WBS is a common microdeletion syndrome characterized by a recognizable facial feature, known as “Elfin Face”, intellectual disability, a particular behavioural and cognitive profile characterized by hyper sociability and sympathetic personality, and often cardiovascular disease. This study aims to establish a genotype-phenotype correlation in a cohort of 31 Tunisian patients and to study the differences in the size of the deletion and its involvement in the spectrum and severity of WBS clinical features (Fig. 1).
Previous studies have identified a critical region on 7q11.2 responsible for the syndrome (WBSCR) ranging from 1.5 million to 1.8 million base pairs and involving 26 to 29 genes. However, the implication of these genes in the clinical features of the syndrome is still unknown. Up to now, the advent of molecular cytogenetic techniques has made it possible to progressively identify the critical regions that may be responsible for the different clinical features of a syndrome.
Our study focuses on the variable features in 31 cases of 7q11.2 deletions. Among this cohort
6 patients have benefited from a CGH array to study the different sizes of deletions and the genes involved to establish an adequate genotype-phenotype correlation. The results revealed a partial loss of 7q with different breakpoints and different size deletions (Fig. 2). The patients who benefited from a CGH array presented atypical clinical features compared to the other WBS patients. The first patient, in addition to the facial dysmorphia and the hyper-sociable compartmental profile, presented two congenital heart defects: lung and aortic stenosis.
For the second patient, she presented a bone malformation: bilateral clinodactyly 5. A deletion of about 1.7MB was objectified in this case with the presence of almost the same clinical signs in his brother. Moreover, the paternal hereditary character for this deletion has been asserted for these 2 children. Despite the confirmation of the ELN gene deletion by FISH in all patients
included in this study and given the implication of this gene in cardiac malformations, the heart ultrasound performed on patient 3 came back without defects. The 4th patient presented a severe clinical feature compared to the other patients, characterized by the presence of renal agenesis and hypothyroidism. However, the last two patients showed an opposite phenotype marked by autism spectrum disorders, contrasting with the main behavioural traits seen in WBS patients characterized by severe difficulties in the visuospatial domain and rather specific behaviour, of the hyper sociable type, but also anxious, associated with a hypersensitivity to noise and a disposition for music.
The CGH array exploitation targeted the 7q loss of 1,43 Mb in the first patient, 1,71 Mb in the second, 1,4MB in the third, 1,39 MB in the fourth, and about 1,7MB for the latest (Fig. 3).
We suspect that the different deletion sizes and variability of the genes implicated may play an important role in the development of the disease in each patient.
The commonly deleted region in all patients involves five important genes: ELN, CLIP2, LIMK1, GTF2I, and GTF2iRD implicated in the development of the main characteristics of WBS.
The ELN gene is considered since 1993 as the gene responsible for Williams syndrome and is the most characterized in this region [4]. The Elastin (ELN) gene [OMIM#130160] located in 7q11.23 codes for the protein elastin. The role of this protein in arterial development and disease has been proven by generating mice lacking elastin [7]. Besides, elastin is a vital protein in the aorta and large arteries. ELN-/- mice die immediately after birth due to vascular obstruction caused by enhanced smooth muscle cell proliferation[8]. Nevertheless, supravalvular aortic stenosis constitutes the prototypical cardiovascular abnormality of Williams–Beuren syndrome and it is found in about 70% of patients [9]. In the present study, the cardiac phenotype occurs in 55% of cases. Yet, atypical defects were found in 4 patients, 3 with septal ventricular defect and one with mitral valvar insufficiencies. However, no specific genetic pattern is significantly associated with these cardiac malformations. Furthermore, cardiovascular complications are the leading cause of death in patients with Williams-Beuren syndrome [9]. Even though deletion of the ELN gene is responsible for the syndrome and its involvement in the cardiac phenotype, no relationship with the neurodevelopmental and dysmorphic phenotype has been shown. We support the hypothesis that Williams syndrome is a multisystem disorder and that many genes are involved in the core phenotype.
Young infants with Williams-Beuren syndrome have delays in the acquisition of initial motor skills and the completion of language. This is due to the loss of function of many genes involved in neurodevelopment and cognitive functions, mainly the gene Lim Domain Kinase1 (LIMK1) [OMIM#601329]. This gene is expressed in the central nervous system during embryogenesis, including the inner nuclear layer of the retina, the cortex, the developing spinal cord, and the cranial nerve and dorsal root ganglia[10]. In the context of WBS LIMK1 is considered a strong candidate for the unexplained neurologic features and is involved in impaired visuospatial constructive cognition. Functional studies in knockout mice have shown that an alteration in this gene results in significant abnormalities in spine morphology and synaptic function, including enhanced hippocampal long-term potentiation [11]. We suggest that the LIMK1 gene
plays a critical role in the development of neurological disorders, which explains the behavioural profile of these patients, which is marked by intellectual disability, delayed acquisition of cognitive functions, and specific behaviour characterized by hyper sociability but also anxiety. On the proximal side of WBSCR, additional genes may act to complete the pleiotropic WBS phenotype such as the General transcription Factor Ⅱ-Ⅰ (GTF2I) [OMIM#601679] and GTF2IRD Repeat Domain-Containing Protein1[OMIM#604318]. These two genes encode transcription factors and they‘re strongly implicated in mental delay[11]. Besides 43% of the patients in the present study suffer from mental retardation. It has been demonstrated that GTF2I interacts with GTF2IRD1 [12]. Recent studies on the phenotypic features of patients with partial deletions of the WBS region suggest that GTF2I and GTF2IRD1 have an overlapping function and are involved in the development of the cognitive-behavioral profile of WBS including severe visuospatial impairment and hyper sociability [13] [14]. Immunohistological staining of brain tissue from patients with WBS patients revealed an absence of GTF2I binding in neurons of the posterior parietal lobe which includes parts of the dorsal parietal visual pathway[15].
Interestingly, the common deleted interval in all patients encompasses the Cap-Gly Domain-Containing Linker Protein 2 (CLIP2)[OMIM#603432] gene which belongs to a family of membrane–microtubule interacting proteins that are highly enriched in neurons of the hippocampus, piriform cortex and olfactory bulb[16]. CLIP2 knockout mice show both brain morphological and behavioural abnormalities that could be attributed to deficits in cerebellar and hippocampal functioning [16]. In addition, there are several reports of partial deletions of the WBS region that does not include the CLIP2 gene. The clinical results from this group suggest a major role for CLIP2 in the motor and cognitive characteristics of WBS. These patients with a partial deletion excluding the CLIP2 gene, among others, had only a mild or no visual-spatial impairment and they performed better in tasks testing perception, fine motor coordination, and gross motor skills [16] [17].
Nevertheless, WBS patients with an uncommon deletion size at 7q11.23 are of particular interest for the genotype-phenotype correlation of WBS. The less frequent deletion reported in the literature involves the Neutrophil Cytosolic Factor1 gene (NCF1) [OMIM#608512] encoding the subunit of NADPH oxidase. The findings suggest that losing a functional copy of NCF1 protects a subset of WBS patients from hypertension, most likely through lifelong reduced angiotensin II-mediated oxidative stress. The study of Del Campo et al [18]speculated that antioxidant therapy that reduces NADPH oxidase activity may benefit identifiable patients with WBS who have serious hypertension complications, as well as forms of essential hypertension mediated by a similar pathogenic mechanism.
Larger WBS deletions extending distal to the common deletion interval were associated with a more severe developmental delay involving several genes. Among these, HIP1 has been proposed as the most potent candidate gene for susceptibility to autistic traits [19]. In this study, we studied 4-year-old and 2-year-old boys, who are suffering from lung shrinking. In addition, they present a specific dysmorphic feature suggestive of the WBS and language and mental delay. Curiously, autism spectrum disorders were noted in contrast to the behavioural profile generally observed in other WBS patients, which is characterized by good contact despite a delay in the acquisition and development of certain cognitive skills, two cases with almost the same phenotype were reported with a large deletion of about 3MB including HIP1 gene (Table 1)
Indeed, Huntingtin-Interacting Protein 1(HIP1) [OMIM#601767] is located at 7q11.2 upstream of the proximal limit of the deletion region studied and is strongly expressed in the brain. In a loss-of-function model, mice with a targeted mutation in the HIP1 gene developed a neurological phenotype characterized by growth retardation, gait ataxia, and epilepsy[20].
We suggest that the more the deleted region is important, the more likely the patient is to develop autism, we oriented our hypothesis towards the fact that other mechanisms, such as variable expressivity or the positional effect of neighbouring genes with distant regulatory regions might also be involved. This may be due to alteration of the HIP1 gene which is a potential candidate. HIP1 is also required for a-amino-3-hydroxy-5-methyl-4-N-methyl-D-aspartate (NMDA) and isoxazole propionic acid (AMPA) main hippocampal neurons with abnormalities in receptor trafficking and is linked to significant and progressive neurological impairments[21].
Thus, HIP1 haploinsufficiency is sufficient to alter neuronal homeostasis, predisposing the human brain to epilepsy via a mechanism involving abnormal trafficking of AMPA and NMDA receptors. However, other hypotheses may be at the origin of this atypical clinical sign for WBS such as a mutation in another gene responsible for autism that is not located in the region studied in this case. As well as other CNVs can be involved. Certainly, genetic factors might be largely responsible for the occurrence of autism, but they cannot fully account for all cases, and it is likely that in addition to a certain combination of autism-related genes, specific environmental factors especially prolonged exposure and dependence on screens might act as risk factors triggering the development of this traits.
This study highlights the importance of accurately mapping the deletion boundaries of WBS genes to reassess the importance of these genes and their implication in the clinical features. It cannot be ignored that a patient with this syndrome is likely to develop autism and the indication for these cases of a CGH array is to establish an adequate genotype-phenotype correlation for better management. We assume that other factors, whether genetic, epigenetic, or environmental, play an important role in determining phenotypic variations and disease severity.
Given all these facts, functional studies or high throughput technologies are strongly recommended to better characterize the genetic and environmental interactions of Williams-Beuren syndrome.