Nine WBS patients have atypical deletions, 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 sufficient 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, i.e., high-resolution molecular testing, such as Whole Exome Sequencing (WES), Whole Genome Sequencing (WGS) and targeted region sequencing (TRS))(16, 17) is recommend to obtain more accurate and complete genetic information.
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 five 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 significantly 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 influence 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 sufficient 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).
In contrast, patient No. 6 mainly deleted genes from the centromere side of WBSCR (from FKBP6 to ELN) and showed typical WBS cognitive characteristics. Analysis of the molecular and phenotypic relationship of patient No. 6 as well as previous case reports(27–30) with similar deletion positions and mental retardation (as shown in Figure. 1) showed that the centromeric side gene of WBSCR also plays an important role in the phenotype of patients with WBS. BAZ1B, FZD9, and STX1A genes are particularly important in this regard, according to previous studies(9, 31–34) and the GeneCards Human Gene Database (https://www.genecards.org/).
BAZ1B (the bromodomain adjacent to zinc finger domain, 1B) gene, also known as Williams Syndrome Transcription Factor (WSTF), plays an important role in the differentiation and migration of nerve cells. It also participates in the neural crest specific transcription loop and remote regulation(31). Wnt signal plays an important role in the regulation of the balance between proliferation and differentiation of neural progenitor cells. Inhibition or overexpression of the Wnt signal function can lead to decrease or proliferation of neural progenitor cells, respectively. The BAZ1B gene is enriched in the Wnt signal transduction pathway, and this pathway is activated because of the deletion of this gene in WBS 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 influencing 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 influencing 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 deficit hyperactivity disorder(42, 43). However, the contribution and underlying mechanism of its deficiency 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 fibers, 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.