Prenatal genetic diagnosis of Williams-Beuren syndrome with atypical and complex phenotypes using SNP array and whole exome sequencing

Prenatal genetic diagnosis of Williams-Beuren syndrome with atypical and complex phenotypes using SNP array and whole exome sequencing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prenatal genetic diagnosis of Williams-Beuren syndrome with atypical and complex phenotypes using SNP array and whole exome sequencing weiqiang Liu, Dingya Cao, Jinshuang Song, Tong Zhang, Shuxian Zeng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4261789/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Williams-Beuren syndrome (WBS) is a severe congenital disorder. Prenatal diagnosis of WBS is difficult because the phenotypes of WBS fetuses are atypical or incomplete. This study used ultrasound, SNP array, and whole exome sequencing to analyze the association between genotype and complex phenotype in fetuses with WBS. Methods Chromosomal microarray analysis (CMA) and whole genome sequencing were performed in pregnant women with prenatal diagnosis. Genome-wide copy number variants (CNVs), regions of homozygosity (ROH), single nucleotide variants (SNVs), small insertions and deletions, and splice sites were analyzed. Results The 7q11.23 deletion was identified in seven fetuses out of 6718 prenatal diagnostic samples; ultrasound revealed that two fetuses had apparent cardiovascular anomalies; one fetus had persistent left superior vena cava and intrauterine growth retardation (IUGR). Two fetuses had polycystic kidney dysplasia, one of which was associated with a small amount of tricuspid regurgitation; the other two fetuses had no apparent ultrasound abnormalities. Detailed genetic analysis revealed CNVs ranging in size from 1.43 megabase pairs (Mb) to 1.66 Mb, affecting 34 to 41 genes, respectively. On average, 1.0 additional CNVs larger than 100 kilobase pairs of unknown significance and 0.43 ROH larger than 5 Mb were detected in these cases. The pathogenic or likely pathogenic SNV or splice site with the function of renal development and cardiovascular development was also identified in these cases. Conclusion The phenotype of WBS fetuses is atypical and complex, and the complex phenotype does not exclude association with other variants within the genome. Williams-Beuren syndrome prenatal diagnosis phenotype chromosome microarray analysis whole exome sequencing Figures Figure 1 Figure 2 Figure 3 Background Williams-Beuren syndrome (WBS; also called Williams syndrome, WS; OMIM 194.050) is a distinct and rare neurodevelopmental disorder with a prevalence of approximately 1 in 7,500 to 1 in 20,000 live births [ 1 , 2 ] . The major clinical features of WBS are cardiovascular disease (such as coarctation of the aorta, peripheral pulmonary stenosis, and hypertension), mild to moderate mental retardation, developmental delay, growth retardation, physical features, cognitive profile, facial features, end profile, facial features, endocrine abnormalities, and abnormal neurocognitive profile [ 3 , 4 ] . The genetic diagnosis of WBS is made by identifying a heterozygous microdeletion in the WBS chromosomal region (WBSCR) located on chromosome 7q11.23, with a deletion size ranging from 1.55 megabase pairs (Mb) to 1.83 Mb [ 2 , 3 ] . Deletions of significant size within this region can result in clinically distinct phenotypes [ 5 – 7 ] . The critical pathogenic region of WBS has been shown to contain 31 known genes. The cardiovascular and other phenotypes of WBS correlate with the size of its deletion and with several genes such as ELN , FZD9 , BAZ1B , STX1A , LIMK1 , CLIP2 , GTF2I are known to contribute to the complex phenotypes [ 3 , 8 ] . However, no single gene has been identified in which a pathogenic variant is the cause of WBS. The understanding of how these genes contribute to the WBS phenotype is still evolving. To date, the majority of patients with WBS are diagnosed postnatally [ 9 ] . The primary indication for prenatal diagnosis of WBS is mainly based on ultrasound anomalies. However, fetuses with WBS may not exhibit the typical WBS phenotype during pregnancy, and some phenotypes are non-specific, such as increased amniotic fluid, intrauterine growth retardation (IUGR), short fetal femur length, and polycystic kidney dysplasia [ 10 , 11 ] . The association of these phenotypes with the deletion of the 7q11.23 region is unclear and requires further elucidation. With the widespread use of CMA as a first-tire technique for prenatal diagnosis, an increasing number of fetuses with WBS are being diagnosed prenatally. About 100 prenatal cases of WBS with various atypical phenotypes have been reported in the literature, but more attention needs to be paid to information on genetic variants other than the 7q11.23 deletion in these studies. In this study, we synthesized multidimensional evidence from CMA, whole exome sequence analysis, and imaging data to analyze the correlation between complex phenotypes and genotypes of WBS and provide a more accurate diagnosis for the precise clinical targets for treatment. Materials and Methods Subject The CMA outcomes of 6718 pregnant women with CMA, who were diagnosed prenatally at our hospital and the Third Affiliated Hospital of Guangzhou Medical University from January 2019 to August 2023, were retrospectively analyzed. The pregnant women were aged 20 to 45 years, and their gestational weeks ranged from 11 + 2 to 35 + 4 weeks. The medical ethics committee of Longgang District Maternal and Child Health Hospital approved the study (No. LGFYYXLLQF-2022-003), and all subjects signed an informed consent form. Ultrasound examination Each case underwent a routine ultrasound examination at a median gestational age of 24 (range 11–35) weeks. The ultrasound machine used for the examinations was a Voluson E10 (GE Medical Systems). The scanning frequency ranged from 1 MHz to 5 MHz. A Level III fetal anatomic scan was performed in each case, and any associated abnormal findings were documented and recorded. A Level III fetal anatomic scan was performed in each case, and any associated abnormal findings were documented and recorded. Amniotic fluid sampling and DNA extraction A 5 ml amniotic fluid sample was collected according to instructions and used for DNA extraction using the QIAamp DNA Blood Mini Kit (No. 51106, Qiagen, Germany). CMA analysis An Affymetrix Cytoscan 750K chip (Thermo Fisher, USA) was used. The extracted DNA was digested, ligated, PCR amplified, purified, fragmented, and labeled for hybridization according to the instructions, and then the washed and stained chip was scanned, and the data were collected and analyzed using Chromosome Analysis Suite (ChAS 4.3) software. Aneuploidy, copy number variation (CNV), and region of homozygosity (ROH) were analyzed, and CNV and ROH were filtered with a threshold of 100 kilobase pairs (Kb) and 5 Mb, respectively. The CNV scoring criteria were based on the interpretation guideline [ 12 ] . Whole exome sequence analysis Four cases of prenatal WBS were sequenced by whole-exome sequencing. DNA extraction and purification of the samples were performed using a kit (DP705, TIANamp Genomic DNA Kit, China); library construction was performed according to the manufacturer's instructions (ND617, Vazyme, China). The library was sequenced on a GenoLab M (Genemind, China) sequencer with a pair-end (PE) read length of 150bp and an average sequencing depth of not less than 20 folds. After decontamination and trimming, the raw sequencing reads were subjected to BWA-GATK ( https://software.broadinstitute.org/gatk/ ) with ClinSV ( https://bio.tools/clinsv ) for bioinformatics analysis. Then, all SNPs and INDELs were annotated using ANNOVAR software ( http://annovar.openbioinformatics.org/en/latest/ ), and all structural variants were annotated using AnnotSV software ( https://lbgi.fr/AnnotSV/ ). Results CNV analysis focused on the 7q11.23 region 7 cases with heterozygous deletions in the 7q11.23 region were detected. The deletion size ranged from 1.43 Mb to 1.66 Mb, affecting 34 to 41 genes (Fig. 1 , Table 1 ). Table 1 CNVs Recognized with CMA and Genes Involved Case no. Microarray Nomenclature Size (kbp) Number of genes (protein-coding genes) Genes contains classification 1 7q11.23 (72692113_74154209)x1 1,462 37(25) gtf2ird2p1, pom121b, nsun5, trim50, rpl7ap77, fkbp6, rnu6-1080p, fzd9, baz1b, rnu6-1198p, bcl7b, tbl2, mlxipl, vps37d, dnajc30, bud23, stx1a. mir4284, rn7sl265p, bicdl3p, abhd11, cldn3, cldn4, mettl27, tmem270, eln, eln-as1, limk1, eif4h, mir590, lat2, rfc2, clip2, gtf2ird1, rna5sp233. gtf2i,gtf2i-as1 P 2 2q37.1 (233917755_234137480)x3 220 1(1) INPP5D VUS 7q11.23 (72624167_74288694)x1 1,665 41(27) NCF1B , ..(same as case 1).... phb1p15,ncf1,gtf2ird2 P 8q21.13(82088743_82229807)x3 141 1(1) FABP5 VUS 3 7q11.23 (72669481_74154209)x1 1,485 37(25) Same as case 1 P 4 7q11.23 (72664089_74154209)x1 1,490 37(25) Same as case 1 P 18p11.32 (283770_462315)x3 179 1(1) COLEC12 VUS 5 7q11.23 (72669481_74146927)x1 1,477 37(25) Same as case 1 P 6 6q14.2 (84606383_84776946)x3 171 2(2) CYB5R4, MRAP2 VUS 7q11.23 (72723371_74154209)x1 1,431 34(24) TRIM50, ..(same as case 1).. GTF2I-AS1 P 9p21.1 (30587632_30697418)x1 110 0 / VUS Xq28(148128198_148571167)x2 443 1(1) IDS VUS 7 7q11.23 (72669481_74154209)x1 1,485 37(25) Same as case 1 P 6q21(110802069_110934453)x3 132 1(1) CDK19 VUS *Abbreviation P: pathogenic; VUS: variants unknown significance. Genes name marked as red means protein-coding genes. CNV detections outside the 7q11.23 region In the 7 cases, 6 duplications and one deletion were observed outside the 7q11.23 region. According to the CNV interpretation guideline, all additional CNVs detected were classified as having unknown clinical significance, 2, 1, 2, and 1 additional duplicated CNVs were detected in cases 2, 4, 6, and 7, respectively, and 1 additional deletion was detected in case 6 (Table 1 ). Analysis of ROH ROH analysis was performed using 5 Mb and 50 markers. Each ROH was detected in cases 2, 3, and 7, and no ROHs were detected in the remaining cases (Table 2 ). Table 2 ROH detection in WBS fetal samples Case ROH Size Mb Number of genes included (Number of protein-coding genes) 1 / / / 2 17q11.1q11.2 (25309337_30511274)x2 hmz 5.2 148(70) 3 11p11.2p11.12 (45546761_51550787)x2 hmz 6.0 117 (57) 4 / / / 5 / / / 6 / / / 7 3p21.31p21.1(46700713_52738165)x2 hmz 6.0 222 (141) Ultrasound findings Ultrasound was performed in seven cases of WBS, and cardiac or cardiovascular anomalies were detected in four fetal ultrasound findings (57.14%, 4/7). Of these, one suggested pulmonary artery anomalies (case 1), one had significant cardiovascular anomalies such as an enlarged right heart with a thin aortic isthmus (case 5), one suggested a slightly smaller left heart with a small amount of tricuspid regurgitation (case 2), and one suggested a permanent left superior vena cava with intrauterine growth retardation (IUGR) (case 6). In cases 2 and 7, ultrasound suggested the presence of polycystic renal dysplasia (28.57%, 2/7). The other two fetuses (cases 3 and 4, 28.57%, 2/7) had no significant cardiovascular abnormalities on ultrasound, except for the suggestion of intense light in the left heart of case 3. The ultrasound findings of the seven fetuses are shown in Table 3 and Fig. 2 . Table 3 Fetal ultrasound findings or prenatal indications in 7 cases of WBS Case Maternal age Gestational week Ultrasound phenotype or indication for prenatal diagnosis 1 37 23 + 2 Enlarged right fetal heart, permanent left superior vena cava, left aortic arch with right clavicle; inferior arterial vagus, aortic isthmus distortion. 2 27 28 + 0 Left polycystic dysplastic kidney; left heart slightly smaller, mild tricuspid regurgitation 3 36 18 + 3 NIPT high risk for CNV in 7q11.23, strong light spot in left heart 4 22 23 + 2 NIPT high risk for CNV in 7q11.23 5 29 32 + 2 Pulmonary Artery Anomalies 6 44 31 + 1 Intrauterine growth retardation, permanent left superior vena cava 7 30 32 + 6 Left polycystic dysplastic kidney Whole exome sequencing To further analyze the reasons for the differences in the phenotypes of the WBS fetuses, whole exome sequencing analysis was performed in four samples. No homozygous variants were identified in the 7q11.23 region, while the other 7q11.23 allele was deleted. Only one homozygous variant was found in the ROH region of case 2, but the clinical significance of this variant was unknown. For phenotype-genotype relationship analysis, the variants associated with atrioventricular septal defect or congenital heart defects were identified in case 1, case 2, and case 3. In addition, the pathogenic or likely pathogenic incidental identified variants were also found in these cases (Fig. 3 , Supplementary Table). Discussion The clinical phenotypes of WBS mainly include malformations of the cardiovascular system, peculiar facial features, connective tissue abnormalities, endocrine abnormalities, growth, and mental retardation, and cognitive difficulties [ 13 ] . Only about 100 cases of prenatally diagnosed WBS have been reported in the literature [ 10 , 11 , 14 – 17 ] , and most of the WBS cases were diagnosed after birth. An important reason is that the phenotypes of WBS cases are atypical or incomplete in the fetal period, resulting in missed prenatal diagnosis. Although cardiovascular abnormalities are an essential phenotype of WBS, only 42.9–58.82% of WBS fetuses have ultrasound-detectable abnormalities [ 10 , 11 ] . There are a higher number of other non-specific phenotypes in WBS fetuses, such as IUGR, abnormal fetal placental Doppler indices, thickened NT/ NF, polyhydramnios, fetal hydrops, duodenal atresia, echogenic bowel, intracardiac echogenic focus (IEF), and other less common phenotypes [ 10 , 11 , 15 ] . The reasons for the complex phenotype of WBS remain to be elucidated. In WBS patients, complex phenotypes may be associated with differences in the size of CNVs and their affected genes in WBS fetuses. It has been shown that ninety percent of deletions in the 7q11.21 region of WBS patients are ~ 1.5 Mb, 5%-8% are ~ 1.8 Mb, and only 2%-5% are atypical [ 18 , 19 ] . The variable size of CNVs leads to inconsistencies in the number of genes involved, especially protein-coding genes. Besides the prominent ELN gene, genotype-phenotype evidence suggests that the transcription factor genes GTF2I and GTF2IRD1 may lead to intellectual functioning, social functioning, and anxiety. There is also increasing evidence that BAZ1B , LIMK1 , STX1A , and MLXIPL deletions have phenotypic consequences. However, more work is needed to understand how these genes contribute to clinical outcomes [ 3 ] . Five of the seven cases in this study shared the same deleted genes. Four additional genes, including two protein-coding genes, NCF1 and GTF2IRD2 , were deleted in case 2. Case 6 had a slightly smaller CNV fragment involving 34 genes, 24 of which are protein-coding genes, and it retained the protein-coding gene NSUN5 without deletion compared to the other cases. Functional analysis of the above differential genes suggests that the NCF1 gene encodes a component of the NADPH oxidase complex [ 20 ] . Although NCF1 is associated with chronic granulomatous disease (CGD), an autosomal recessive disorder, heterozygous deletion of NCF1 may be relevant in the form of essential hypertension and vascular stiffness in WBS individuals [ 21 , 22 ] . Although deletion of GTF2IRD2 has been shown to result in more impaired visuospatial abilities and more significant behavioral problems [ 23 ] , the function of GTF2IRD2 and NSUN5 and their relationship to complex WBS phenotypes require further investigation. Interestingly, although cases 1, 3, 4, 5, and 7 had the same gene deletion, they had different phenotypes. For example, case 7 was diagnosed prenatally because of ultrasound findings of fetal polycystic kidney, whereas case 4 was diagnosed because NIPT suggested high risk, and the cardiovascular abnormality phenotype was not observed at 23 weeks of gestation. The discrepancy in the in-utero phenotypes may be due to limited and incomplete prenatal evaluation or suggest clinical heterogeneity in the WBS individuals and may also be related to other unknown genetic variants. In a detailed genetic analysis of these cases, we found that in addition to the deletion of the 7q11.23 region detected in each WBS fetus, additional CNVs were detected on other chromosomes, all of which are of uncertain clinical significance according to the ACMG/ClinGen 2019 guideline. For example, the duplicated CNV on chromosome 6 in case 7 included a CDK19 gene; loss of function for CDK19 can cause mental retardation and epileptic encephalopathy (OMIM 614720) [ 24 , 25 ] but is unrelated to the triplet duplication. Case 4 had an additional deletion CNV on chromosome 18, including the COLEC12 gene (OMIM 607621), a class of c-type lectins with collagen-like sequences and carbohydrate-recognizing structural domains [ 26 ] , but its relationship to disease is currently unclear. There is no evidence to suggest whether these additional CNVs have a second hit or co-expression effect on the complex phenotype of WBS. However, the cumulative genotypic and phenotypic information will contribute to understanding the complex phenotype of WBS. We also analyzed ROH in this study for a more detailed analysis of the relationship between genotyping and phenotyping. Although three ROHs were detected in three cases, none were disease-causing regions. Whole exome sequencing was performed to exclude the homozygous variants that occurred in the protein-coding genes in these ROH regions, and except for a VUS variant detected in the ADAP2 gene in case 2, no additional homozygous pathogenic variants were identified within these ROH regions. Although haploinsufficiency of the ADAP2 gene has been implicated in the occurrence of cardiovascular malformations in NF1 microdeletion patients [ 27 ] , the clinical correlation between the ADAP2 gene and disease is currently unknown. Therefore, ROH may not influence the phenotypic complexity of WBS. Our analysis of whole-exome sequencing in four cases found no homozygous variants in approximately 25 protein-coding genes in the 7q11.23 region. Furthermore, WES sequencing suggests that other variants in the genome might also contribute to the complex phenotype observed in WBS patients. In case 1, who had classic cardiovascular abnormalities, we found the CRELD1 variant. Although the clinical significance of this variant is unknown, CRELD1 variants can cause atrial septal defects [ 28 ] and are associated with bicuspid aortic valve anomalies [ 29 ] . In case 2, with a polycystic dysplastic renal phenotype, we found a variant in the PKHD1 gene in case 2, which can lead to polycystic kidney disease [ 30 ] . Thus, the complexity of the phenotype in WBS fetuses seems to be related to other variants within the genome, and larger sample sizes are needed to validate this conclusion. Another interesting observation is that we found polycystic kidney dysplasia in two out of seven cases. It has been reported that the detection rate of renal cysts or multicystic dysplastic kidneys in fetuses with WBS is about 4.7–12.5% [ 11 , 15 ] , whereas the detection rate of polycystic kidney dysplasia in the present study is about 28.6% (2/7). The reason why so many WBS patients have the polycystic dysplastic renal phenotype needs more detailed analysis. Conclusions In summary, the prenatal ultrasound phenotype of WBS fetuses is atypical and incomplete. The phenotypic variation of WBS is influenced by the size of the microdeletion, the genes involved in the breakpoint region, the background genotype, and possibly by other unknown variants. Therefore, a more in-depth genome-wide analysis of WBS patients would be beneficial to elucidate the relationship between complex phenotypes and genotypes. Abbreviations WBS Williams-Beuren syndrome CMA Chromosomal microarray analysis CNVs copy number variants ROH Regions of homozygosity WES Whole exome sequencing IUGR Intrauterine growth retardation IEF Intracardiac echogenic focus LSVC Left superior vena cava DA Duct arterial AA Aortic arch RSVC Right superior vena cava T:Trachea ARSA Aberrant right subclavian artery P pathogenic VUS variants unknown significance. Declarations Acknowledgments We want to thank all the members of the Central Laboratory, Longgang District Maternity & Child Healthcare Hospital of Shenzhen City, who collaborated in this study. Author contributions WQ. Liu conceived and designed the study. Data analysis and manuscript writing: WQ.Liu and M.Chen; clinical data collection: DY. Cao, T. Zhang, and SX. Zeng; sample sequencing and data analysis: XY. Cong, XJ. Luo, L. Hu, and YY. Pei; ultrasound data collection: JS.S. All authors read and approved the manuscript and are accountable for all study aspects. Funding This research was supported by grants from the Medical and Health Technology Research Project, Longgang District of Shenzhen City (LGKCYLWS2021000024), (LGWJ2021-143), Medical and Health Technology Research Project, Special Funds for Science and Technology, Innovation Longgang District of Shenzhen City (LGKCYLWS2022013), Natural Science Foundation of Shenzhen City (JCYJ20230807141908017). Data availability statement Data can be made available upon reasonable request. Declarations Ethics approval and consent to participate The medical ethics committee of Longgang District Maternal and Child Health Hospital approved the study (No. LGFYYXLLQF-2022-003), and all subjects signed an informed consent form. All methods were performed according to the ethical standards in the Declaration of Helsinki and its later amendments or comparable ethical standards. Consent for publication Not applicable. Competing interests The authors have no conflict of interest to declare. References Ferrero GB, Biamino E, Sorasio L, Banaudi E, Peruzzi L, Forzano S, et al. Presenting phenotype and clinical evaluation in a cohort of 22 Williams-Beuren syndrome patients. Eur J Med Genet. 2007;50(5):327–37. 10.1016/j.ejmg.2007.05.005 . Pober BR. Williams-Beuren syndrome. N Engl J Med. 2010;362(3):239–52. 10.1056/NEJMra0903074 . Kozel BA, Barak B, Kim CA, Mervis CB, Osborne LR, Porter M, et al. Williams syndrome. Nat Rev Dis Primers. 2021;7(1):42. 10.1038/s41572-021-00276-z . Wilson M, Carter IB. Williams Syndrome. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Iverson Carter declares no relevant financial relationships with ineligible companies. 2024. Twite MD, Stenquist S, Ing RJ. Williams syndrome. Paediatr Anaesth. 2019;29(5):483–90. 10.1111/pan.13620 . Xia Y, Huang S, Wu Y, Yang Y, Chen S, Li P, et al. Clinical application of chromosomal microarray analysis for the diagnosis of Williams-Beuren syndrome in Chinese Han patients. Mol Genet Genomic Med. 2019;7(2):e00517. 10.1002/mgg3.517 . Williams syndrome. Nat Rev Dis Primers. 2021;7(1):43. 10.1038/s41572-021-00283-0 . Zhou J, Zheng Y, Liang G, Xu X, Liu J, Chen S, et al. Atypical deletion of Williams-Beuren syndrome reveals the mechanism of neurodevelopmental disorders. BMC Med Genomics. 2022;15(1):79. 10.1186/s12920-022-01227-7 . Honjo RS, Monteleone VF, Aiello VD, Wagenfuhr J, Issa VS, Pomerantzeff PMA, et al. Cardiovascular findings in Williams-Beuren Syndrome: Experience of a single center with 127 cases. Am J Med Genet A. 2022;188(2):676–82. 10.1002/ajmg.a.62542 . Yuan M, Deng L, Yang Y, Sun L. Intrauterine phenotype features of fetuses with Williams-Beuren syndrome and literature review. Ann Hum Genet. 2020;84(2):169–76. 10.1111/ahg.12360 . Wang Y, Liu C, Hu R, Geng J, Lu J, Zhao X, et al. Prenatal phenotype features and genetic etiology of the Williams-Beuren syndrome and literature review. Front Pediatr. 2023;11(1141665). 10.3389/fped.2023.1141665 . Mikhail FM, Biegel JA, Cooley LD, Dubuc AM, Hirsch B, Horner VL, et al. Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). Genet Med. 2019;21(9):1903–16. 10.1038/s41436-019-0545-7 . Wilson M, Carter IB. Williams Syndrome. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Iverson Carter declares no relevant financial relationships with ineligible companies. 2023. Courdier C, Boudjarane J, Malan V, Muti C, Sperelakis-Beedham B, Odent S, et al. Antenatal ultrasound features of isolated recurrent copy number variation in 7q11.23 (Williams syndrome and 7q11.23 duplication syndrome). Prenat Diagn. 2023;43(6):734–45. 10.1002/pd.6340 . Huang R, Zhou H, Fu F, Li R, Lei T, Li Y, et al. Prenatal diagnosis of Williams-Beuren syndrome by ultrasound and chromosomal microarray analysis. Mol Cytogenet. 2022;15(1):27. 10.1186/s13039-022-00604-2 . Li C, Zhang J, Li J, Qiao G, Zhan Y, Xu Y, et al. BACs-on-Beads Assay for the Prenatal Diagnosis of Microdeletion and Microduplication Syndromes. Mol Diagn Ther. 2021;25(3):339–49. 10.1007/s40291-021-00522-w . Tsagkas N, Katsanevakis E, Karagioti N, Perdikaris P, Billis M. Prenatal Diagnosis of Williams-Beuren Syndrome Based on Suspected Fetal Hypotonia in Early Pregnancy. Cureus. 2023;15(2):e34841. 10.7759/cureus.34841 . Ramirez-Velazco A, Dominguez-Quezada MG. [Atypical deletions in Williams-Beuren syndrome]. Rev Med Inst Mex Seguro Soc. 2017;55(5):615–20. Serrano-Juarez CA, Prieto-Corona B, Rodriguez-Camacho M, Sandoval-Lira L, Villalva-Sanchez AF, Yanez-Tellez MG, et al. Neuropsychological Genotype-Phenotype in Patients with Williams Syndrome with Atypical Deletions: A Systematic Review. Neuropsychol Rev. 2023;33(4):891–911. 10.1007/s11065-022-09571-2 . Stasia MJ, Mollin M, Martel C, Satre V, Coutton C, Amblard F, et al. Functional and genetic characterization of two extremely rare cases of Williams-Beuren syndrome associated with chronic granulomatous disease. Eur J Hum Genet. 2013;21(10):1079–84. 10.1038/ejhg.2012.310 . Del Campo M, Antonell A, Magano LF, Munoz FJ, Flores R, Bayes M, et al. Hemizygosity at the NCF1 gene in patients with Williams-Beuren syndrome decreases their risk of hypertension. Am J Hum Genet. 2006;78(4):533–42. 10.1086/501073 . Kozel BA, Danback JR, Waxler JL, Knutsen RH, de Las Fuentes L, Reusz GS et al., Williams syndrome predisposes to vascular stiffness modified by antihypertensive use and copy number changes in NCF1. Hypertension. (2014) 63(1):74 – 9. 10.1161/HYPERTENSIONAHA.113.02087 . Serrano-Juarez CA, Venegas-Vega CA, Yanez-Tellez MG, Rodriguez-Camacho M, Silva-Pereyra J, Salgado-Ceballos H, et al. Cognitive, Behavioral, and Adaptive Profiles in Williams Syndrome With and Without Loss of GTF2IRD2. J Int Neuropsychol Soc. 2018;24(9):896–904. 10.1017/S1355617718000711 . Zarate YA, Uehara T, Abe K, Oginuma M, Harako S, Ishitani S, et al. CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants. Genet Med. 2021;23(6):1050–57. 10.1038/s41436-020-01091-9 . Chung HL, Mao X, Wang H, Park YJ, Marcogliese PC, Rosenfeld JA, et al. De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy. Am J Hum Genet. 2020;106(5):717–25. 10.1016/j.ajhg.2020.04.001 . Grabert K, Engskog-Vlachos P, Skandik M, Vazquez-Cabrera G, Murgoci AN, Keane L, et al. Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate. Cell Death Dis. 2023;14(3):192. 10.1038/s41419-023-05714-2 . Venturin M, Carra S, Gaudenzi G, Brunelli S, Gallo GR, Moncini S, et al. ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome. J Med Genet. 2014;51(7):436–43. 10.1136/jmedgenet-2013-102240 . Mass E, Wachten D, Aschenbrenner AC, Voelzmann A, Hoch M. Murine Creld1 controls cardiac development through activation of calcineurin/NFATc1 signaling. Dev Cell. 2014;28(6):711–26. 10.1016/j.devcel.2014.02.012 . Pinnaro CT, Beck CB, Major HJ, Darbro BW. CRELD1 variants are associated with bicuspid aortic valve in Turner syndrome. Hum Genet. 2023;142(4):523–30. 10.1007/s00439-023-02538-0 . Burgmaier K, Brinker L, Erger F, Beck BB, Benz MR, Bergmann C, et al. Refining genotype-phenotype correlations in 304 patients with autosomal recessive polycystic kidney disease and PKHD1 gene variants. Kidney Int. 2021;100(3):650–59. 10.1016/j.kint.2021.04.019 . Supplementary Files SupplementaryTableWESresultsoffetalsamples.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4261789","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":298822779,"identity":"3417eca7-5b81-4663-b707-ee3c164811c9","order_by":0,"name":"weiqiang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYBACPmYQaWAjx8befIA4LWxgLRVpxnw8xxKI1AImzxxKnCeRo0CkFnYeM4m3bQfS2xhyGBh+VGwjxmE8ZpJz2+7ktjGcPcDYc+Y2cVpu87Y9y21j7EtgZmwjXsvhdCDDgAQtPGcOJ7CxEa+FrfznnIo0wzYetoSDRPmFn//wZoM3Bjby8vMfH3zwo4IILWDAA6UPEKkeScsoGAWjYBSMAqwAAPXRNKQF/WJxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2536-9176","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":true,"prefix":"","firstName":"weiqiang","middleName":"","lastName":"Liu","suffix":""},{"id":298822781,"identity":"160a6567-0b0a-4dd4-bb98-de8f1f34c7e8","order_by":1,"name":"Dingya Cao","email":"","orcid":"","institution":"The Third Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dingya","middleName":"","lastName":"Cao","suffix":""},{"id":298822782,"identity":"f5833680-bdc0-40b1-95a3-816ca2eba452","order_by":2,"name":"Jinshuang Song","email":"","orcid":"","institution":"longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinshuang","middleName":"","lastName":"Song","suffix":""},{"id":298822783,"identity":"85c3e069-33bc-4a9b-8adf-fe4a00b4f6ef","order_by":3,"name":"Tong Zhang","email":"","orcid":"","institution":"Longgang distirct maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Zhang","suffix":""},{"id":298822784,"identity":"8f895b04-ae08-4375-a094-e56f41ea69a5","order_by":4,"name":"Shuxian Zeng","email":"","orcid":"","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuxian","middleName":"","lastName":"Zeng","suffix":""},{"id":298822785,"identity":"77463ff3-35ae-4b2a-abdf-fd2446736ce3","order_by":5,"name":"Xiaoyi Cong","email":"","orcid":"","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyi","middleName":"","lastName":"Cong","suffix":""},{"id":298822786,"identity":"ece7c018-37b4-4143-aec4-046089ba8eef","order_by":6,"name":"Xiaojin Luo","email":"","orcid":"","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaojin","middleName":"","lastName":"Luo","suffix":""},{"id":298822787,"identity":"400bffef-a4bb-4301-a7e5-5f0713cb540d","order_by":7,"name":"Liang Hu","email":"","orcid":"","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Hu","suffix":""},{"id":298822788,"identity":"f64e5152-88ad-44c6-be71-0143ed7619e1","order_by":8,"name":"Yuanyuan Pei","email":"","orcid":"","institution":"Longgang district maternity and child healthcare hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Pei","suffix":""},{"id":298822789,"identity":"d833d11f-8133-46d1-aea7-35a6238d2763","order_by":9,"name":"Min Chen","email":"","orcid":"","institution":"The Third Affiliated Hospital of Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-04-13 12:38:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4261789/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4261789/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56410406,"identity":"83b3071b-f293-46be-af67-f7f9273e802b","added_by":"auto","created_at":"2024-05-13 20:20:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75223,"visible":true,"origin":"","legend":"\u003cp\u003e7q11.23 deletions identified in 7 fetuses\u003c/p\u003e\n\u003cp\u003eThe CMA results indicated that seven fetuses had heterozygous deletions in the 7q11.23 region, and all deletions involved the ELN gene. There were differences in the size of the deletion fragments and the genes involved in different samples (the lower green square columns represent OMIM-causing genes, and the gray square columns represent OMIM genes).\u003c/p\u003e","description":"","filename":"OnlineFIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-4261789/v1/d8b6881b48bfbbc70146afc0.png"},{"id":56410404,"identity":"51907097-33e6-40ba-94ae-d7e7a609fb94","added_by":"auto","created_at":"2024-05-13 20:20:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1801200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrasound findings on WS fetuses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCase 1: Ultrasound suggests (A): In the three-vessel tracheal section (3VT section), the persistent left superior vena cava (LSVC) is observed on the left side of the arterial duct (DA). The right subclavian artery (ARSA) is seen anteriorly on the right side of the spine, originating from the descending aorta and looping behind the trachea to the right side.\u003c/p\u003e\n\u003cp\u003e(B) and (C) show a persistent left superior vena cava (LSVC) on the left side of the ductus arteriosus (DA) in both cases 1 and 6.\u003c/p\u003e\n\u003cp\u003e(D) In case 2, the fetal left kidney is enlarged with increased echogenicity, and multiple cysts are visible within the left kidney.\u003c/p\u003e\n\u003cp\u003e*Abbreviation\u003c/p\u003e\n\u003cp\u003eLSVC: left superior vena cava; DA: arterial duct; AA: aortic arch;\u003c/p\u003e\n\u003cp\u003eRSVC: right superior vena cava; T:trachea; ARSA: aberrant right subclavian artery\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4261789/v1/56968953f89e5082629c8737.png"},{"id":56410407,"identity":"1decf51c-7d04-4786-ac7b-92db78ef90b5","added_by":"auto","created_at":"2024-05-13 20:20:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":96568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole exome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) A heterozygous variant was observed in case 1; the mutation in the CRELD1 gene was associated with an atrioventricular septal defect. (B) A probable pathogenic variant in the FPGT-TNNI3K gene was identified in case 2, associated with cardiac conduction disease. (C) A VUS variant in the PKHD1 gene was found in case 2, who had multiple cysts on ultrasound. A homozygous variant in the ADAP2 gene was identified in the ROH region of case 2.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4261789/v1/9729a16f0f61d1c0d4739ec7.png"},{"id":60440995,"identity":"de939c7c-f80a-492c-858b-68a2a750fb21","added_by":"auto","created_at":"2024-07-16 19:12:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2997805,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4261789/v1/98cee1c4-6af4-47e3-a777-75d2a7d87399.pdf"},{"id":56410405,"identity":"2b2fb31e-7046-4f23-b204-732c89ff2698","added_by":"auto","created_at":"2024-05-13 20:20:08","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":19193,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableWESresultsoffetalsamples.docx","url":"https://assets-eu.researchsquare.com/files/rs-4261789/v1/bcce3fc47bd2767e639a4a9b.docx"}],"financialInterests":"","formattedTitle":"Prenatal genetic diagnosis of Williams-Beuren syndrome with atypical and complex phenotypes using SNP array and whole exome sequencing","fulltext":[{"header":"Background","content":"\u003cp\u003eWilliams-Beuren syndrome (WBS; also called Williams syndrome, WS; OMIM 194.050) is a distinct and rare neurodevelopmental disorder with a prevalence of approximately 1 in 7,500 to 1 in 20,000 live births\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The major clinical features of WBS are cardiovascular disease (such as coarctation of the aorta, peripheral pulmonary stenosis, and hypertension), mild to moderate mental retardation, developmental delay, growth retardation, physical features, cognitive profile, facial features, end profile, facial features, endocrine abnormalities, and abnormal neurocognitive profile \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genetic diagnosis of WBS is made by identifying a heterozygous microdeletion in the WBS chromosomal region (WBSCR) located on chromosome 7q11.23, with a deletion size ranging from 1.55 megabase pairs (Mb) to 1.83 Mb\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Deletions of significant size within this region can result in clinically distinct phenotypes \u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The critical pathogenic region of WBS has been shown to contain 31 known genes. The cardiovascular and other phenotypes of WBS correlate with the size of its deletion and with several genes such as \u003cem\u003eELN\u003c/em\u003e, \u003cem\u003eFZD9\u003c/em\u003e, \u003cem\u003eBAZ1B\u003c/em\u003e, \u003cem\u003eSTX1A\u003c/em\u003e, \u003cem\u003eLIMK1\u003c/em\u003e, \u003cem\u003eCLIP2\u003c/em\u003e, \u003cem\u003eGTF2I\u003c/em\u003e are known to contribute to the complex phenotypes \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. However, no single gene has been identified in which a pathogenic variant is the cause of WBS. The understanding of how these genes contribute to the WBS phenotype is still evolving.\u003c/p\u003e \u003cp\u003eTo date, the majority of patients with WBS are diagnosed postnatally\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The primary indication for prenatal diagnosis of WBS is mainly based on ultrasound anomalies. However, fetuses with WBS may not exhibit the typical WBS phenotype during pregnancy, and some phenotypes are non-specific, such as increased amniotic fluid, intrauterine growth retardation (IUGR), short fetal femur length, and polycystic kidney dysplasia\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The association of these phenotypes with the deletion of the 7q11.23 region is unclear and requires further elucidation.\u003c/p\u003e \u003cp\u003eWith the widespread use of CMA as a first-tire technique for prenatal diagnosis, an increasing number of fetuses with WBS are being diagnosed prenatally. About 100 prenatal cases of WBS with various atypical phenotypes have been reported in the literature, but more attention needs to be paid to information on genetic variants other than the 7q11.23 deletion in these studies. In this study, we synthesized multidimensional evidence from CMA, whole exome sequence analysis, and imaging data to analyze the correlation between complex phenotypes and genotypes of WBS and provide a more accurate diagnosis for the precise clinical targets for treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubject\u003c/h2\u003e \u003cp\u003eThe CMA outcomes of 6718 pregnant women with CMA, who were diagnosed prenatally at our hospital and the Third Affiliated Hospital of Guangzhou Medical University from January 2019 to August 2023, were retrospectively analyzed. The pregnant women were aged 20 to 45 years, and their gestational weeks ranged from 11\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e to 35\u003csup\u003e+\u0026thinsp;4\u003c/sup\u003e weeks. The medical ethics committee of Longgang District Maternal and Child Health Hospital approved the study (No. LGFYYXLLQF-2022-003), and all subjects signed an informed consent form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound examination\u003c/h2\u003e \u003cp\u003eEach case underwent a routine ultrasound examination at a median gestational age of 24 (range 11\u0026ndash;35) weeks. The ultrasound machine used for the examinations was a Voluson E10 (GE Medical Systems). The scanning frequency ranged from 1 MHz to 5 MHz. A Level III fetal anatomic scan was performed in each case, and any associated abnormal findings were documented and recorded. A Level III fetal anatomic scan was performed in each case, and any associated abnormal findings were documented and recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAmniotic fluid sampling and DNA extraction\u003c/h2\u003e \u003cp\u003eA 5 ml amniotic fluid sample was collected according to instructions and used for DNA extraction using the QIAamp DNA Blood Mini Kit (No. 51106, Qiagen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCMA analysis\u003c/h2\u003e \u003cp\u003eAn Affymetrix Cytoscan 750K chip (Thermo Fisher, USA) was used. The extracted DNA was digested, ligated, PCR amplified, purified, fragmented, and labeled for hybridization according to the instructions, and then the washed and stained chip was scanned, and the data were collected and analyzed using Chromosome Analysis Suite (ChAS 4.3) software. Aneuploidy, copy number variation (CNV), and region of homozygosity (ROH) were analyzed, and CNV and ROH were filtered with a threshold of 100 kilobase pairs (Kb) and 5 Mb, respectively. The CNV scoring criteria were based on the interpretation guideline \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWhole exome sequence analysis\u003c/h2\u003e \u003cp\u003eFour cases of prenatal WBS were sequenced by whole-exome sequencing. DNA extraction and purification of the samples were performed using a kit (DP705, TIANamp Genomic DNA Kit, China); library construction was performed according to the manufacturer's instructions (ND617, Vazyme, China). The library was sequenced on a GenoLab M (Genemind, China) sequencer with a pair-end (PE) read length of 150bp and an average sequencing depth of not less than 20 folds.\u003c/p\u003e \u003cp\u003eAfter decontamination and trimming, the raw sequencing reads were subjected to BWA-GATK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/gatk/\u003c/span\u003e\u003cspan address=\"https://software.broadinstitute.org/gatk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with ClinSV (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bio.tools/clinsv\u003c/span\u003e\u003cspan address=\"https://bio.tools/clinsv\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for bioinformatics analysis. Then, all SNPs and INDELs were annotated using ANNOVAR software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://annovar.openbioinformatics.org/en/latest/\u003c/span\u003e\u003cspan address=\"http://annovar.openbioinformatics.org/en/latest/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and all structural variants were annotated using AnnotSV software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lbgi.fr/AnnotSV/\u003c/span\u003e\u003cspan address=\"https://lbgi.fr/AnnotSV/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCNV analysis focused on the 7q11.23 region\u003c/h2\u003e \u003cp\u003e7 cases with heterozygous deletions in the 7q11.23 region were detected. The deletion size ranged from 1.43 Mb to 1.66 Mb, affecting 34 to 41 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCNVs Recognized with CMA and Genes Involved\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMicroarray Nomenclature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSize (kbp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of genes (protein-coding genes)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenes contains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eclassification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72692113_74154209)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003egtf2ird2p1, pom121b, nsun5, trim50, rpl7ap77, fkbp6, rnu6-1080p, fzd9, baz1b, rnu6-1198p, bcl7b, tbl2, mlxipl, vps37d, dnajc30, bud23, stx1a. mir4284, rn7sl265p, bicdl3p, abhd11, cldn3, cldn4, mettl27, tmem270, eln, eln-as1, limk1, eif4h, mir590, lat2, rfc2, clip2, gtf2ird1, rna5sp233. gtf2i,gtf2i-as1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2q37.1 (233917755_234137480)x3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eINPP5D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72624167_74288694)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41(27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNCF1B\u003c/em\u003e, ..(same as case 1).... \u003cem\u003ephb1p15,ncf1,gtf2ird2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8q21.13(82088743_82229807)x3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eFABP5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72669481_74154209)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSame as case 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72664089_74154209)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSame as case 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18p11.32 (283770_462315)x3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCOLEC12\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72669481_74146927)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSame as case 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6q14.2 (84606383_84776946)x3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCYB5R4, MRAP2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72723371_74154209)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34(24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRIM50, ..(same as case 1).. \u003cem\u003eGTF2I-AS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9p21.1 (30587632_30697418)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXq28(148128198_148571167)x2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eIDS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7q11.23 (72669481_74154209)x1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37(25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSame as case 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6q21(110802069_110934453)x3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCDK19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*Abbreviation\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eP: pathogenic; VUS: variants unknown significance.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eGenes name marked as red means protein-coding genes.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCNV detections outside the 7q11.23 region\u003c/h2\u003e \u003cp\u003eIn the 7 cases, 6 duplications and one deletion were observed outside the 7q11.23 region. According to the CNV interpretation guideline, all additional CNVs detected were classified as having unknown clinical significance, 2, 1, 2, and 1 additional duplicated CNVs were detected in cases 2, 4, 6, and 7, respectively, and 1 additional deletion was detected in case 6 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of ROH\u003c/h2\u003e \u003cp\u003eROH analysis was performed using 5 Mb and 50 markers. Each ROH was detected in cases 2, 3, and 7, and no ROHs were detected in the remaining cases (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eROH detection in WBS fetal samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase ROH Size Mb Number of genes included\u003c/p\u003e \u003cp\u003e(Number of protein-coding genes)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 / / /\u003c/p\u003e \u003cp\u003e2 17q11.1q11.2 (25309337_30511274)x2 hmz 5.2 148(70)\u003c/p\u003e \u003cp\u003e3 11p11.2p11.12 (45546761_51550787)x2 hmz 6.0 117 (57)\u003c/p\u003e \u003cp\u003e4 / / /\u003c/p\u003e \u003cp\u003e5 / / /\u003c/p\u003e \u003cp\u003e6 / / /\u003c/p\u003e \u003cp\u003e7 3p21.31p21.1(46700713_52738165)x2 hmz 6.0 222 (141)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound findings\u003c/h2\u003e \u003cp\u003eUltrasound was performed in seven cases of WBS, and cardiac or cardiovascular anomalies were detected in four fetal ultrasound findings (57.14%, 4/7). Of these, one suggested pulmonary artery anomalies (case 1), one had significant cardiovascular anomalies such as an enlarged right heart with a thin aortic isthmus (case 5), one suggested a slightly smaller left heart with a small amount of tricuspid regurgitation (case 2), and one suggested a permanent left superior vena cava with intrauterine growth retardation (IUGR) (case 6). In cases 2 and 7, ultrasound suggested the presence of polycystic renal dysplasia (28.57%, 2/7). The other two fetuses (cases 3 and 4, 28.57%, 2/7) had no significant cardiovascular abnormalities on ultrasound, except for the suggestion of intense light in the left heart of case 3. The ultrasound findings of the seven fetuses are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFetal ultrasound findings or prenatal indications in 7 cases of WBS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase Maternal age Gestational week Ultrasound phenotype or indication for prenatal diagnosis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 37 23\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e Enlarged right fetal heart, permanent left superior vena cava, left aortic arch with right clavicle; inferior arterial vagus, aortic isthmus distortion.\u003c/p\u003e \u003cp\u003e2 27 28\u003csup\u003e+\u0026thinsp;0\u003c/sup\u003e Left polycystic dysplastic kidney; left heart slightly smaller, mild tricuspid regurgitation\u003c/p\u003e \u003cp\u003e3 36 18\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e NIPT high risk for CNV in 7q11.23, strong light spot in left heart\u003c/p\u003e \u003cp\u003e4 22 23\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e NIPT high risk for CNV in 7q11.23\u003c/p\u003e \u003cp\u003e5 29 32\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e Pulmonary Artery Anomalies\u003c/p\u003e \u003cp\u003e6 44 31\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e Intrauterine growth retardation, permanent left superior vena cava\u003c/p\u003e \u003cp\u003e7 30 32\u003csup\u003e+\u0026thinsp;6\u003c/sup\u003e Left polycystic dysplastic kidney\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWhole exome sequencing\u003c/h2\u003e \u003cp\u003eTo further analyze the reasons for the differences in the phenotypes of the WBS fetuses, whole exome sequencing analysis was performed in four samples. No homozygous variants were identified in the 7q11.23 region, while the other 7q11.23 allele was deleted. Only one homozygous variant was found in the ROH region of case 2, but the clinical significance of this variant was unknown. For phenotype-genotype relationship analysis, the variants associated with atrioventricular septal defect or congenital heart defects were identified in case 1, case 2, and case 3. In addition, the pathogenic or likely pathogenic incidental identified variants were also found in these cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Table).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe clinical phenotypes of WBS mainly include malformations of the cardiovascular system, peculiar facial features, connective tissue abnormalities, endocrine abnormalities, growth, and mental retardation, and cognitive difficulties\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Only about 100 cases of prenatally diagnosed WBS have been reported in the literature \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and most of the WBS cases were diagnosed after birth. An important reason is that the phenotypes of WBS cases are atypical or incomplete in the fetal period, resulting in missed prenatal diagnosis.\u003c/p\u003e \u003cp\u003eAlthough cardiovascular abnormalities are an essential phenotype of WBS, only 42.9\u0026ndash;58.82% of WBS fetuses have ultrasound-detectable abnormalities \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. There are a higher number of other non-specific phenotypes in WBS fetuses, such as IUGR, abnormal fetal placental Doppler indices, thickened NT/ NF, polyhydramnios, fetal hydrops, duodenal atresia, echogenic bowel, intracardiac echogenic focus (IEF), and other less common phenotypes \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The reasons for the complex phenotype of WBS remain to be elucidated.\u003c/p\u003e \u003cp\u003eIn WBS patients, complex phenotypes may be associated with differences in the size of CNVs and their affected genes in WBS fetuses. It has been shown that ninety percent of deletions in the 7q11.21 region of WBS patients are ~\u0026thinsp;1.5 Mb, 5%-8% are ~\u0026thinsp;1.8 Mb, and only 2%-5% are atypical \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The variable size of CNVs leads to inconsistencies in the number of genes involved, especially protein-coding genes. Besides the prominent \u003cem\u003eELN\u003c/em\u003e gene, genotype-phenotype evidence suggests that the transcription factor genes \u003cem\u003eGTF2I\u003c/em\u003e and \u003cem\u003eGTF2IRD1\u003c/em\u003e may lead to intellectual functioning, social functioning, and anxiety. There is also increasing evidence that \u003cem\u003eBAZ1B\u003c/em\u003e, \u003cem\u003eLIMK1\u003c/em\u003e, \u003cem\u003eSTX1A\u003c/em\u003e, and \u003cem\u003eMLXIPL\u003c/em\u003e deletions have phenotypic consequences. However, more work is needed to understand how these genes contribute to clinical outcomes \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFive of the seven cases in this study shared the same deleted genes. Four additional genes, including two protein-coding genes, \u003cem\u003eNCF1\u003c/em\u003e and \u003cem\u003eGTF2IRD2\u003c/em\u003e, were deleted in case 2. Case 6 had a slightly smaller CNV fragment involving 34 genes, 24 of which are protein-coding genes, and it retained the protein-coding gene \u003cem\u003eNSUN5\u003c/em\u003e without deletion compared to the other cases. Functional analysis of the above differential genes suggests that the \u003cem\u003eNCF1\u003c/em\u003e gene encodes a component of the NADPH oxidase complex \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Although \u003cem\u003eNCF1\u003c/em\u003e is associated with chronic granulomatous disease (CGD), an autosomal recessive disorder, heterozygous deletion of \u003cem\u003eNCF1\u003c/em\u003e may be relevant in the form of essential hypertension and vascular stiffness in WBS individuals \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Although deletion of \u003cem\u003eGTF2IRD2\u003c/em\u003e has been shown to result in more impaired visuospatial abilities and more significant behavioral problems \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, the function of \u003cem\u003eGTF2IRD2\u003c/em\u003e and \u003cem\u003eNSUN5\u003c/em\u003e and their relationship to complex WBS phenotypes require further investigation.\u003c/p\u003e \u003cp\u003eInterestingly, although cases 1, 3, 4, 5, and 7 had the same gene deletion, they had different phenotypes. For example, case 7 was diagnosed prenatally because of ultrasound findings of fetal polycystic kidney, whereas case 4 was diagnosed because NIPT suggested high risk, and the cardiovascular abnormality phenotype was not observed at 23 weeks of gestation. The discrepancy in the in-utero phenotypes may be due to limited and incomplete prenatal evaluation or suggest clinical heterogeneity in the WBS individuals and may also be related to other unknown genetic variants.\u003c/p\u003e \u003cp\u003eIn a detailed genetic analysis of these cases, we found that in addition to the deletion of the 7q11.23 region detected in each WBS fetus, additional CNVs were detected on other chromosomes, all of which are of uncertain clinical significance according to the ACMG/ClinGen 2019 guideline. For example, the duplicated CNV on chromosome 6 in case 7 included a \u003cem\u003eCDK19\u003c/em\u003e gene; loss of function for \u003cem\u003eCDK19\u003c/em\u003e can cause mental retardation and epileptic encephalopathy (OMIM 614720) \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e but is unrelated to the triplet duplication. Case 4 had an additional deletion CNV on chromosome 18, including the \u003cem\u003eCOLEC12\u003c/em\u003e gene (OMIM 607621), a class of c-type lectins with collagen-like sequences and carbohydrate-recognizing structural domains \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, but its relationship to disease is currently unclear. There is no evidence to suggest whether these additional CNVs have a second hit or co-expression effect on the complex phenotype of WBS. However, the cumulative genotypic and phenotypic information will contribute to understanding the complex phenotype of WBS.\u003c/p\u003e \u003cp\u003eWe also analyzed ROH in this study for a more detailed analysis of the relationship between genotyping and phenotyping. Although three ROHs were detected in three cases, none were disease-causing regions. Whole exome sequencing was performed to exclude the homozygous variants that occurred in the protein-coding genes in these ROH regions, and except for a VUS variant detected in the \u003cem\u003eADAP2\u003c/em\u003e gene in case 2, no additional homozygous pathogenic variants were identified within these ROH regions. Although haploinsufficiency of the \u003cem\u003eADAP2\u003c/em\u003e gene has been implicated in the occurrence of cardiovascular malformations in NF1 microdeletion patients \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, the clinical correlation between the \u003cem\u003eADAP2\u003c/em\u003e gene and disease is currently unknown. Therefore, ROH may not influence the phenotypic complexity of WBS.\u003c/p\u003e \u003cp\u003eOur analysis of whole-exome sequencing in four cases found no homozygous variants in approximately 25 protein-coding genes in the 7q11.23 region. Furthermore, WES sequencing suggests that other variants in the genome might also contribute to the complex phenotype observed in WBS patients. In case 1, who had classic cardiovascular abnormalities, we found the \u003cem\u003eCRELD1\u003c/em\u003e variant. Although the clinical significance of this variant is unknown, \u003cem\u003eCRELD1\u003c/em\u003e variants can cause atrial septal defects \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e and are associated with bicuspid aortic valve anomalies \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In case 2, with a polycystic dysplastic renal phenotype, we found a variant in the \u003cem\u003ePKHD1\u003c/em\u003e gene in case 2, which can lead to polycystic kidney disease \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Thus, the complexity of the phenotype in WBS fetuses seems to be related to other variants within the genome, and larger sample sizes are needed to validate this conclusion.\u003c/p\u003e \u003cp\u003eAnother interesting observation is that we found polycystic kidney dysplasia in two out of seven cases. It has been reported that the detection rate of renal cysts or multicystic dysplastic kidneys in fetuses with WBS is about 4.7\u0026ndash;12.5% \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, whereas the detection rate of polycystic kidney dysplasia in the present study is about 28.6% (2/7). The reason why so many WBS patients have the polycystic dysplastic renal phenotype needs more detailed analysis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the prenatal ultrasound phenotype of WBS fetuses is atypical and incomplete. The phenotypic variation of WBS is influenced by the size of the microdeletion, the genes involved in the breakpoint region, the background genotype, and possibly by other unknown variants. Therefore, a more in-depth genome-wide analysis of WBS patients would be beneficial to elucidate the relationship between complex phenotypes and genotypes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWilliams-Beuren syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChromosomal microarray analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNVs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecopy number variants\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRegions of homozygosity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWES\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole exome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIUGR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntrauterine growth retardation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIEF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntracardiac echogenic focus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSVC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeft superior vena cava\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDuct arterial\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAortic arch\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRSVC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRight superior vena cava T:Trachea\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eARSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAberrant right subclavian artery\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epathogenic\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVUS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariants unknown significance.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe want to thank all the members of the Central Laboratory, Longgang District Maternity \u0026amp; Child Healthcare Hospital of Shenzhen City, who collaborated in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWQ. Liu conceived and designed the study. Data analysis and manuscript writing: WQ.Liu and M.Chen; clinical data collection: DY. Cao, T. Zhang, and SX. Zeng; sample sequencing and data analysis: XY. Cong, XJ. Luo, L. Hu, and YY. Pei; ultrasound data collection: JS.S. All authors read and approved the manuscript and are accountable for all study aspects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the\u0026nbsp;Medical and Health Technology Research Project, Longgang District of Shenzhen City (LGKCYLWS2021000024), (LGWJ2021-143), Medical and Health\u0026nbsp;Technology Research Project, Special Funds for Science and Technology, Innovation Longgang District of Shenzhen City (LGKCYLWS2022013), Natural Science Foundation of Shenzhen City (JCYJ20230807141908017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be made available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe medical ethics committee of Longgang District Maternal and Child Health Hospital approved the study (No. LGFYYXLLQF-2022-003), and all subjects signed an informed consent form. All methods were performed according to the ethical standards in the Declaration of Helsinki and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerrero GB, Biamino E, Sorasio L, Banaudi E, Peruzzi L, Forzano S, et al. Presenting phenotype and clinical evaluation in a cohort of 22 Williams-Beuren syndrome patients. Eur J Med Genet. 2007;50(5):327\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ejmg.2007.05.005\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmg.2007.05.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePober BR. Williams-Beuren syndrome. N Engl J Med. 2010;362(3):239\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMra0903074\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra0903074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozel BA, Barak B, Kim CA, Mervis CB, Osborne LR, Porter M, et al. Williams syndrome. Nat Rev Dis Primers. 2021;7(1):42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41572-021-00276-z\u003c/span\u003e\u003cspan address=\"10.1038/s41572-021-00276-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson M, Carter IB. Williams Syndrome. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Iverson Carter declares no relevant financial relationships with ineligible companies. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTwite MD, Stenquist S, Ing RJ. Williams syndrome. Paediatr Anaesth. 2019;29(5):483\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pan.13620\u003c/span\u003e\u003cspan address=\"10.1111/pan.13620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia Y, Huang S, Wu Y, Yang Y, Chen S, Li P, et al. Clinical application of chromosomal microarray analysis for the diagnosis of Williams-Beuren syndrome in Chinese Han patients. Mol Genet Genomic Med. 2019;7(2):e00517. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/mgg3.517\u003c/span\u003e\u003cspan address=\"10.1002/mgg3.517\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams syndrome. Nat Rev Dis Primers. 2021;7(1):43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41572-021-00283-0\u003c/span\u003e\u003cspan address=\"10.1038/s41572-021-00283-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Zheng Y, Liang G, Xu X, Liu J, Chen S, et al. Atypical deletion of Williams-Beuren syndrome reveals the mechanism of neurodevelopmental disorders. BMC Med Genomics. 2022;15(1):79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12920-022-01227-7\u003c/span\u003e\u003cspan address=\"10.1186/s12920-022-01227-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHonjo RS, Monteleone VF, Aiello VD, Wagenfuhr J, Issa VS, Pomerantzeff PMA, et al. Cardiovascular findings in Williams-Beuren Syndrome: Experience of a single center with 127 cases. Am J Med Genet A. 2022;188(2):676\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ajmg.a.62542\u003c/span\u003e\u003cspan address=\"10.1002/ajmg.a.62542\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan M, Deng L, Yang Y, Sun L. Intrauterine phenotype features of fetuses with Williams-Beuren syndrome and literature review. Ann Hum Genet. 2020;84(2):169\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/ahg.12360\u003c/span\u003e\u003cspan address=\"10.1111/ahg.12360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Liu C, Hu R, Geng J, Lu J, Zhao X, et al. Prenatal phenotype features and genetic etiology of the Williams-Beuren syndrome and literature review. Front Pediatr. 2023;11(1141665). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fped.2023.1141665\u003c/span\u003e\u003cspan address=\"10.3389/fped.2023.1141665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikhail FM, Biegel JA, Cooley LD, Dubuc AM, Hirsch B, Horner VL, et al. Technical laboratory standards for interpretation and reporting of acquired copy-number abnormalities and copy-neutral loss of heterozygosity in neoplastic disorders: a joint consensus recommendation from the American College of Medical Genetics and Genomics (ACMG) and the Cancer Genomics Consortium (CGC). Genet Med. 2019;21(9):1903\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41436-019-0545-7\u003c/span\u003e\u003cspan address=\"10.1038/s41436-019-0545-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson M, Carter IB. Williams Syndrome. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Iverson Carter declares no relevant financial relationships with ineligible companies. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCourdier C, Boudjarane J, Malan V, Muti C, Sperelakis-Beedham B, Odent S, et al. Antenatal ultrasound features of isolated recurrent copy number variation in 7q11.23 (Williams syndrome and 7q11.23 duplication syndrome). Prenat Diagn. 2023;43(6):734\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/pd.6340\u003c/span\u003e\u003cspan address=\"10.1002/pd.6340\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang R, Zhou H, Fu F, Li R, Lei T, Li Y, et al. Prenatal diagnosis of Williams-Beuren syndrome by ultrasound and chromosomal microarray analysis. Mol Cytogenet. 2022;15(1):27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13039-022-00604-2\u003c/span\u003e\u003cspan address=\"10.1186/s13039-022-00604-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Zhang J, Li J, Qiao G, Zhan Y, Xu Y, et al. BACs-on-Beads Assay for the Prenatal Diagnosis of Microdeletion and Microduplication Syndromes. Mol Diagn Ther. 2021;25(3):339\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s40291-021-00522-w\u003c/span\u003e\u003cspan address=\"10.1007/s40291-021-00522-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsagkas N, Katsanevakis E, Karagioti N, Perdikaris P, Billis M. Prenatal Diagnosis of Williams-Beuren Syndrome Based on Suspected Fetal Hypotonia in Early Pregnancy. Cureus. 2023;15(2):e34841. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.34841\u003c/span\u003e\u003cspan address=\"10.7759/cureus.34841\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamirez-Velazco A, Dominguez-Quezada MG. [Atypical deletions in Williams-Beuren syndrome]. Rev Med Inst Mex Seguro Soc. 2017;55(5):615\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerrano-Juarez CA, Prieto-Corona B, Rodriguez-Camacho M, Sandoval-Lira L, Villalva-Sanchez AF, Yanez-Tellez MG, et al. Neuropsychological Genotype-Phenotype in Patients with Williams Syndrome with Atypical Deletions: A Systematic Review. Neuropsychol Rev. 2023;33(4):891\u0026ndash;911. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11065-022-09571-2\u003c/span\u003e\u003cspan address=\"10.1007/s11065-022-09571-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStasia MJ, Mollin M, Martel C, Satre V, Coutton C, Amblard F, et al. Functional and genetic characterization of two extremely rare cases of Williams-Beuren syndrome associated with chronic granulomatous disease. Eur J Hum Genet. 2013;21(10):1079\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ejhg.2012.310\u003c/span\u003e\u003cspan address=\"10.1038/ejhg.2012.310\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDel Campo M, Antonell A, Magano LF, Munoz FJ, Flores R, Bayes M, et al. Hemizygosity at the NCF1 gene in patients with Williams-Beuren syndrome decreases their risk of hypertension. Am J Hum Genet. 2006;78(4):533\u0026ndash;42. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1086/501073\u003c/span\u003e\u003cspan address=\"10.1086/501073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozel BA, Danback JR, Waxler JL, Knutsen RH, de Las Fuentes L, Reusz GS et al., \u003cem\u003eWilliams syndrome predisposes to vascular stiffness modified by antihypertensive use and copy number changes in NCF1. Hypertension. (2014) 63(1):74\u0026thinsp;\u0026ndash;\u0026thinsp;9.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/HYPERTENSIONAHA.113.02087\u003c/span\u003e\u003cspan address=\"10.1161/HYPERTENSIONAHA.113.02087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerrano-Juarez CA, Venegas-Vega CA, Yanez-Tellez MG, Rodriguez-Camacho M, Silva-Pereyra J, Salgado-Ceballos H, et al. Cognitive, Behavioral, and Adaptive Profiles in Williams Syndrome With and Without Loss of GTF2IRD2. J Int Neuropsychol Soc. 2018;24(9):896\u0026ndash;904. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/S1355617718000711\u003c/span\u003e\u003cspan address=\"10.1017/S1355617718000711\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZarate YA, Uehara T, Abe K, Oginuma M, Harako S, Ishitani S, et al. CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants. Genet Med. 2021;23(6):1050\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41436-020-01091-9\u003c/span\u003e\u003cspan address=\"10.1038/s41436-020-01091-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung HL, Mao X, Wang H, Park YJ, Marcogliese PC, Rosenfeld JA, et al. De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy. Am J Hum Genet. 2020;106(5):717\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajhg.2020.04.001\u003c/span\u003e\u003cspan address=\"10.1016/j.ajhg.2020.04.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrabert K, Engskog-Vlachos P, Skandik M, Vazquez-Cabrera G, Murgoci AN, Keane L, et al. Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate. Cell Death Dis. 2023;14(3):192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-023-05714-2\u003c/span\u003e\u003cspan address=\"10.1038/s41419-023-05714-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenturin M, Carra S, Gaudenzi G, Brunelli S, Gallo GR, Moncini S, et al. ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome. J Med Genet. 2014;51(7):436\u0026ndash;43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/jmedgenet-2013-102240\u003c/span\u003e\u003cspan address=\"10.1136/jmedgenet-2013-102240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMass E, Wachten D, Aschenbrenner AC, Voelzmann A, Hoch M. Murine Creld1 controls cardiac development through activation of calcineurin/NFATc1 signaling. Dev Cell. 2014;28(6):711\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.devcel.2014.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.devcel.2014.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinnaro CT, Beck CB, Major HJ, Darbro BW. CRELD1 variants are associated with bicuspid aortic valve in Turner syndrome. Hum Genet. 2023;142(4):523\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00439-023-02538-0\u003c/span\u003e\u003cspan address=\"10.1007/s00439-023-02538-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurgmaier K, Brinker L, Erger F, Beck BB, Benz MR, Bergmann C, et al. Refining genotype-phenotype correlations in 304 patients with autosomal recessive polycystic kidney disease and PKHD1 gene variants. Kidney Int. 2021;100(3):650\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.kint.2021.04.019\u003c/span\u003e\u003cspan address=\"10.1016/j.kint.2021.04.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Williams-Beuren syndrome, prenatal diagnosis, phenotype, chromosome microarray analysis, whole exome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-4261789/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4261789/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWilliams-Beuren syndrome (WBS) is a severe congenital disorder. Prenatal diagnosis of WBS is difficult because the phenotypes of WBS fetuses are atypical or incomplete. This study used ultrasound, SNP array, and whole exome sequencing to analyze the association between genotype and complex phenotype in fetuses with WBS.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eChromosomal microarray analysis (CMA) and whole genome sequencing were performed in pregnant women with prenatal diagnosis. Genome-wide copy number variants (CNVs), regions of homozygosity (ROH), single nucleotide variants (SNVs), small insertions and deletions, and splice sites were analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe 7q11.23 deletion was identified in seven fetuses out of 6718 prenatal diagnostic samples; ultrasound revealed that two fetuses had apparent cardiovascular anomalies; one fetus had persistent left superior vena cava and intrauterine growth retardation (IUGR). Two fetuses had polycystic kidney dysplasia, one of which was associated with a small amount of tricuspid regurgitation; the other two fetuses had no apparent ultrasound abnormalities. Detailed genetic analysis revealed CNVs ranging in size from 1.43 megabase pairs (Mb) to 1.66 Mb, affecting 34 to 41 genes, respectively. On average, 1.0 additional CNVs larger than 100 kilobase pairs of unknown significance and 0.43 ROH larger than 5 Mb were detected in these cases. The pathogenic or likely pathogenic SNV or splice site with the function of renal development and cardiovascular development was also identified in these cases.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe phenotype of WBS fetuses is atypical and complex, and the complex phenotype does not exclude association with other variants within the genome.\u003c/p\u003e","manuscriptTitle":"Prenatal genetic diagnosis of Williams-Beuren syndrome with atypical and complex phenotypes using SNP array and whole exome sequencing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-13 20:20:02","doi":"10.21203/rs.3.rs-4261789/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba9b1c57-d3b5-447e-863c-d859e7b2393c","owner":[],"postedDate":"May 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-16T19:04:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-13 20:20:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4261789","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4261789","identity":"rs-4261789","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}