His sister, female, 8 years old, weighed 19.0 kg (3th percentile), 115.2 cm tall (3th percentile), and low bone density (11th percentile). Main clinical manifestations consist of intellectual disability, severely impaired speech (can only call dad and mom), high-arched palate, dental abnormalities (crowding and abnormal shape), small jaw, short tongue strap (received surgery), hypotonia, poor appetite, and salivation. She started walking at the age of 2. She started showing epilepsy since 6 years of age, even though her head MRI showed normal results at the age of 6. Her intellectual abilities were below an average 7 years old.
The karyotype analysis of G-banding in the peripheral blood of two siblings and parents showed a standard chromosomal pattern (data not shown). CMA result of both the proband and his sister using CytoScan  HD whole genome SNP array (Affymetrix, USA) showed two copy number variations (CNVs): arr[hg19] 2q33.1(200,192,328-200,197,269)x1,2q35(218,105,663-218,816,675)x3. They have a 4.9-kb deletion in the 2q33.1 region of chromosome 2 (Figure S1). The deletion spans the exon 9 of the SATB2 (OMIM: 608148) gene. They also have a 711-kb duplication (arr[hg19] 2q35(218,105,663-218,810,908)x3) in the 2q35 region, which contains DIRC3 (OMIM: 608262) and TNS1 (OMIM: 600076) genes. There has been no clear disease-related report on the duplication of this fragment or on these two genes. The mother's CMA result: arr[hg19] 5q31.3q33.1 (141,383,108-151,756,016)x2 hmz. The father's CMA results: arr[hg19] 2q35 (218,105,663-218,810,908)x3. The couple did not carry the 4.9-kb deletion at 2q33.1, while the 711-kb duplication at 2q35 of the children should have been inherited from their father. However, the possibility of a low-level somatic or gonadal mosaicism was not excluded. Meanwhile, the exact breakpoints were yet to be determined.
The pregnant woman received amniocentesis at 19 weeks of gestation. The karyotype of the fetus was normal, but CMA result was the same as those of the affected children. In addition, the B-ultrasound showed that the fetus had ventricular septal defect. After genetic counseling and through consideration, the couple chose to terminate the pregnancy at 23 weeks of gestation.
This study was carried out in accordance with the recommendations of the Ethics Committee of Women's Hospital, School of Medicine Zhejiang University. All subjects gave written informed consent in accordance with the Declaration of Helsinki, and written informed consent was also obtained from the guardians of the two affected siblings for the publication of this case report. The protocol was approved by the Ethics Committee of Women's Hospital, School of Medicine Zhejiang University.
Peripheral blood samples were collected from the family members including the husband's father (I1), the couple (II1 and II2), and their two children bearing SAS (III1 and III2) ( Figure 1A ). Genomic DNA was extracted from peripheral blood samples using the QIAGEN spin columns on a QIACube (QIAGEN GmbH) according to the manufacturer's instructions. All DNA samples were dissolved in water and stored at -20 C.
Gap-PCR was chosen for locating exact breakpoints of the deletion. Three sets of primers (Gap2500-F/R, Gap1000-F/R, Gap500-F/R,  Table 1 ) were designed according to the CMA results. Gap2500-F/R primer set worked well using TaKaRa LA Taq  (TaKaRa Bio Inc.) and two-step procedure. The procedure of the PCR was as follows: 94 C for 1 min followed by 30 cycles at 98 C for 10 s, 68 C for 8 min, and a final extension step at 72 C for 10 min. Based on this result, another four primer sets (6666-F/R, 5335-F/R, 4334-F/R, 3442-F/R,  Table 1 ) were designed to amplify more suitable size of fragments for sequencing. The procedure of the PCR was as follows: 94 C for 1 min followed by 30 cycles at 98 C for 10 s, 68 C for 4 min, and a final extension step at 72 C for 10 min.
The semen sample of the father was obtained by masturbation following 5 days of sexual abstinence and then was allowed to liquefy for 60 min at 37 C before processing. Semen sample was diluted 1:2 in Earle's solution, centrifuged at 500xg for 5 min. Then sperm pellets were washed twice with Earle's solution, and the final pellets were preserved immediately in liquid nitrogen for DNA extraction. Pellets were homogenized, and genomic DNA was extracted using the QIAGEN spin columns on a QIACube (QIAGEN GmbH) according to the manufacturer's instructions.
Three pairs of primers ( Figure 2A ) were designed to detect potential deletions in this family, including two primer sets outside the deletion region and one primer set inside the deletion. The design of primers is in accordance with the following standard: the length of the amplified product is less than 300 bp, and annealing temperature is close to 60 C. The three pairs of primers were designed by Primer-BLAST software () (see  Table 2 ). Specificity and effectiveness have been examined in preliminary experiment. Furthermore, beta-globin was chosen as reference gene (forward primer: 5'-ACACAACTGTGTTCACTAGC-3'; reverse primer: 5'-CAACTTCATCCACGTTCACC-3'). The reaction was performed on a LightCycler 480 II real-time PCR system (Roche) in a final volume of 20 microl containing 10 microl of SYBR Premix Ex Taq Polymerase (TaKaRa), 0.4 microl each of the forward and reverse primers (10 micromol/l), and 2 microl of the genomic DNA (20 ng). Each sample was tested in triplets. The procedure of the PCR was as follows: 95 C for 10 s, followed by 40 cycles at 95 C for 5 s, 58 C for 30 s, and a final extension step at 72 C for 10 min. The dosage ratio of the tested samples was calculated by the DeltaDeltaCt method for each test sample according to.
The somatic and gonadal mosaicism levels of the father were estimated using the Pilot Droplet Digital PCR system [Pilot Gene Technologies (Hangzhou) Co., Ltd. China]. The primer set SATB2Del-F: CAGAAAGTATCTGGGCCTATCAT/SATB2Del-R: GTACAAAAGAGCTGAAAACAAATACA, which was located on each side of the breakpoint, were used along with the 6-carboxyl-x-rhodamine (ROX)-labeled fluorescence probe SATB2Del-P: ROX-AGGTGACTTCTGCTTGTT-MGB, which spanned the deletion region to detect the copy number of the mutation. Another primer-probe set PARP2-F: GCGGAGGGAAGCTCATCAGTG/PARP2-R: CCCTAGTCTCAGACCTTCCCAA/PARP2-P: 6-FAM-ACATGGGAGTGGAGTGACAGG-BHQ1 was employed to detect the single copy reference gene Poly (ADP-ribose) polymerase 2 (PARP2) in this study. The mosaic ratio was calculated as the copy number of mutation/the copy number of the reference gene.
The droplet generation, PCR amplification, and chip scanning were performed according to the manufacturer's instructions. Briefly, 20 microl of PCR mixture containing 1x PCR buffer, 1 microl each of the genomic DNA, 150 nM of 6-FAM-labeled probe, 600 nM of ROX-labeled probe, and 600 nM of each primer were loaded into Pilot droplet chips. Droplets were automatically generated in the Pilot droplet generator. Chips were then transferred into the Pilot iThermal1.0, and samples were amplified using the following cycling settings: initial denaturation at 95 C for 10 min, followed by 40 cycles of 95 C for 30 s, and 55 C for 60 s. Post-PCR chips were scanned using Pilot iScanner5 chip scanner, and results were analyzed using the GenePMSv1.1 software.