This project is a combined retrospective (data from medical records) and prospective longitudinal study of a single centre cohort, which aims to consecutively include all patients (infants, children, adolescents and adults) with KS (47,XXY) including other rare variants of KS with more than one supernumerary X-chromosome (e.g. 48,XXXY; 48,XXYY; 49,XXXXY), mosaicism (e.g. 47,XXY/46,XY) and SRY-positive 46,XX-males (ICD: DQ980, DQ981, DQ982, DQ983, DQ984, DQ987).
Data Collection The data base will consist of clinical and biochemical data and results obtained at the routine control visits and admittance at a hospital. Data will be collected both retrospectively (from the medical record) and prospectively. Retrospective data from the medical record will include data collected at previous visits at the Department of Growth and Reproduction (e.g. anthropometry, DXA scans, bone age (including bone health index, hormone concentrations, karyotype, biomarkers of general health, blood pressure, pulse, semen quality, results of microTESE and testicular histology as well as medical treatment in general and other diagnoses/co-morbidity). All data and paraclinical parameters that will be collected prospectively are listed in the following. Some of the points are only relevant for children whereas others are only relevant for adolescents or adults as noted at each point. All points have been marked with an a or b to specify which evaluations are part of the standard care (a) and which are extra evaluations as part of the study (b).
1\. Medical history (a): medical history (including karyotype, pubertal development (Tanner stages, gynecomastia, serum concentrations of reproductive hormones and growth factors), growth, DXA scans, testosterone replacement therapy (type, date, dose, effects and side-effects), family history, co-morbidities, medical treatment in general (type, date, dose), allergies, blood pressure, diet, exercise, education, and in adolescents and adults: semen quality, result of mTESE, testicular histology, alcohol consumption, smoking, drug abuse, employment/job.
2\. Bone age including bone health index (a): x-ray of left hand is performed in growing boys to evaluate final height and bone health index.
3\. Pubertal development and virilization:
(a) Tanner staging (pubic hair stage, gonadal stage), testicular volume by palpation, gynecomastia (b)Acne stage, voice frequency, penile length and penile width.
4\. Anthropometry:
1. Height (cm) and sitting height (cm) as measured using a wall-mounted stadiometer and body weight (kg) by using an electronic scale. Target height will be calculated from biological parental heights (if available).
2. Arm span, head, thoracal, hip and waist circumferences (cm). Sitting height (cm) in adults.
5\. Clinical evaluation (a): Evaluation of tremor (yes/no), skeletal deformities (clinodactyly, pectus excavatum/carinatum, scoliosis, kyphosis, pes planus) and presence of hypermobility.
6\. Grip-strength (b): Evaluated by Digital hand dynamometer
7\. Body composition/bioimpedance (b) measured by Tanita MC780 MAS
8\. Blood samples:
* Reproductive and adrenal hormones (a): follicle stimulating hormone (FSH), Luteinizing hormoner (LH), steroid metabolites (testosterone, dihydrotestosterone, androstendione, 17-hydroxyprogesterone, progesterone, 17-alfa-hydroxypregnenolone, estradiol, estrone, estrone sulfate, cortisol, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, aldosterone), sex hormone binding globulin (SHBG), inhibin B, anti-Müllerian hormone (AMH), insulin like factor 3 (INSL3)
* Thyroid hormones (a): free thyroxin (T4) and thyroid stimulating hormone (TSH)
* Thyroid antibodies (b)
* Growth factors (b): Insulin-like growth factor-1 (IGF-1), IGF-2 and insulin-like growth factor binding protein-1 (IGFBP-1) to 6 and acid-labile subunit (ALS)
* Bone turnover markers (a): 25-OH-vitamin D and calcium
* Bone turnover markers (b): Phosphate, magnesium, PTH, alkaline phosphatase (liver and bone), osteocalcin, PINP (procollagen type I N-terminal propetide), CTX (carboxy terminal telopeptide of collagen type I), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprogeterin), Klotho, sclerostin and TRACP 5b (tartrate-resistant acid phosphatase 5b) in serum and urine
* Insulin resistance (a): glucose and hemogolbin A1c (HbA1C),
* Insulin resistance (b): Proinsulin and lipocalin-2
* Inflammation (b): Mesoscale
* Immune response (b): antibody titer of vaccines
* Lipids (a): cholesterol and triglyceride
* Lipdis (b): Free fatty acids and glycerol
* Fat tissue (b): adiponectin and leptin
* Haematology (a): Hematocrit and hemoglobin
* Organ markes (a): Liver, kidney parameters and prostate specific antigen (PSA)
* DNA purification (b) for genetic analysis (see separate section on genetics below)
* RNA (b) (analysis of circulating small non-coding RNAs)
Hormonal analyses and DNA purification are carried out at our own laboratories and standard biochemical analyses are performed at the hospital's central laboratory, all of which are DANAK certified.
7\. Genetic analyses A targeted approach will be used, and whole-genome or whole-exome sequencing will not be performed. A selected set of genetic polymorphisms in target genes with established or theoretic effects on metabolism, overweight, growth and infertility will be analyzed either by PCR genotyping or targeted sequencing (max. 200 selected genes). In addition, SNP-arrays that exclusively target common variants and not any rare variants will be used to determine the influence of common genetic variation on the observed associations.
"Omfattende kortlægning af arvemassen" according to the list provided by National Ethics Committee (NVK) will not be performed. Since only commonly occurring variants are investigated, no random findings are expected.
DNA methylation patterns will be analyzed by applying Illumina methylation arrays which do NOT reveal sequence information. The Illumina methylation array only reveal the level of methylation at common CpG sites and epigenetic analyses by sequencing will NOT be performed.
8\. Dual-energy x-ray absorptiometry scan (DXA) (a) A whole body (all) and regional (adults) DXA scan is performed to evaluate bone mineral density (BMD), total and regional body fat percentage and body lean mass. This scan is performed on an individual basis. Typically, the first time around 8-10 years if the boy can cooperate. Thereafter before starting TRT and one year after stating TRT. In adults a DXA scan is performed when the diagnosis is made and one year after starting TRT. Thereafter the patient is scanned on an individual basis depending on the results of the first scans. If the result is normal a scan will typically be performed every 5 years. If the scan shows reduced bone mineralization the patient will have another scan after 1, 2 or 3 years depending on the reduction.
9\. Blood pressure (a) The blood pressure is measured at every visit after the participants have been sitting and relaxing for at least five minutes. This will be carried out at the upper arm of the dominant side using a calibrated automatic sphygmomanometer. Blood pressure and pulse will be registered.
10\. Electrocardiography (ECG) (b) Standard ECG with 12 leads.
11\. Gonadal ultrasound (a) Ultrasound of the testicles is performed at the first visit after referral in adolescents and adults with KS.
12\. Breast ultrasound (b) Ultrasound of the breast will be performed to quantify the size of a gynecomastia, if present.
13\. Semen analysis (a) As part of the standard care of patients with KS, adolescents and adults can deliver a semen sample for analysis and if spermatozoa are present, they will have the possibility to cryopreserve the cells. The patient will be informed that most patients with KS have azoospermia and only very few cases with spermatozoa in the ejaculate have been reported. This is to prepare the patient for the most likely result.
Sexual abstinence for 48 hours to 7 days is recommended prior to collection of a semen sample. The sample will be collected by masturbation, primarily in a designated room at the department using standard equipment.
14\. Micro TESE (a) Patients with KS older than 18 years with a fertility wish can have an operation performed (micro TESE (testicular sperm extraction)) with the goal to cryopreserve potential intratesticular spermatozoa for future fertility treatment. This is part of the standard care of patients with KS. If the patient has a history of cryptorchidism a standard testicular biopsy for histological evaluation is performed as part of the operation. This biopsy is performed to rule out the presence of precursors for testicular cancer (germ cell neoplasia in situ (GCNIS)).
15\. Questionnaires (b) A variety of questionnaires will be used to measure psychopathology and mental health in our samples. Depending on the cut-off for the individual rating scales described below, self-report forms will be used for ages 16-18 and older. For children and adolescents younger than this, parents or caregivers will respond. None of the questionnaires are diagnostic and will NOT reveal severe psychopathology that needs to be handled acutely. If concerns arise regarding issues such as poor well-being or potential signs of ADHD the family or the patient will be contacted and guided to seek appropriate help, primarily through the general practitioner who can refer for relevant follow-up care.
To ensure valid responses in the adult self-report questionnaires assessing psychopathology and mental health, participants will be screened with a short form of the Weschler Adult Intelligence Scale, ed. 4 (WAIS-IV; (Wechsler, 2008)). On the basis of the two subtasks chosen (Vocabulary and Block Design), a mean scale score will be calculated. Mean scale scores ≥ 7 will be taken to reflect overall intellectual capacity within the normal range.
BASC-3 Bess (Reynolds, 2015) is a 28-item rating scale designed to screen the social and psychological functioning of children and adolescents.
ADHD-rs (DuPaul, 1998) and ASRS v.1.1 (Kessler, 2005)are brief rating scales to assess symptom characteristics of ADHD (inattention and impulsivity) in children, adolescents, and adults.
Peds-Ql Multidimentional Fatigue Scale (Varni, 2002; Varni \& Limbers, 2008) measure perceived lack of energy (fatigue) in children, adolescents, and adults.
Brief-2 and Brief-A (Gioia, 2015) assess executive function in children, adolescents, and adults. Brief-domains address the ability to regulate emotion, attention, and behavior.
Symptom Checklist-90 revised (SCL-90-R, (Derogatis, 1994) is a self-report inventory for adults that assesses a broad range of psychological problems and symptoms of psychopathology.
The Autism Spectrum Quotient (Baron-Cohen et al., 2001)is a self-report questionnaire for adults designed to measure the degree to which an adult has traits associated with the autism spectrum.
The World Health Organization Quality of Life - BREF (WHO, 1997) is a self-report questionnaire for adults which assesses 4 domains of quality of life (QOL): physical health, psychological health, social relationships, and environment
16\. 3D-scan of face (b) Clinical photo including 3D-scan of face for detailed description of the facial characteristics will be performed by a LifeViz camera from QuantifiCare.
17\. Collection of biological material for a research biobank: Serum, purified DNA and urine will be stored at -20 degrees Celcius or -80 degrees Celcius after each visit. Analyses that are not part of the routine evaluation will be performed simultaneously to avoid influence of interassay variation. DNA will be purified before freezing. The research biobank consists of -20 and -80 degrees Celcius degree freezers located at Rigshospitalet in a locked room. The freezers are equipped with an alarm.
18\. Collection of biological material for a research biobank for future research: When the above-mentioned analyses have been run the exceeding material will be transferred to a biobank for future studies. The investigators aim at examining endocrinological, biochemical, metabolic, inflammatory, genetic, and epigenetic biomarkers during mini-puberty, pubertal transition as well as before and during TRT. After transferring the exceeding material to the biobank for future research the material will be stored at the Department of Growth and Reproduction for 20 years and any remaining material will be destroyed thereafter.
The biobank can only be used for future research after new approval by the local ethics committee. When consenting to participate in the study the patient and/or family will be asked permission to save the biological material in the biobank for future studies. If the patient or the parents do not agree on saving the material in the biobank for future studies, no extra material will be saved.
The patient or family can at any time decide that they do no longer want to have biological material stored or that they do no longer want to collect material for the biobank. In the first case all collected material will be destroyed. In the latter case no biological material will be collected in the future. Such decision will not have any consequences for the patients follow up or treatment in the department.
