A total of 8,572 patients with DD/ID and/or suspected PWS features were included from July 2013 to December 2021 through outpatients in pediatrics and inpatients in neonatal ward of Shengjing Hospital of China Medical University. The inclusion criteria were: < 70 of development quotient (DQ) and/or intelligent quotient (IQ) for DD/ID; and ≥ 1 item of ① neonatal hypotonia with poor suck and feeding problems, ② male cryptorchidism, ③ short stature with small hands/feet, ④ obesity with hyperphagia and extreme food seeking, for suspected PWS features. Cases with other definitive genetic aetiology (such as Fragile X syndrome, Down syndrome, etc.), abnormal neuroimaging and metabolic screening have been excluded. Written informed consents for participation and publication were obtained from a legal guardian. We followed the Declaration of Helsinki, and all protocols were approved by the Ethics Committee of Shengjing Hospital of China Medical University.
Subject demographics were recorded including maternal age, age, sex, height, weight, and BMI. Clinical features were collected including neonatal hypotonia, feeding problems, dysmorphic faces, light-colored hair and skin, sticky saliva, undescended testes, central and/or obstructive sleep apnea, seizures, hyperphagia, delayed developmental milestones and language skills, learning disabilities, psychosocial and behavioral problems, reduced sensitivity to pain, and medical interventions. DQ was assessed using Gesell Developmental Schedules . IQ was assessed in cooperative children using Wechsler Intelligence Scale for Children . Autistic traits were evaluated using Childhood Autism Rating Scale . Laboratory parameters of patients with recombinant human GH treatment for more than one year were collected from the hospital laboratory center, including serum insulin-like growth factor I (IGF-I), serum thyroid stimulating hormone (TSH), free thyroxine (fT4), free triiodothyronine (fT3), fasting glucose and insulin, total triglyceride, total cholesterol, and low density lipoprotein (LDL). Standard deviation scores (SDS) values were calculated for height, weight, BMI, and IGF-I according to reference values for the Chinese population [17,18,19].
Multiplex ligation-dependent probe amplification (MLPA) assay
Genomic DNA was isolated from peripheral blood samples using the Blood Genomic DNA Miniprep Kit (Axygen, CA, USA). MLPA P245 kits (MRC-Holland, Amsterdam, the Netherlands) were used to screen common microdeletion syndromes following the manufacturer’s instructions. Briefly, DNA was denatured at 98 °C for 5 min and hybridized with the probes at 60 °C for 16 h. Ligation was performed at 54 °C for 15 min, and ligated probes were subsequently amplified by PCR using universal fluorescent primers. The fragments were separated by capillary electrophoresis using the 3730 Genetic Analyzer (Applied Biosystems, CA, USA) and analyzed using Coffalyser software (MRC-Holland).
Methylation-specific MLPA (MS-MLPA) assay
MS-MLPA ME028 kits (MRC-Holland) were used for the diagnosis of PWS following the manufacturer’s instructions. Denaturing and hybridization were the same as the MLPA assay. The hybridized samples underwent ligation with or without methylation-sensitive restriction enzyme Hha I at 48 °C for 30 min, and were amplified by PCR following the same procedure as MLPA. The fragments were separated using the 3730 Genetic Analyzer (Applied Biosystems) and assayed with the Coffalyser software (MRC-Holland).
Atypical deletions were defined by genomic CNV-sequencing as previously described . Briefly, 50 ng of genomic DNA was fragmented to construct DNA libraries by end filling, adapter ligation, and PCR amplification. The DNA libraries were constructed using library preparation kits (Berry Genomics, China) and sequenced by the NextSeq 500 platform (Illumina, San Diego, CA, USA) to generate about 8 million raw reads with 36 bp in length. All the sequences were aligned to the GRCh7/hg19 genome using the Burrows-Wheeler algorithm. Mapped reads were allocated progressively to the chromosomes, and copy number changes were evaluated by comparing bin counts between all test samples run in the same flow cell.
Genotyping of microsatellite markers
Cases with maternal UPD underwent genotyping of 10 polymorphic microsatellite markers on chromosome 15q11-q13 by multiplex PCR using fluorescent primers listed in supplementary Table 1. Amplicons were purified (Axygen) and separated by capillary electrophoresis using the 3730 Genetic Analyzer (Applied Biosystems), and analyzed by the GeneMapper 4.1 software (Applied Biosystems).
All statistical analyses were performed in SPSS-V-23.0 (IBM Corporation, USA). Normal data were expressed as means ± SD or number (percentage). The Shapiro–Wilk test was employed to examine continuous variation. Comparisons between two groups were performed by chi-square test, nonparametric Mann–Whitney U test or Student t test accordingly. P < 0.05 was considered to indicate statistical significance.