Clinical Presentation and Genetic Profiles of Chinese Patients With

Journal of Genetics (2019) 98:42 © Indian Academy of Sciences https://doi.org/10.1007/s12041-019-1090-5

RESEARCH ARTICLE

Clinical presentation and genetic proﬁles of Chinese patients with velocardiofacial syndrome in a large referral centre

DANDAN WU1,2, YANG CHEN1,2, QIMING CHEN1,2, GUOMING WANG1,2∗, XIAOFENG XU1,2,A.PENG3, JIN HAO4, JINGUANG HE5∗,LIHUANG6∗ and JIEWEN DAI1,2∗

1Department of Oral and Cranio-maxillofacial Surgery, National Clinical Research Center for Oral Disease, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, People’s Republic of China 2Shanghai Key Laboratory of Stomatology, Shanghai 200011, People’s Republic of China 3State Key Laboratory of Oral Diseases, West China School of Stomatology, Chengdu 610041, People’s Republic of China 4Harvard School of Dental Medicine, Harvard University, Boston 02125, USA 5Department of Plastic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, People’s Republic of China 6Department of Oral and Maxillofacial Surgery, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China *For correspondence. E-mail: Jiewen Dai, [email protected]; Guoming Wang, [email protected]; Jinguang He, [email protected]; Li Huang, [email protected].

Received 29 November 2017; revised 8 January 2019; accepted 11 January 2019; published online 4 May 2019

Abstract. Diagnosis and treatment of velocardiofacial syndrome (VCFS) with variable genotypes and phenotypes are considered to be very complicated. Establishing an exact correlation between the phenotypes and genotypes of VCFS is still a challenging. In this paper, 88 Chinese VCFS patients were divided into five groups based on palatal anomalies and one or two of other four common phenotypes, and copy number variations (CNVs) were detected using multiplex ligation-dependent probe amplification (MLPA), array comparative genomic hybridization (aCGH) and quantitative polymerase chain reaction. The findings showed that palatal anomalies and characteristic malformation of face were important indicators for 22q11.2 microdeletion, and there was difference in the phenotypic spectrum between the duplication and deletion of 22q11.2. MLPA was a highly cost-effective, sensitive and preferred method for patients with 22q11.2 deletion or duplication. Our results also firstly reported that all three patients who simultaneously exhibited palatal anomalies and cognitive disorder, without other phenotypes, have Top3b duplication, which strongly suggested that Top3b may be a pathogenic gene for these patients. Further, the findings showed that patients with palatal anomalies and congenital heart disease or immune deficiency, with or without other uncommon phenotypes, exhibited heterogeneity in CNVs, including 4q34.1- qter, 6q25.3, 4q23, Xp11.4, 13q21.1, 17q23.2, 7p21.3, 2p11.2, 11q24.3 and 16q23.3, and some possible pathogenic genes, including BCOR, PRR20A, TBX2, SMYD1, KLKB1 and TULP4 have been suggested. For these patients, aCGH, whole genomic sequencing, combined with references and phenomics database to find pathogenic gene, may be choices of priority. Taking these findings together, we offered an alternative method for diagnosis of Chinese VCFS patients based on this phenotypic strategy.

Keywords. diagnosis; Chinese patients; velocardiofacial syndrome patients; phenotypic strategy.

Introduction

Dandan Wu, Yang Chen and Qiming Chen contributed equally to Velocardiofacial syndrome (VCFS) (MIM: 192430, also this work. named DiGeorge syndrome, MIM: 188400), affects ∼1

Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12041-019-1090-5) contains supplementary material, which is available to authorized users.

1 42 Page 2 of 11 Dandan Wu et al. in 2000–7000 individuals (Kobrynski and Sullivan 2007; Materials and methods Wu et al. 2013; Kruszka et al. 2017). Phenotypes in every patient with VCFS exhibited great variety, and more Patients than 180 clinical features involving almost all organs and systems have been reported. The five most common Eighty-eight patients, diagnosed as VCFS based on clini- symptoms for VCFS patients include palatal anomalies, cal and auxiliary examinations, from the Center for Cleft congenital heart disease (CHD), characteristic malfor- Lip and Palate, Shanghai Ninth People’s Hospital, Shang- mation of face (CMF), immune deficiency and cognitive hai Jiao Tong University School of Medicine were enrolled or behavioural disorder. However, no single phenotype in this study. Some patients’ samples and materials from occurs in all patients, and no patient would exhibit all our previous study also were included in this study (Wu reported phenotypes (Perez and Sullivan 2002; Kobryn- et al. 2013). Patients who exhibited at least two of the ski and Sullivan 2007; Lopez-Rivera et al. 2017). five most common phenotypes, including palatal abnor- Previous evidence showed that an autosomal dom- mality, CHD, CMF, immune deficiency and cognitive or inant microdeletion on the long arm of chromosome behavioural disorder were clinically diagnosed as VCFS. 22(22q11.2) was the most common pathogenic factor for Except for these five common phenotypes, all the other VCFS, and the vast majority of individuals with 22q11.2 phenotypes were defined as uncommon phenotypes. In microdeletion carry a typical 3-Mb deletion. In addition, this study, palatal abnormalities mean patients exhibited some patients carry a proximal 1.5-Mb deletion nested to submucosal cleft palate, occult submucous cleft palate, the typically deleted region (TDR), and only a few patients complete cleft palate (CCP) or congenital velopharyngeal exhibited atypical deletions overlapping or nonoverlap- insufficiency (CVPI). CMF mean patients exhibited all ping with the TDR (Perez and Sullivan 2002; Ensenauer of the following phenotypes: vertically long face, narrow et al. 2003; Kobrynski and Sullivan 2007; Panamonta palpebral fissures, fleshy nose with a broad nasal root, flat- et al. 2016a, b). However,except for 22q11.2 microdeletion, tened malar region and retrognathia. CHD mean patients previous findings also showed that some cases exhib- with one or several conotruncal CHDs, aortic arch anoma- ited clinical phenotypes similar to those of VCFS having lies or other phenotypes: tetralogy of fallot, atrial septal 22q11.2 duplication or copy number variations (CNVs) defect, interrupted aortic arch, patent ductus arteriosus on other chromosomes. These patients were also clinically and ventricular septal defect. Immune deficiency means diagnosed as VCFS by some geneticists and doctors (Wu patients had thymic hypoplasia, T-cell deficiency or history et al. 2013). of recurrent infections. Cognitive or behavioural disorders Establishing an exact correlation between the pheno- mean mild mental retardation or intellectual disability. types and genotypes of VCFS is still a challenge. With Every enrolled patient would receive a general physical the development of biotechnology, such as whole-genome examination in detail, and the phenotypes were diagnosed sequencing, array comparative genomic hybridization by specialist physicians. The basic information and clin- (aCGH) and multiplex ligation-dependent probe ampli- ical phenotypes of these patients are listed in table 1 in fication (MLPA), detection of genetic variation in patients electronic supplementary material at http://www.ias.ac.in/ has become easier (Jalali et al. 2008; Poirsier et al. 2016). jgenet/. However, narrowing the detecting area and focus on some In this study, we tried to classify VCFS patients based on potential target based on the phenotypes may increase the five most common phenotypes. All enrolled 88 patients the precision of diagnosis and reduce patients’ cost. in this study were divided into five groups (table 2 in elec- Human phenotype ontology offers a computational bridge tronic supplementary material): (i) group 1 (patients 1 to between genotypes and phenotypes (Zemojtel et al. 2014; 66): patients exhibited palatal anomalies and CMF, with Kohler et al. 2017). Our previous preliminary findings also or without other phenotypes; (ii) group 2 (patients 67 to showed that all VCFS patients with palatal anomalies and 69): patients exhibited palatal anomalies and cognitive CMF would have a 22q11.2 microdeletion (Wu et al. 2013). or behavioural disorder, with or without other uncom- All of these findings inspired us that VCFS patients may be mon phenotypes; (iii) group 3 (patients 70 to 80): patients divided into different groups based on clinical phenotypes, exhibited palatal anomalies and CHD, with or without and these different groups may exhibit different genetic other uncommon phenotypes; (iv) group 4 (patients 81 to variations. 86): patients exhibited palatal anomalies and immune defi- In this paper, 88 Chinese patients with clinical diagnosis ciency, with or without other uncommon phenotypesand of VCFS were divided into five groups based on palatal (v) group 5 (patients 87 and 88): there were two patients anomalies and one or two of other four common pheno- exhibiting palatal anomalies, cognitive or behavioural types, and these groups exhibited different CNVs. Further, disorder and immune deficiency, with or without other some new possible pathogenic genes also were found based uncommon phenotypes. on this strategy, which offered a useful method for classi- One hundred normal patients were included in this fication diagnosis of Chinese VCFS patients based on this study as a control. The summary of these control patients’ phenotypic strategy. demographic data is listed in tables 3 and 4 in electronic Clinical and genetic profiles of Chinese VCFS patients Page 3 of 11 42

Figure 1. Algorithm for CNV testing in VCFS patients and controls. All VCFS patients and controls would ﬁrstly undergo MLPA analysis, and the patients without 22q11.2 microdeletion, a most common pathogenic factor of VCFS, being detected by MLPA would undergo aCGH analysis. Further, if the internal reference probe sites were similar to the sample’s sites, sample probes array ﬂoating and irregular, or there were less sample probes in the aCGH image, the aCGH results should be further evaluated by qPCR. CNVs, copy number variations.

supplementary material. Thirty available parent pairs (60 10–50 ng/μL DNA was prepared for MLPA analysis, parents) of subjects with typical 22q11 deletion were also aCGH or qPCR. included in this study to determine whether 22q11 deletion was inherited or de novo. The VCFS patients, controls and parent pairs would MLPA analysis undergo MLPA, aCGH or quantitative polymerase chain reaction (qPCR) analyses following the planed arrange- The protocols for MLPA analysis is described in detail ment (figure 1). in the previous literature. Briefly, the SALSA MLPA KIT P250-B1 DiGeorge (MRC-Holland, Amsterdam, the Netherlands) was used for MLPAanalysis according to the DNA extraction manufacturer’s instructions. Capillary electrophoresis on an ABI 3130XL Genetic Analyser (Applied Biosystems, Whole peripheral blood was collected, and genomic DNA Foster City, USA) was used to detect and quantify the was extracted from the peripheral blood using a Qiagen amplification products, and the files of electropherograms DNA blood kit (Qiagen, Hilden, Germany) following the were analysed using GeneMarker software v1.8 (Softge- manufacturer’s instructions. Then the concentration was netics, State College, USA). Forty-eight probes for 48 detected using a spectrophotometry method (NanoDrop different genes were included in this MLPA kit, and the 1000, Thermo Scientific, USA), and a concentration of detailed information on these probes is described in the 42 Page 4 of 11 Dandan Wu et al. previous literature (Wu et al. 2013). Each MLPA analysis on the VCFS-related tissues, including cardiovascular was carried out in triplicate. system, brain, haemolymphoid system, branchial arch and limb. Array comparative genomic hybridization (aCGH) Prediction of CNVs and possible pathogenic genes based on The MLPA analysis results showed that there were 16 phenomics patients exhibiting no CNVs (patients 70–76, 78–80, 81–86), four patients with atypical 22q11.2 duplication We used the on-line database, phenolizer (http://phenolyze (patients 77, 67–69) and one patient (patient 88) with r.wglab.org/) and phenomizer (http://compbio.charite.de/ 4q34 loss of heterozygosity. High-density probe microar- phenomizer/), to make a prediction of CNVs or possi- ray analyses were performed to evaluate the complete ble pathogenic genes based on the patients’ phenotypes. genomes of these 21 patients, who had no 22q11.2 Whenever there were many genes located in CNVs, the microdeletion, to determine the other possible CNVs. phenolizer database was also used to sort the possible Genomic DNA was extracted from whole blood using pathogenicity for these genes based on the patients’ phe- the QIAamp DNA Blood Maxi kit following the instruc- notypes. tions described above. CNVs were analysed using the CytoScan HD array (Affymetrix, Santa Clara, USA) and Statistical analysis Affymetrix Chromosome Analysis Suite (Affymetrix) software according to the manufacturer’s instructions. Briefly, The χ 2 test was used to compare the CNVs and clinical the protocols included the following eight procedures characteristics among the five groups using SPSS 17.0 soft- before scanning the chip: genomic DNA digestion, Nsp ware, and P < 0.05 was considered significant. I adapter ligation, fragment amplification by PCR, purifi- cation of PCR product, fragmentation of PCR product, Ethics statement end-labelling, hybridization and washing. The obtained CNVs were compared with the CNVs in the database of The protocols used in this study were carried out according genomic variants (DGV). to the principles of the Declaration of Helsinki, and were approved by the Ethics Committee in Shanghai Ninth Peo- CNVs validation by qPCR ple’s Hospital, Shanghai Jiao Tong University, School of Medicine. Written informed consent was obtained from all If the internal reference probe sites were similar to the participants or from their parents. sample’s sites, sample probes array floating and irregular or there were less sample probes in the aCGH image, Results the aCGH results should be further evaluated. In this study, the detected CNVs in patients 70, 71, 75, 79–86 CNVs and clinical characteristics of VCFS patients in group 1 and 88 by aCGH were selected for further validation by qPCR. SYBR Green analysis on an ABI 7900 HT The MLPA results showed that all 66 patients (patients Sequence Detection System (Applied Biosystems) was 1 to 66) who simultaneously exhibited CMF and palatal used to quantify copy numbers. Primer 5.0 was used to anomalies have 22q11.2 heterozygous deletion, of whom design primers to amplify the region of interest, and the 62 (93.93%) exhibited typical 3-Mb deletion, and four sequences of these primers are listed in table 5 in elec- (6.07%) displayed proximal 1.5-Mb deletion (table 1 in tronic supplementary material. Each qPCR run included electronic supplementary material). No case was found amplification of an endogenous control with known copy with atypical deletion and duplication of 22q11.2. μ number (RPP30-71 and RPPH1-63). In total, 10 L reac- For common phenotypes, 32 patients (48.48%) exhib- tion solutions were used with 10 ng DNA according to ited incomplete cleft palate (ICP) or occult submucous the manufacturer’s recommended protocols. qPCR was cleft palate, whereas 34 patients had CVPI among these conducted in duplicate. The CNVs were calculated as 1−(C samples−C internal reference) 66 patients. Twenty-three patients (34.85%) suffered from 2 . CHDs, 50 patients (75.76%) exhibited cognitive or behavioural disorder and 38 patients (57.58%) had immune Expression patterns of possible pathogenic genes deficiency (table 1 in electronic supplementary material). For uncommon phenotypes, 33 patients (50%) showed The expression patterns of possible pathogenic genes were other uncommon phenotypes, such as epilepsy, funnel obtained from the database of Mouse Genome Infor- chest and enamel hypomineralization. Among these matics (http://www.informatics.jax.org/), Gene Expression uncommon phenotypes, seven patients (10.61%) exhibited Atlas (http://www.ebi.ac.uk/rdf/services/atlas/) and NCBI epilepsy, and 16 (24.24%) showed growth retardation or (https://www.ncbi.nlm.nih.gov/gene/). Wemainly focussed short stature. No patient exhibited CCP or cleft lip in Clinical and genetic profiles of Chinese VCFS patients Page 5 of 11 42

Figure 2. Images of MLPA analysis using the P250-B1 DiGeorge kit. (a) Patients exhibited normal copy numbers. (b) Patients 67–69 exhibited TOP3B heterozygous duplications within 22q11.2. (c) Patient 77 exhibited LZTR1 heterozygous deletion within 22q11.2. (d) Patient 88 exhibited 4q34 (SLC25A4, KLKB1) heterozygous deletion (red spots). all these 66 patients (table 1 in electronic supplementary process (figure 3, b–e) (Wamstad and Bardwell 2007). material). Previous studies showed that Top3b plays a crucial role No chromosomal abnormalities were detected by using in catalysing RNA transesterification, and works with the the MLPA kit in all 325 controls (figure 2a; tables 3 and fragile X syndrome protein to promote synapse forma- 4 in electronic supplementary material), which further tion (Stoll et al. 2013; Xu et al. 2013). Knock out of confirmed that nonsyndromic palate anomalies or CHD Top3b in mice would lead to reduced mean lifespan, and would exhibit no 22q11.2 deletion or duplication, and results in inflammation or autoimmunity in many impor- most 22q11.2 deletion in patients was de novo. tant organs (table 6 in electronic supplementary material) (Kwan and Wang 2001; Kwan et al. 2003, 2007). Further, previous studies also reported Top3b duplication or dele- CNVs and clinical characteristics of VCFS patients in group 2 tion in a small number of cases when performed a screening in a relatively large samples of CHD or schizophrenia The MLPA results showed that all the three patients patients (table 6 in electronic supplementary material) had 22q11.2 duplication, and this region only includes (Kaufman et al. 2016). In this study, we firstly reported Top3b(figure 2b; table 1 in electronic supplementary mate- that patients simultaneously having palatal anomalies and rial). The aCGH results further confirmed the findings mild-cognitive disorder (without other phenotypes) would (figure 3a). The Gene Expression Atlas on-line database exhibit a higher ratio with Top3b duplication (3/3, 100%). showed that Top3b expressed in the brain and branchial Further, we also find that patient 67 has 7p21.3 dupli- arch tissues during the mouse embryonic development cation (figure 1 in electronic supplementary material; 42 Page 6 of 11 Dandan Wu et al.

Figure 3. (a) aCGH detection results showed that one 264 kb copy duplication of 22q11.2 (22,315,092–22,578,983) in patients 67, 68 and 69. (b) Top3b expression patterns in the mouse brain obtained from the Gene Expression Atlas on-line database. (c–e) Top3b expression patterns in the E8.5 (c), E9.5 (d) and E10.5 (e) mouse tissues from first branchial arch obtained from the Gene Expression Atlas on-line database. E, embryonic. table 1), which may contribute to growth retardation and The MLPA results showed that patient 77 exhibited morphology anomalies. Previous study reported a case LZTR1 duplication (figure 2c). But aCGH results did not with 7p21.3-p22 duplication exhibiting slow response, detect this variation, and excluded this false-positive result. facial anomalies, hypoimmunity and pulmonary hyperten- Although all patients in group 3 exhibited no deletion sion. Although, patient 67 exhibited no such phenotypes or duplication of 22q11.2, the aCGH and qPCR results at the current stage, we should pay attention in future showed that four patients (40.0%) exhibited other CNVs follow-up. in these patients (table 1). We firstly showed that patient 70 has a deletion in 4q23 (figure 2a in electronic supplementary material), CNVs and clinical characteristics of VCFS patients in group 3 and only C4orf37 (also named STPG2) located in this region (table 1). Although no STPG2 variation related For common phenotypes, all 11 patients (100%) exhib- to human disease has been reported, previous findings ited cleft palate, and three patients exhibited CCP,whereas showed that SRCAP mutation contributed to Floating- no patients with CVPI. For uncommon phenotypes, eight Harbour syndrome and would lead to STPG2 hyperme- patients (72.73%) have uncommon phenotypes, and three thylate (Hood et al. 2016). Previous records also showed patients (27.27%) exhibited cleft lip (table 1 in electronic that STPG2 expressed in the cardiovascular system, brain supplementary material). and haemolymphoid system, which implied that STPG2 Clinical and genetic profiles of Chinese VCFS patients Page 7 of 11 42 , , needs to be taken into consideration (table 7 in electronic , ,

, supplementary material).

, There was also Xp11.4 deletion in patient 72 (ﬁgure FRG2 , SPATA4 TBX4 , CASP3 , , , 2b in electronic supplementary material), and BCOR was FOXI3 , LOC285441 , , the only gene in this region (table 1). Previous studies IRF2 C4orf47

F11 showed that BCOR variation contributes to oculofacio- , , , MGC45800 WDR17 TUBB4Q C17orf82 , , CLDN24 , cardiodental syndrome, an X-linked syndrome, which was , , THNSL2

, also similar to this patient (Horn et al. 2005; Davoody UFSP2 ENPP6 KLKB1 FRG1 , , et al. 2012). Further, previous study also demonstrated , TBX2 , , GPM6A

, that BCOR expressed in the mice eye, brain, neural tube FABP1 CLDN22 , , and branchial arches correlates with tissues affected in STOX2 NCRNA00290 , , BCAS3 CYP4V2 oculofaciocardiodental patients (table 7 in electronic sup- , , ANKRD37 , WWC2 ADAM29 plementary material). To further conﬁrm our ﬁndings, LOC401164 SMYD1 , , , , we input these phenotypes into the Phenolizer and Phe- C4orf41 ,

PPM1D nomizer on-line phenomics database to obtain genetic , PRR20E FAM149A LOC285501 LRP2BP GLRA3 , C4orf38 , diagnosis, and the results showed that BCOR was one , , , KRCC1 , , TRIML1 ,

, of the most possible pathogenic genes contributed to this TBC1D3P2 RWDD4 , AGA , TLR3

, patient (ﬁgure 3 in electronic supplementary material). , APPBP2 SNX25 SLED1 HPGD , PRR20B , , , aCGH results also showed that patient 74 exhibited RGPD2 , TULP4 , ANKRD36BP2 ING2 , FAM92A3 TRIML2 ,

, 13q21.1 duplication (ﬁgure 2c in electronic supplementary , , MED13 LOC729852 NEIL3 , , , material), and there were ﬁve genes located in this region SORBS2 ACSL1 , , C17orf64 RPIA MIR4276 RGPD1

PRR20A (table 1). These genes encoded proteins that were rich in , , KIAA1430 DCTD , , ZFP42 , RPA3 GTF2H5 , , INTS2 , ,

, proline and D4 dopamine receptor, and previous cases , VEGFC ,

LOC389247 reported that PRR20A may contribute to cleft lip (table 7 , MLF1IP PDLIM3 FAT1 ODZ3 , , MIOS , in electronic supplementary material) (Chengle et al. 2010; , EIF2AK3 PLGLB1 , PRR20D BRIP1 , , MIR3182 , , SLC25A4 KIAA1712 SERAC1 SCARNA20 SPCS3 Song et al. 2015). , , , , , , Further, patient 78 had 17q23.2 duplication (ﬁgure 2d in electronic supplementary material), and there were 14 ASB5 MIR1305 CDKN2AIP NACA2 CCDC111 HELT MTNR1A C2orf51 CCDC110 COL28A1 C4orf37 BCOR PRR20C SYNJ2 USP32 PLGLB2 TMEM45B CDH13 FBXO8 genes located in this domain (table 1). Among these genes, previous studies showed that TBX2 and TBX4 play crucial roles in heart and craniofacial development, and TBX2 duplication would lead to mild mental retardation, growth retardation, cerebellar hypoplasia, complex heart defect and minor skeletal anomalies (table 7 in electronic supplementary material), which were similar to the phenotypes in this patient (Radio et al. 2010; Mesbah et al. 2012). The possible pathogenicity for these 14 genes was sorted by using the phenolizer database based on the patients’ phenotypes, and the results showed that TBX2 and TBX4 were related to the patients’ phenotypes (ﬁgure 4a in electronic supplementary material).

CNVs and clinical characteristics of VCFS patients in group 4

For common phenotypes, ﬁve patients (83.33%) exhibited CVPI, and only one patient exhibited occult submucous cleft palate. No CCP/ICP was observed. For uncommon phenotypes, two patients (33.33%) had uncommon phenotypes (table 1 in electronic supplementary material). aCGH results showed that there was a 2p11.2 dele- 6q25.3 158,505,474 158,759,951 254.5 kb 3 11q24.3 129,525,300 129,690,730 165.4 kb 3 tion in patient 85 (ﬁgure 5a in electronic supplementary material), and previous study reported that 2p11.2 dele-

Clinical pathogenic CNVs detected by aCGH chip and qPCR. tion may contribute to Potocki–Lupski syndrome that exhibited recognitive impairment, growth retardation, CHD and morphology anomaly (table 8 in electronic Table 1. Case no. Chromosomal region Initiation site Termination site67 Sizes Copy number 7p21.3 7,532,349 7,913,761 381.4 kb Contained genes 3 707274 4q23 Xp11.4 13q21.1 99,058,770 3,895,253 57,216,730 99,170,809 40,023,503 57,758,257 112.0 kb 1.1 541.5 Mb kb 1 3 1 78 17q23.2 58,301,487 60,373,562 2.1 Mb 3 85 2p11.2 88,046,608 89,129,064 1.1 Mb 1 88 4q34.1-qter 174,561,232 190,957,473 16.4 Mb 1 86 16q23.3 82,884,663 83,781,604 896.9supplementary kb 3 material). There were 13 genes located in 42 Page 8 of 11 Dandan Wu et al. this region (table 1), and previous findings showed that anomalies, intellectual disability, development retardation SMYD1 plays crucial roles in cardiac, skeleton muscle and and limb deformities. The ISCA database (https://www. T-cell development (table 8 in electronic supplementary iscaconsortium.org) showed that 6q25.3 duplication may material) (Rasmussen et al. 2015; Doughan et al. 2016). contribute to development retardation and abnormal mor- The possible pathogenicity for these 13 genes was sorted phology. There were four genes located in this region by using the phenolizer database based on the patients’ (table 1), and TULP4 had been proved to be a candidate phenotypes, and the results showed that SMYD1 were gene for craniofacial cleft and short stature. Further, pre- related to the patients’ phenotypes (figure 4b in electronic vious study showed that 6q25.3 deletion may contribute to supplementary material). Additionally, previous findings patient’s facial anomalies and abnormal behaviour (table also demonstrated that FOXI3 plays a crucial role in pha- 9 in electronic supplementary material) (van Duyvenvo- ryngeal arch, molar, ectodermal development, and FOXI3 orde et al. 2014; Vieira et al. 2015). These finding further duplication may contribute to congenital aural atresia supported that 4q34-qter deletion and 6q25.3 duplication, (table 8 in electronic supplementary material) (Jussila et al. especially KLKB1 and TULP4, may be contributed factors 2015; Tassano et al. 2015). These findings implied that for this patient. SMYD1 and FOXI3 may be the possible pathogenic genes for this patient. In addition, this patient also had 11q24.3 duplication (figure 5b in electronic supplementary mate- Comparison of CNVs and clinical characteristics among the five rial), and there was only TMEM45B located in this region. groups aCGH and qPCR results also showed that patient 86 had 16q23.3 duplication (figure 5c in electronic sup- Although there were a small number of patients in groups plementary material; table 1), and the ISCA database 2 and 5, the gender combination showed that there were (https://www.iscaconsortium.org) showed that 16q23.3 more female patients in groups 1, 3 and 4 (figure 4a). For duplication may contribute to development retardation palatal anomalies, there was a similar ratio of ICP and and facial anomalies. In addition, previous study reported CVPI patients in group 1, and a higher ratio of ICP patients that patients with 16q23.3 duplication combined with in group 3, but there were a higher ratio of CVPI patients 5p15.32 deletion exhibited facial anomalies, intellectual in group 4 (figure 4b). There were no CVPI patients in disability and speech disorder, which was similar to this group 3 but only group 3 has CCP patients. For uncom- patient, and further confirm that 16q23.3 duplication may mon phenotypes, there was a similar ratio of patients with be a contributed factor. There were only two genes in this or without uncommon phenotypes in group 1, and a higher region, and previous study showed that CHD13 may con- ratio of patients with uncommon phenotypes in group 3, tribute to schizophrenia, metabolic syndrome and tumour but a lower ratio of patients with uncommon phenotypes in (table 8 in electronic supplementary material) (Fava et al. group 4 (figure 4c). For CNVs, there was high heterogene- 2011). ity for CNVs in groups 2 to 5 and no same CNVs in these groups, but homogeneity for CNVs in group 1. Further, the ratio of patients with CNVs in groups 1, 2 and 5 was CNVs and clinical characteristics of VCFS patients in group 5 100%, whereas the ratios of patients with CNVs in groups 3 and 4 were 33.36 and 33.33%, respectively (figure 4d). The MLPAresults showed that patient 87 had 22q11 duplication (table 1 in electronic supplementary material). The MLPA results also showed that patient 88 had 4q34.1 dele- Discussion tion, and SLC25A4 and KLKB1 located in this region (figure 2d; table 1 in electronic supplementary material). Rare, recurrent copy number variants of pathogenic Previous findings showed that KLKB1 may contribute to significance termed genomic disorders (Girirajan et al. heart disease, submucosal cleft palate, hypernasal speech 2012), and VCFS is one of the most common human and learning difficulties (table 9 in electronic supplemen- genomic disorders with extreme phenotypic heterogeneity tary material) (Gittleman et al. 2016). The aCGH results (McDonald-McGinn et al. 2015). Molecular diagnosis further confirmed that patient 88 had 4q34.1 deletion offered some choices for VCFS patients, and is important (figure 6a in electronic supplementary material), but there for avoiding missed diagnosis, early intervention, treat- was a long region and 64 genes located in this region ment, genetic counselling and prognosis. However, the (table 1), and the possible pathogenicity for these 64 interpretation of CNVs associated with phenotypic vari- genes was sorted by using the phenolizer database based ation is still challenging (Jalali et al. 2008; Guo et al. on the patients’ phenotypes (figure 4c in electronic sup- 2015; Poirsier et al. 2016). In this study, we offered some plementary material). Further, patient 88 also exhibited new genetic information for VCFS, and presented a step 6q25.3 duplication (figure 6b in electronic supplemen- towards classification diagnosis for Chinese VCFS patients tary material). Previous study showed that patients with based on a phenotypic strategy, which is helpful for molec- 4q34-qter deletion would exhibit cleft palate, CHD, facial ular diagnosis and avoiding missed diagnosis, as well Clinical and genetic profiles of Chinese VCFS patients Page 9 of 11 42

Figure 4. Comparison of CNVs and clinical characteristics among the five groups. (a) There were more female patients in groups 1, 3 and 4. (b) There was a similar ratio of ICP and CVPI patients in group 1, and a higher ratio of ICP patients in group 3, but a higher ratio of CVPI patients in group 4 and no CVPI patients in group 3, whereas only group 3 had CCP patients. (c) There was a similar ratio of patients with or without uncommon phenotypes in group 1, and a higher ratio of patients with uncommon phenotypes in group 3, but a lower ratio of patients with uncommon phenotypes in group 4. (d) Ratio of patients with CNVs in groups 1, 2 and 5 was 100%, whereas the ratios of patients with CNVs in groups 3 and 4 were 33.36 and 33.33%, respectively. ICP, incomplete cleft palate; CCP, complete cleft palate; CVPI, congenital velopharyngeal insufficiency; UP, uncommon phenotypes; CNVs, copy number variations; **P < 0.01. as early intervention, treatment, genetic counselling and that patients simultaneously having palatal anomalies and prognosis for patients with VCFS. mild-cognitive disorder (without other phenotypes) would The phenotypes of VCFS exhibited heterogeneity exhibit a higher ratio with Top3b duplication (3/3, 100%). among different populations, different patients and inter- Previous finding also showed that Top3b may works generational or intrafamilial members (Kobrynski and with the fragile X syndrome protein to promote synapse Sullivan 2007; Kruszka et al. 2017). Many possible mech- formation (Stoll et al. 2013; Xu et al. 2013), which strongly anisms may explain the heterogeneity, such as deletions suggested that Top3b was a pathogenic gene for these of different sizes and location and the extension of an patients. In addition, the phenotypes of patients 67 to unstable mutation at the 22q11.2 locus, CNVs as well as 69, combined with the phenotypes of patient 87 who has other chromosomal anomalies have molecules in a com- 22q11.2 duplication showed that there was difference in the mon genetic pathway or in functionally related pathways phenotypic spectrum between the duplication and deletion to pathogenic gene in 22q11.2 (Perez and Sullivan 2002). of 22q11.2. Our previous preliminary findings showed that all VCFS Although there was homogeneity for CNVs in groups patients with palatal anomalies and CMF would have a 1 and 2, CNVs in other three groups exhibited het- 22q11.2 microdeletion (Wu et al. 2013), which implied erogeneity, and not all patients could be detected with that there may be some regularity among these com- CNVs. Further, only one patient in these groups had mon phenotypes and CNV patterns. In this study, we 22q11.2 duplication, whereas no 22q11.2 microdeletion further confirmed that palatal anomalies and CMF were had been detected, which implied that these patients may absolute indicators for 22q11.2 microdeletion, which sug- have different pathogenic mechanisms when compared gested that patients should take the 22q11.2 microdeletion to patients with 22q11.2 microdeletion. Except for sin- into consideration. In this study, we also firstly reported gle CNVs, there may also be other mechanisms such as 42 Page 10 of 11 Dandan Wu et al. second copy-number variants, single-nucleotide changes, with palatal anomalies should be classified in detail as gene mutation, epigenetic or stochastic factors altering velopharyngeal insufficiencies, submucous cleft palate or the expression of genes within functionally relevant path- isolated cleft palate, would be a beneficial complement for ways, contribute to these phenotypes (Girirajan et al. this study. 2012). Thus, for these patients, MLPA may not be the first choice for examination, and aCGH, even whole exon Acknowledgements or genomic sequencing may be priority choice for these patients. This work was supported by the Interdisciplinary Program of In this study, we also reported many possible pathogenic Shanghai Jiaotong University (nos. YG2016MS08 and CNVs and genes, such as BCOR, PRR20A, TBX2, YG2016QN11), the National Key Research and Development SMYD1, KLKB1 and TULP4, in groups 3, 4 and 5. Program of China (no. 2016YFC1000502), the National Natu- ral Science Foundation of China (no. 81300842) and Morning Previous studies also reported that some of these genes Star Rewarding Fund of Shanghai Jiaotong University. related to different phenotypes. For example, BCOR in group 3 contributes to oculofaciocardiodental syndrome that exhibited many uncommon phenotypes of VCFS References (Horn et al. 2005; Davoody et al. 2012). KBKL1 and TULP4 in group 5 contributed to craniofacial cleft, Chengle H., Kaihong D. and Fuzhi B. 2010 Association anal- heart disease, hypernasal speech and learning difficul- ysis of the poliovirus receptor related-2 gene in patients with nonsyndromic cleft lip with or without cleft palate. DNA Cell ties (Vieira et al. 2015; Gittleman et al. 2016). In this Biol. 29, 681–685. study, we could also find that patients in groups 3 Davoody A., Chen I. P., Nanda R., Uribe F. and Reichenberger and 5 exhibited more phenotypes, whereas patients in E. J. 2012 Oculofaciocardiodental syndrome: a rare case and group 4 exhibited relatively less phenotype. Phenomics review of the literature. Cleft Palate Craniofac. J. 49, e55–e60. databases, such as phenolizer and phenomizer on-line Doughan M., Spellmon N., Li C. and Yang Z. 2016 SMYD proteins in immunity: dawning of a new era. 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Corresponding editor: Indrajit Nanda