Atypical Deletion of Williams-beuren Syndrome Reveals the Mechanism of Neurodevelopmental Disorders

Background The Williams-Beuren syndrome (WBS) is a multiple phylogenetic disorder, caused by the hemizygous deletion of 1.55 to 1.84 Mb on chromosome 7q11.23, which encodes a fragment of 26 to 28 genes. Among these genes, the deletion of the elastin (ELN) gene haplotype is the main cause of cardiovascular abnormalities. Other genes, such as CLIP2, GTF2IRD1, and GTF2I, may be associated with specic cognitive and craniofacial features. However, genes associated with specic neurocognitive phenotypes are still controversially discussed. The purpose of this study is to further explore the mechanism of neurodevelopmental disorders in patients with Williams-Beuren syndrome. Patients and methods Patients who had been diagnosed with WBS were recruited. The deletion was precisely dened by chromosome microarray analysis and the clinical phenotype was evaluated. Results This study identied nine patients with atypical deletions from 111 patients with WBS. One patient had normal neurodevelopmental with deletion of Williams-Beuren syndrome chromosomal region (WBSCR) telomere side genes, including GTF2I and GTF2IRD1, while another patient retained these genes but showed neurodevelopmental abnormalities. Seven of the eight patients with the WBSCR22 gene deletion developed growth restriction. Conclusions By comparing the genotype and phenotype of patients with typical deletions and atypical deletions, the deletion of GTF2I and GTF2IRD1 genes alone insucient to induce typical neurocognitive phenotypes in WBS patients. The BAZ1B, FZD9, and STX1A genes may play an important role in the neurodevelopment of patients with WBS. Furthermore, the deletion of the WBSCR22 gene may be the main cause of physical growth restriction in WBS patients. one of which shows normal neurocognitive development. These results show that if only the genes on the telomere side of WBSCR are deleted, especially the GTF2I and GTF2IRD1 genes, the effects are not sucient to cause a neurodevelopmental delay in patients with WBS. This seems to contrast with previous reports(6, 7), which showed that the genes on the telomere side of WBSCR play a major role in the WBS phenotype. At the same time, according to patient No. 6, the deletions of BAZ1B, FZD9, and STX1A, located on the centromeric side of WBSCR, can also cause the typical neurocognitive phenotype of WBS. This suggests that the deletion of genes on the centromeric side of WBSCR also exerts an equally important effect on the phenotype of WBS. Furthermore, it can be inferred that the main target gene that causes growth retardation in patients with WBS is WBSCR22. WBS patients with smaller and larger deletions may not have a typical clinical phenotype, which detrimentally affects clinicians' precise diagnosis and treatment. At this time, complete genetic testing is particularly important. This is not only important for genetic counseling purposes, but also to avoid expensive and repetitive routine examinations, which can establish targeted follow-up. At the same time, atypical deletions play an important role in the study of gene function. Nine (8%) out of 111 Chinese patients with WBS had atypical deletions, which exceeded the ratio of 2–5% reported in previous literature. Possible reasons include the advancement of genetic testing technology, which has increased the detection rate of atypical deletions. A further reason is the deepening of people’s understanding of the disease, which has led to the diagnosis of many WBS patients with non-classical clinical phenotypes. Because of the economics of FISH technology, a number of laboratories still focus on this detection technology. However, with the development of sequencing technology and the reduction of price more modern technologies have become broadly available. For patients with suspected WBS, especially those with non-classical clinical phenotypes, the CMA or next-generation sequencing (NGS, Z-scores of weight-for-age (WAZ), body mass index (BMI), Z-scores of BMI-for-age (BAZ), Gesell development schedule (GDS), development quotient (DQ), Williams Syndrome Transcription Factor (WSTF).


Introduction
The Williams-Beuren syndrome (WBS; OMIM number 194050), also known as the Williams syndrome, is a multiple phylogenetic disorder caused by the hemizygous deletion of 1.55 to 1.84 Mb on chromosome 7q11.23, a fragment containing approximately 26 to 28 genes(1). Genome rearrangement often occurs in this region because low-copy repeats are located on both sides of the common deletion region, which leads to nonallelic recombination during meiosis (2). This region is also referred to as the Williams-Beuren syndrome chromosomal region (WBSCR). It has been estimated that the prevalence of WBS is about 1/7,500 to 1/20,000 (3). Although the phenotype features extensive heterogeneity in severity and performance, it usually shows facial deformities, cardiovascular abnormalities, mental retardation, speci c cognitive characteristics, developmental limitations, hypothyroidism, infantile hypercalcemia, and other clinical symptoms that affect multiple organs and systems (4).
However, it remains unclear how these gene deletions cause the characteristic phenotype of WBS, which may be related to the low expression of gene products.
Most patients have the same deletion span but few individuals have smaller or larger deletion fragments. To study the relationship between genotype and phenotype, patients with atypical deletions are promising research objects. However, since WBS is a rare disease and since the proportion of atypical deletions is low (about 2-5%) (1,2), the number of subjects available for research is extremely limited. So far, the deletion of the elastin (ELN) gene has been identi ed as the main cause of cardiovascular disorders in WBS patients, especially arterial stenosis (5). Moreover, several researchers have used atypical deletion patients and animal experiments to show that the genes located on the telomere side of WBSCR (i.e.,GTF2I, GTF2IRD1, and CLIP2) are the main causes of patients' behavior and cognitive phenotypes (6,7). Other scholars suggested that the deletion of WBSCR centromere-side genes also contributes to the speci c phenotype of WBS patients (8,9).
This study describes nine cases of Chinese WBS patients with atypical deletions, one of which showed normal neurocognitive development. The clinical phenotypic characteristics and genotypes of these atypical deletion patients are used to verify the ndings of previous literature and expand these. At the same time, we try to discuss the contribution of several gene deletions in WBSCR to the symptoms of WBS patients. Highresolution molecular testing is recommended for WBS patients, especially those with non-classical clinical symptoms. In this way, more comprehensive and accurate genetic information can be obtained, which can enable accurate diagnosis and treatment.

Patient subjects
The research plan was approved by the Research Ethics Committee of Guangdong Provincial People's Hospital [No. GDREC2019587H(R1)].
Informed written consent was obtained from the patients' parents.
Patients who had been diagnosed with WBS or were clinically suspected by the Lowery scoring system(10) were recruited. Their clinical data, including medical records, gestational age, birth weight, birth length, echocardiography, heart catheterization ndings, gene test reports, and family history, were reviewed.

Genetic testing
Chromosomal microarray analysis (CMA) was used to detect genes in patients with suspected WBS using the Lowery scoring system. Approximately 2.0 ml of peripheral venous blood was collected from patients and parents. Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) according to the manufacturer's instructions.
DNA samples (250 ng) were hybridized with Affymetrix Cytoscan 750 K array (Affymetrix, Santa Clara, CA, USA), which contains more than 750000 markers for copy number analysis and 200000 single nucleotide polymorphism (SNP) probes for genotyping. After hybridization, Chromosome Analysis Suite software (Affymetrix, USA) and human genome version GRCh37 (hg19) were used to analyze the results. The detected copy number variation (CNV) was compared with internal and national public CNV databases, such as the Database of Genomic Variants (DGV), the International Standards for Cytogenomic Arrays Consortium, and the Online Mendelian Inheritance in Man.
According to the latest standards and guidelines for sequence variations, as developed by the American College of Medical Genetics and Genomics, the CMA results were divided into ve grades: "pathogenicity", "possible pathogenicity", "uncertain signi cance", "possible benign", and "benign".

Cardiovascular status assessment
Not only were clinical cardiovascular data collected from all treated hospitals, but also, WBS patients who voluntarily came to the center for physical examination, were subjected to cardiac ultrasound examination by pediatric cardiologists. Supravalvular aortic stenosis (SVAS) was diagnosed by echocardiography when the pressure gradient (PG) exceeded 10 mm Hg. Pulmonary stenosis (PS) was diagnosed if the main or branch of the pulmonary artery showed local stenosis or diffuse stenosis with a PG exceeding 10 mm Hg (11). Coarctation of the aorta (CoA) was de ned by echocardiography as peak PG exceeding 40 mm Hg at the distal aortic arch (11).

Physical development assessment
The height (accurate to 0.1 cm) and weight (accurate to 0.1 kg) were measured by electronic height and weight meter without shoes and in light clothes. According to the patients' sex, date of birth, and date of the visit, the Z-scores of height-for-age (HAZ), weight-for-age (WAZ), and body mass index (BMI, i.e., the weight in kg divided by the height in m 2 )-for-age (BAZ) were calculated by WHO Anthro software (https://www.who.int/childgrowth/software/en/). Based on the WHO 2006 and 2007 growth reference standards (12), stunting was de ned as HAZ < -2; underweight was de ned as WAZ < -2, and emaciation was de ned as BAZ < -2.

Neurodevelopmental assessment
The Gesell development schedule (GDS) is one of the commonly used methods to assess the neurological and intellectual development of infants and children in China (13,14). In the present study, a version of the GSD was used, that had been adjusted by the Chinese Pediatric Association (15), to evaluate the neurodevelopment of WBS patients. The assessment was conducted by trained rehabilitation doctors. The assessment contents included gross motor, ne motor, adaptive behavior, language, and social behavior. Each test obtained a development quotient (DQ) and the total average DQ was obtained by calculating the average of the ve DQs. The correspondence between the individual neurocognitive development status and DQs is shown in Table 1.

Results
Deletion mapping in patients with atypical deletions In this study, a total of 111 patients with WBS were recruited, including nine patients with atypical deletions. As shown in Figure. 1, all nine patients had heterozygous microdeletions in chromosome 7q11. 23 centromeric side. Moreover, the deleted fragment of patient No. 9 was the longest, and several genes in the 7q11.22 region were also deleted. No other chromosomal CNV or mutated gene related to clinical symptoms was found in any of these patients. All of them are de novo cases without family history.  The symbol used are "+" (exist) and "-" (not exist).  Table 3.

Characteristics of participants
Neuropsychological testing According to the information provided by the patient's parents, except for patient No. 7, all other patients suffered from signi cant delays in developmental milestones, such as standing and walking independently, saying the rst word, and the rst sentence.
According to their parents' recollection, most patients could walk without support at about 16 to 18 months, and they could only say a few simple words after 20 months, such as father and mother. They were not able to say a complete sentence consciously until the visit. However, the development of patient No. 7 was normal. He could sit at 7 months, walk alone at 12 months, and speak short sentences with 6-10 words when interviewed.
The patients' GDS scores are shown in Table 4. The results of 20 patients with typical deletion of WBS in the same age group are also shown for comparison. All patient test data were obtained before any medical intervention. The results of the assessment were almost consistent with the information provided by parents. Except for patient No. 7, the total average GDS scores of all patients remained below 85. Neurologic and mental retardation is one of the most important and common features in patients with WBS (1,18,19). This study used GDS to assess patients' neurodevelopmental status, which includes the ve main functional areas of the human body. All patients with atypical deletions (except patient No. 7) showed mild to moderate developmental retardation similar to those with typical deletions. The age and size of chromosome deletions were similar between patients No. 6 and No. 7, but their GDS results were signi cantly different (as shown in Figure. 2).
This suggests that the difference between them is mainly caused by the different positions of chromosome breakage and gene deletions.
GTF2I and GTF2IRD1 belong to the transcription factor family. They interact with a variety of proteins and DNA to in uence neurophysiology and developmental processes (20). Previously, the deletion of heterozygosity of GTF2I and GTF2IRD1 genes has been reported as the main cause of neurocognitive characteristics, special facial features, and motor dysfunction in WBS patients (7,21,22). However, the deleted genes of patient No.
7 in this study included these two genes but showed normal neurological development. As recently reported (23), the symptoms of neurodevelopmental delay in WBS cannot be explained by the deletion of functional heterozygosity of GTF2I and GTF2IRD1. In-depth study of the molecular and phenotypic characteristics of patient No. 7 showed that the language and adaptive development of the patient were in a marginal state. This may be because the genes GTF2I and GTF2IRD1 mainly affect the neurodevelopment related to these two functional regions (24,25), but they are not su cient to cause the neurodevelopmental retardation symptoms typical for WBS patients. However, it cannot be ruled out that CNV size-related position effects, non-deletion allele variants, epigenetic mechanisms, regulatory sequences, or other factors may affect the patient's phenotype (26). patients (35,36). This affects the proliferation and differentiation of nerve cells in patients with corresponding neurocognitive phenotype. Studies (31,37) have shown that the BAZ1B gene is associated with facial features and behavioral phenotypes of WBS patients.
Furthermore, a recent study (38) has suggested that through the PTEN-mediated pathway, the deletion of BZA1B gene heterozygosity reduces both the viability and survival of thyroid cells, thereby causing hypothyroidism in patients with WBS. At the same time, the BAZ1B gene is also involved in the development of sperm, and its deletion may be one of the in uencing factors of infertility in WBS patients. Knockout of the BAZ1B gene can cause changes in the time of chromosome aggregation in cells and errors in the process. This may lead to delays in the prophase of mitosis, which may affect sperm development (39). Therefore, its deletion may be one of the in uencing factors of infertility in WBS patients.
It should be noted that the FZD9 gene also plays a role in the Wnt signaling pathway (40). By increasing the doubling time and apoptosis of nerve cells, deletion of the FZD9 gene can affect the development of a patient's nervous system and cause cognitive impairment (9).
The STX1A gene encodes a neuronal soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, which promotes the nerve function of the central nervous system by regulating the release of transmitters (41). Recent studies have shown that mutations or deletions of STX1A are related to human neuropsychological diseases, such as autism spectrum disorder and attention de cit hyperactivity disorder (42,43). However, the contribution and underlying mechanism of its de ciency to the neurocognitive symptoms of WBS still remain unclear. These studies will enable a deeper understanding of the genotype-phenotype correlation of WBS microdeletion, and help to understand the molecular mechanisms of diseases and the human social brain.
Previous reports have shown that patients' clinical symptoms are also affected by the size of the deletion (6,44). In comparison to other patients with atypical deletions, the chromosome deletions of patient No. 9 are larger and the neurodevelopmental delay is more severe, which is consistent with previous reports (44,45). This suggests that this effect is related to the deletion of the extension genes HIP1 and YWHAG on the telomere side of WBSCR, which inhibits the patient's neurodevelopment (46). The data of this study also showed that patients with large deletions (such as patients No. 2 and No. 9) did not have classical facial features, which may be related to the size and position of the deleted fragments. This prompts to focus on the diagnosis of patients with non-classical atypical WBS.
Growth restriction is another characteristic of WBS patients (4). The WBSCR22 gene encodes a putative methyltransferase protein that is strongly expressed in the heart, skeletal muscle, and kidney. Its hemizygous deletion may lead to growth retardation, myopathy, or premature aging (47). In this study, only patient No. 7 retained the WBSCR22 gene and showed normal physical development, while seven of the eight (87.5%) patients with deletion of this gene showed growth restriction, of which patient No. 3 with low-birth-weight. This suggests that this effect may be caused by the deletion of the WBSCR22 gene. However, the growth of patients is affected by many factors such as diet, endocrine, function, and the environment. Therefore, more research is needed to verify the effect of the WBSCR22 gene on the growth and development of WBS patients.
The ELN gene encodes elastic bers, which are essential elements of the extracellular matrix. Heterozygous deletion of the ELN gene is the main cause of cardiovascular abnormalities in WBS patients, especially SVAS and PS (5). All patients in this study have a deletion of heterozygosity in the ELN gene, and all patients (except for patient No. 9) developed cardiovascular disease, which is consistent with previous reports (48). The possible reason why patient No. 9 has not yet developed a relevant cardiovascular phenotype may be the young age of the patient. Alternatively, the patient's deletion of genes outside the WBSCR region may also be related to the cardiovascular phenotype.

Conclusions
The BAZ1B, FZD9, and STX1A genes may play an important role in the neurodevelopment of patients with WBS. Furthermore, the deletion of the Declarations