript.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

SUPPLEMENTARY INFORMATION Supplementary information is available at EJHG's website

Hyperthermia