Introduction
The advent of the next-generation sequencing (NGS) technology has revolutionized the current approaches in molecular diagnostics of individuals with severe intellectual disabilities (ID), developmental delay (DD), autism spectrum disorders (ASD) and multiple congenital abnormalities (MCA). Recently, >1,000 genes have been identified as the cause of ID and other neurodevelopmental disorders (1).
A previous study showed that >30% of cases of idiopathic ID and 13% of cases of severe ID (IQ<50) can be explained with a specific molecular diagnosis using a targeted NGS approach (2). Moreover, the current molecular diagnostic method uses whole-exome sequencing (WES), which is a highly powerful and cost-effective part of the first-tier standard diagnostic approach, with a diagnostic yield of up to 55% (3).
Bainbridge-Ropers syndrome (BRS; OMIM #615485) is a rare congenital disorder characterized by delayed neuronal, motor and growth development, severe ID accompanied by absent or poor speech, muscular hypotonia, feeding difficulties and facial dimorphism (4). It was first described by Bainbridge et al (5), who identified rare de novo truncating sequence variants in the additional sex-combs like 3 (ASXL3) gene in a group of 4 unrelated affected children presenting similar phenotypic features. The pathogenic sequence variants in the ASXL3 gene lead to the reduced expression of the gene. The haploinsuffiency of the ASXL3 gene with high penetrance is the most likely the underlying and causative mechanism of the disease (6).
In the present study, the case of a child (female, born in 2013) with global developmental delay, central hypotonia, microcephaly and poor speech is described. She was examined using a multi-step molecular diagnostics algorithm, including karyotype and array-comparative genomic hybridization (CGH) analyses. However, no pathogenic chromosomal rearrangements or copy-number variants (CNVs) that could explain the phenotype of the patient were identified. Therefore, the patient and her healthy parents took part in a pilot study using targeted NGS with a commercially available gene-rich panel. This panel contained 2,742 genes catalogued in the Online Mendelian Inheritance in Man (OMIM) database whose pathogenic sequence variants are associated with human inherited diseases. The results of this approach were subsequently validated using consecutive Sanger sequencing.
Discussion
The present study reports the case of a child with severe psychomotor delay, hypotonia, microcephaly and facial dimorphism. Using targeted NGS and following verification analysis by Sanger sequencing, a novel de novo pathogenic sequence variant in the ASXL3 gene, p.R1004Efs*21, was identified.
The ASXL3 gene is located at the 18q12.1 chromosome region and encodes a member of the vertebrate ASX-like protein family. ASXL family members represent epigenetic scaffolding proteins that assemble specific epigenetic regulators and transcription factors to specific genomic loci containing histone modifications (41,42). Polycomb protein ASX (ASX) was first identified in Drosophila melanogaster as a part of the polycomb group of proteins involved in embryonic development, maintaining HOX genes in a transcriptionally repressive state (43,44). The encoded protein ASXL3 contains 12 exons and has 2,248 amino acids. Like other ASX family members, the ASXL3 protein has a conserved domain structure: ASXN and ASXH domains in the N terminus; ASXM1 and ASXM2 domains in the middle region; and the PHD domain in the C terminus (41,42). The ASXN and PHD zinc-finger domains play a role in the regulation of gene transcription, representing putative DNA- or histone-recognition sites. The region around the ASXH domain creates protein-protein interaction sites for association with epigenetic regulators. The ASXM1 and ASXM2 domains are involved in protein-protein interactions. Between ASXH and ASXM1, there is the 5′ mutational cluster region (MCR), where truncated or splice variants tend to cluster, giving rise to aberrant ASXL proteins with intact ASXN and ASXH domains, while pathogenic sequence variants in the 3′MCR lead to variants with changes between the ASXM1 and ASXM2 domains (45).
Recent studies have highlighted important roles for the ASXL3 gene in congenital disorders (45) and cancer (40) through the identification of multiple ASXL3 pathogenic sequence SNVs and indels. Focusing on congenital disorders, the pathogenic variants affecting the ASXL3 gene have been identified as the cause of disorders in patients sharing similar phenotypic features, including delayed psychomotor development with missed milestones, microcephaly, severe persisting feeding difficulties, poor growth, poor or absent speech and dimorphic facial features (4,5,6). As presented, the proband exhibits most of the typical BRS-related phenotypic features (abnormal pregnancy, preterm birth and reduced birth parameters, feeding difficulties, craniofacial abnormalities, psychomotor delay and autistic features). Brain MRI scans in patients with BRS usually indicate mild white matter loss and mild corpus callosum hypoplasia, or mild cerebellar vermis hypoplasia. With the exception of the brain MRI findings, these features are different to those previously described as a consequence of pathogenic variants in the ASXL1 gene, causing Bohring-Opitz syndrome (BOS) (46). A recent study identified a group of six unrelated patients having de novo heterozygous truncating variants in the ASXL2 gene (47). These patients shared specific clinical features, some of which are also present in patients with the ASXL1 gene (BOS) and ASXL3 gene (BRS) truncating or splicing pathogenic variants (developmental and intellectual impairment, facial dimorphism, feeding difficulties in the neonatal period and hypotonia). Since 2013, ~30 patients with ASXL3 pathogenic truncating or splicing pathogenic variants causing BRS have been identified in scientific publications, to the best of our knowledge. The variants are clustered predominantly in two previously reported separated MCRs in exon 11 and exon 12, respectively (45).
In the proband, a novel truncating variant, p.R1004Efs*21, located in the terminal part of exon 11 outside the reported MCRs, was identified. The mutation is located in an amino acid coding region conserved among vertebrates; thus, the MutationTaster2 and VarSome tools predicted a deleterious effect of this variant on protein structure and function due to truncation. Moreover, as mRNAs containing a premature stop codon can undergo degradation through nonsense-mediated decay (NMD) (4), the truncating variants may have a loss-of-function (LoF) impact. Functional studies have suggested that all ASXL3 nonsense variants may be translated to prematurely terminated proteins, which may consequently act in a dominant-negative way (4,5). The correlation between the position of the truncating variant and the severity of the phenotype has also been debated: The disease severity may decrease as the variant occurs further away from the 5′-end of exon 11 and towards the 3′-end (5). A recently described case of a male child with atypical BRS also revealed the occurrence of a novel heterozygous de novo variant, p.P1010Lfs*14, in the ASXL3 gene (48). This variant leads to an identically truncated ASXL3 protein as that seen in the proband of the present study, but the phenotypic features between the cases are different, namely the absence of apparent structural brain abnormalities and less prominent facial dimorphism in the proband.
As an exceptional case of two compound heterozygous variants in the ASXL3 gene, there was a reported case of another patient harboring a missense variant, p.R989G, of maternal origin, and a missense variant, p.K1026N, of paternal origin (49). The patient experienced BRS-like features and primary insulin-like growth factor 1 deficiency. That study predicted that the phenotype was the product of the synergistic or additive effect of these missense variants in the LoF of the ASXL3 gene. The ASXL3 gene has also been previously identified as an ASD risk gene, particularly missense variants of this gene (50). In the ClinVar database (23), there are no reported cases of patients with a BRS phenotype having heterozygous de novo pathogenic missense variants in the ASXL3 gene.
Unfortunately, BRS is not a common clearly recognizable syndromes, especially due to the absence of specific phenotypic features and the low total number of reported cases. Among patients with BRS, it is possible to observe varying degrees of phenotypic severity, especially varying in the degree of ID (5). This range in severity may have an early or late post-zygotic origin, leading to pathogenic variants with somatic mosaicism, which will then have an incomplete penetrance and/or variable phenotypic features (5). Two of the described variants occur at the 3′-end of the gene (p.E1761fs, p. E1824K), indicating escape from NMD and the retention of protein activity. These observations also confirm that a broad allelic heterogeneity may be implicated in the pathogenesis of BRS.
Based on previously reported cases, the penetrance of truncated or spliced pathogenic variants seems to be high, and these have a de novo origin since they are absent in healthy individuals. In the ExAC database (32) there are four reported LoF variants (three exonic truncating variants and one splicing variant), none of which have an allelic frequency higher than 8.34×10−6 (exonic truncating variants) or 1.38×10−5 (splicing variant), and the probability of LoF intolerance is estimated to be 1.00. This value predicts high intolerance of the gene to LoF mutations (32). A total of >670 missense variants have been reported at present, and their allele frequency varies significantly (from 8.281×10−6 to 0.6029), occurring across the whole coding region of the ASLX3 gene. This observation is indicative of the different impact of missense variants on the ASXL3 protein function, from benign polymorphisms to possibly damaging effects (which was evaluated using Polyphen-2 in silico prediction) (35).
In the present study, a novel de novo frameshift variant in the ASXL3 gene, which could lead to a truncated protein in a young patient with severe psychomotor developmental delays and microcephaly, was detected. Based on these findings and previous reported cases, it was proposed that the identified frameshift variant in the ASXL3 gene may have significantly affected ASXL3 protein structure and function. These observations may help in defining and diagnosing BRS in patients with similar phenotypic features.