# Targeted Gene Sequencing, Bone Health, and Body Composition in Cornelia de Lange Syndrome
## 2 Materials and Methods  
Participants: A total of 15 individuals with CdLS (10 females, all Caucasian, aged 5-38 years) participated in the present observational study with a case series design. Most of the individuals were referred by the Spanish Association of Cornelia de Lange Syndrome. They were evaluated by experienced clinicians in the field of CdLS. Clinicians reviewed medical records, took standard medical histories, and performed physical examinations to classify individuals following the international consensus criteria of CdLS [8]. A combination of cardinal and suggestive features was used to diagnose CdLS phenotypes. Classic phenotypes are indicated with a score of \(\geq\)11 points if at least 3 cardinal features are identified; a score of 9-10 indicates nonclassic phenotypes if at least 2 cardinal features are present. In addition, all individuals were subjected to molecular analysis using a custom targeted gene panel consisting of 35 CdLS-related genes via deep sequencing analysis based on Ion Chef and Ion S5 XL Systems (Thermo Fisher Scientific), as described previously [23], including _NIPBL_, _SMC1A_, _SMC3_, _RAD21_, _HDAC8_, _BRD4_, _ANKRD11_, and _MAU2_.  
Written informed consent was obtained from parents or guardians. The ethical guidelines for human research outlined by the Declaration of Helsinki (revision of Fortaleza 2013) [24] and the Declaration of Taipei [25] were followed. The protocol study was approved by the Ethics Committee of Clinical Research of the Government of Aragon (CEICA; PI16/225).  
Measurements: Individuals with CdLS underwent anthropometric examination wearing no shoes and minimal clothing. Height was measured to the nearest 0.1 cm with a stadiometer (SECA 225, SECA, Hamburg, Germany) and weight to the nearest 0.1 kg with an electronic scale (SECA 861, SECA, Hamburg, Germany). BMI, FMI, and LMI were calculated as weight (kg) or fat mass (kg) or lean body mass (kg) divided by height squared (m\({}^{2}\)). Anthropometric Z-scores were calculated based on Centers for Disease Control growth charts for children and adolescents age 2 to 20 years. FMI and LMI Z-scores were calculated based on the body composition reference values published by [PERSON] et al. [26].  
Lumbar spine (LS) and total body less head (TBLH) bone mineral content (BMC) and aBMD were determined by means of DXA scan, evaluated with the pediatric version of QDR-Explorer software, version 12.4 (Hologic Corp., Bedford, MA, USA). All scans were performed by the same operator, who had been fully trained in the operation of the scanner, the positioning of subjects, and the analysis of scans according to the manufacturer's guidelines. Coefficients of variation for the DXA measurements in our laboratory have already been published [27], and were 2.3% for BMC and 1.3% for aBMD. Adjusted BMC and aBMD Z-scores, according to the reference values provided by [PERSON] et al. [28], were calculated.  
Blood samples were collected and centrifuged, and serum was separated into aliquots in October (autumn). Levels of 25-OH-vitamin D, calcium, phosphorus, alkaline phosphatase (ALP), and parathyroid hormone (PTH) were measured by a commercially available kit using the Roche Cobas 6000 autoanalyzer (Roche Diagnostic, Mannheim, Germany). Levels of 25-OH-vitamin D and PTH were determined by electrochemiluminescence. Levels of calcium, phosphorus, and ALP were measured photometrically.