Hair cortisol- a novel method to explore Prader-Willi syndrome.

Background: Prader-Willi syndrome (PWS) is a multisymptomatic, rare, genetic, neurodevelopmental disorder in adults mainly characterized by hyperphagia, cognitive dysfunction, behavioral problems and risk of morbid obesity. Although endocrine insuciencies are common, hypocortisolism is rare and knowledge on long-term cortisol concentrations is lacking. The aim of this study was to evaluate long-term cortisol levels in PWS by measurements of hair cortisol. Methods: Twenty-nine adults with PWS, 15 men and 14 women, median age 29 years, median BMI 27 kg/m 2 , were included. Scalp hair samples were analyzed for cortisol content using liquid-chromatography tandem-mass spectrometry. In addition, a questionnaire on auxology, medication and stress were included. For comparison, 105 age- and sex-matched participants from the population-based Lifelines Cohort study were included as controls. The mean hair cortisol between the groups were compared and associations between BMI and stress were assessed by a generalized linear regression model. Results: In the PWS group large variations in hair cortisol was seen. Mean hair cortisol was 12.8±25.4 pg/mg compared to 3.8±7.3 pg/mg in controls (p=0.001). The linear regression model similarly showed higher cortisol levels in patients with PWS, which remained consistent after adjusting for BMI and stress (p=0.023). Furthermore, hair cortisol increased with BMI (p=0.012) and reported stress (p=0.014). Conclusion: Long-term cortisol concentrations were higher in patients with PWS compared to controls and increased with BMI and stress, suggesting an adequate cortisol response to chronic stress. Hair cortisol demonstrate promising applications in the context of PWS treatment and disease management.


Background:
Prader-Willi syndrome (PWS, OMIM 176270) is a rare, complex neurodevelopmental disorder with a reported incidence of approximately 1 in 15,000-30,000 newborns (1). PWS is caused by a lack of expression of paternally inherited genes in the q11-q13 region of chromosome 15, most commonly caused by a paternal deletion or a maternal disomy (1,2). PWS in adults is characterized by muscular hypotonia, hyperphagia requiring restricted diet and regular physical activity to prevent the development of severe obesity (3). Other features include a mild to moderate intellectual disability, a distinct behavioral phenotype with stubbornness, temper tantrums, obsessive and compulsive behaviors and mood instability (4,5).
Many of the symptoms in PWS are believed to be caused by a dysfunction of the hypothalamus.
Endocrine abnormalities are common including de ciencies in growth hormone, sex hormones and thyroid hormones. Concerning, the function of the hypothalamus-pituitary-adrenal (HPA) axis both normal function and partial central adrenal insu ciency have been reported (6,7,8,9,10,11).
In previous studies, different tests were used to evaluate the function of the HPA-axis. They were all based on acute variations in adrenocorticotropic hormone (ACTH) and/or cortisol, whereas information about cortisol concentrations over a longer period is lacking. Due to the intellectual disability and behavior problems in PWS it is also of great value to nd other approaches for evaluating the HPA axis in PWS than the conventional tests. By measuring cortisol in hair, the average cortisol concentrations over longer periods can be assessable using an easy, non-invasive and convenient procedure (12).
The aim of this study was to examine hair cortisol in a group of adults with PWS in comparison to population-based controls. Self-reported clinical parameters from a semi-structured questionnaire were included for further evaluation.

Participants and procedures:
Twenty-nine adults (older than 18 years of age) with PWS were enrolled in 2015 and 2016 either during routine visits to the Department of Endocrinology, Karolinska University Hospital, Stockholm, or after responding to advertisements in the journal of the Swedish PWS Association.
Data for controls were retrieved from 266 population-based individuals included in the Lifelines Cohort study of whom hair cortisol analysis previously had been performed (13). Lifelines is a multi-disciplinary prospective population-based cohort study examining in a unique three-generation design, the health and health-related behaviors of 167,729 persons living in the North of The Netherlands. It employs a broad range of investigative procedures in assessing the biomedical, socio-demographic, behavioral, physical and psychological factors, which contribute to the health and disease of the general population, with a special focus on multi-morbidity and complex genetics (14,15).

Hair processing and analysis
A scalp hair sample of approximately 100-150 hairs was cut from the posterior vertex of the scalp, as close to the scalp as possible. Hair samples were taped to a piece of paper and stored in envelopes in the dark at room temperature prior to processing.
Hair sample analysis was performed as described previously (13). In summary, from each hair sample, approximately 10 mg of the proximal 3 cm was weighed and cut into 1 cm segments. Hair samples were washed in 2 mL of LC-MS grade isopropanol for 2 min, and left to dry. The hair was then extracted for 18 h at 25 °C in 1.4 mL LC-MS grade methanol and 100 µL of internal standard. Extracted samples were puri ed using solid phase extraction, and cortisol was quanti ed by liquid chromatography − tandem mass spectrometry (LC-MS/MS) using a Xevo TQ S system (Waters, Milford, MA) (16). The geometric mean of hair cortisol analyses in The Lifelines Cohort study (N = 266) was 2.7 pg/mg (13).

Questionnaire
The participants with PWS and/or their caretakers, lled out a questionnaire about their age, sex and anthropometric measurements. The stress was assessed by the question: did any stressful events occur during the last three months? In case there did, what happened?
In addition, the participants were asked in a standardized manner whether they used any product containing corticosteroids in the past three months and the route of administration (i.e., oral, intravenous, nasal, topical, inhaled, joint injections or others).
Data retrieved from the Lifelines Study for the controls included information on the subjects' age, sex, anthropometric and stressful life events. Life events were evaluated using the Dutch version of the List of Threatening Experiences (LTE), including occurrence of twelve major life events in the past twelve months (13).

Matching process and statistical analysis
The PWS patients were categorized into four age-groups [(18-25 years), (26-35 years), (36-45 years) and (46-60 years)], resulting in a fairly balanced age-sex groups for each of the approximately 10 years intervals. Subjects from each group of PWS patients were matched for age and sex to a total of 105 subjects from the corresponding control groups, in a 1:5 ratio. However, due to low number of controls for males in the 18-25 age-group (9 adults with PWS, 23 matched-controls) matching was made in a ratio of 1:2.5 for this particular sex-age group. The matching was programmed using the "seed" command in STATA 15.0 to ensure a random selection of subjects.
Results are presented as mean ± SD. For comparison between the groups, Student's t-test was used. Mean hair cortisol was considered to best capture the clinical reality with large variations in hair cortisol and a generalized linear regression model was used to examine the association of crude levels and levels adjusted for BMI and stress as potential confounders. The Akaike Information Criteria (AIC) test was used to con rm best t model where by both BMI and reported stress were retained in the model, as they predicted the model well. Statistical signi cance was set at p < 0.05.

Results
Twenty-nine adults (15 men and 14 women) with con rmed PWS participated. Mean (SD) for age was 33.4 years (12.7) in the patients with PWS and 42.1 (11.6) years in the controls. By virtue of the matching process based on the sex-age groups, there were no signi cant differences between the patients with PWS and controls with respect to age and sex.
Individual hair cortisol levels in patients with PWS are displayed in Table 1. Five patients with PWS had hair cortisol levels > 10 pg/mg and all but one of them reported occurrence of stressful situations. Fifteen other adults with hair cortisol < 10 pg/mg had not experienced any stress, and the remaining nine adults reported different kinds of stress that did not result in high levels of hair cortisol. Six patients with hair cortisol < 1.3 pg/mg did not report any stress or use of glucocorticoids. Figure 1 shows the hair cortisol in patients with PWS with and without reported stress.

Discussion
In this study of 29 adults with PWS, mean hair cortisol levels were signi cantly higher than hair cortisol in 105 sex and age matched population-based controls. Hair cortisol increased with BMI and reported stress in patients with PWS.
The stress hormone cortisol, produced in the adrenals, is important in maintaining body homeostasis by modulating neuroendocrine functions, the sympathetic nervous system and the immune function. Cortisol levels are regulated by activity in the hypothalamus and pituitary and secreted in a pulsatile circadian rhythm with increased secretion in response to stress. Cortisol is commonly measured in blood, and saliva, but these methods only re ect the time point at which the sampling is done or the past 24 h cortisol production when 24 h urine is collected. Multiple samplings are often needed. However, during the past decade, the measurement of hair cortisol has emerged as a non-invasive method to estimate the cortisol concentrations over months (6,14,17). Hair grows at a relatively stable rate of approximately 1 cm/month, and cortisol accumulates in hair. Thus, cortisol in hair retrospectively represents the average cortisol level over the corresponding period. There is no discomfort associated with collection of the hair sample and no special storage precautions are required for the samples. This is an attractive procedure, especially in a population such as PWS where the psychological and behavioral features might complicate advanced sampling procedures. We found that collection of the hair samples worked out well in our PWS cohort, although some men could not participate because of too short hair for su cient analyses. In addition, four patients declined participation because they felt that even the small hairless spot on the scalp after cutting the hair would stigmatize them further, although the sampling spot at the back of the scalp is usually too small to be visible afterwards.
In our cohort of patients with PWS, hair cortisol levels were clearly increased in patients who had reported signi cant stressful events. This is in accordance with several studies showing that the content of cortisol in hair is higher in individuals exposed to stress and stress-related disorders (12,(17)(18)(19)(20)(21)(22)(23)(24). Some of the patients in the present study with normal to low normal hair cortisol reported nonspeci c stressful situations, but the analysis of hair cortisol was probably not sensitive enough to detect minor or shortlived alterations of the HPA-axis (25).
Cortisol levels have been found to correlate positively with waist to hip ratio and waist circumference (26,27,19) and to be higher in obese individuals compared to normal-weight people (28,29). To retain all patients with PWS in the statistical analyses, matching for BMI between patients with PWS and controls was not done since some severely obese patients with PWS could not be matched with controls. However, similar to previous studies of hair cortisol (30) we found an increase in hair cortisol with BMI in the patients with PWS.
It is well known that some patients with PWS suffer from diseases secondarily to obesity, such as diabetes, cardiovascular diseases, respiratory diseases and arthrosis (31). Chronic stress might exacerbate comorbidity and therefore, it can be of interest to be able to indirectly quantify chronic stress in patients with PWS, by measuring hair cortisol to guide monitoring and early treatment.
Furthermore, PWS is associated with a decreased life expectancy, with an annual death rate reported to be as high as 3% (32,33). Nevertheless, a number of reported deaths were unexpected and unexplained, and it has been speculated if central adrenal insu ciency might have been the cause of some of the deaths (6). While the ACTH stimulation test provides information about the maximum response of the adrenal gland under stress hair cortisol concentrations provide information about average cortisol concentrations over a longer period. Our results warrant for larger and potentially randomized studies, but measurement of hair cortisol in PWS is attractive due to its non-invasive and simple performance. This is of great importance in individuals like patients with PWS, in whom a complicated procedure is a challenge. It is also an advantage that hair cortisol gives information on long-term cortisol exposure and might in the future be a baseline test for evaluation of the adrenal function and consideration for further evaluation.

Limitations
PWS is a rare disease, and the number of participants in the present study was too small for more complex analyses and comparisons with the control group.
Another limitation in our study is the assessment of stress, which in both groups was interpreted from reported stress during different duration of time and different questionnaires, which potentially might have affected the results. Moreover, in the PWS group most of the questionnaires were answered by the patients' caretakers and not directly by themselves and the questions on stress were not validated. Finally, patients with PWS who were able and willing to participate, might represent a selected group of quite healthy and well-functioning adults with PWS.

5.conclusions
In conclusion, in our adult patients with PWS the variation in hair cortisol was large. The mean cortisol levels were signi cantly higher than in the age and sex matched population-based controls. Increased BMI and reported stress were both associated with increased hair cortisol levels. Our results suggest an adequate long-term cortisol level and cortisol response to chronic stress in PWS. Hair cortisol might become a useful tool to guide the monitoring and management efforts for adults with PWS, including follow-up of comorbidities.