Neurodevelopmental disorders, including intellectual disability and language disorder, have become a major public health concern that places a heavy burden on both families and the whole society (Maia et al., 2021). In recent years, the rapid development of genome sequencing technology has provided an effective solution to diagnosing neurodevelopmental disorders, which is conducive to the prevention of birth defects. BCL11B-related disorder has been a recently reported hereditary disease caused by the BCL11B gene de novo variant. Up to now, there have been plenty of reports on the role of the BCL11B gene in the nervous and immune systems (Arlotta et al., 2005; Li L. et al., 2010; Li P. et al., 2010; Kominami, 2012; Yu et al., 2015; Simon et al., 2016, 2020; Fang et al., 2018; Daher et al., 2020), i.e., early studies demonstrate that Bcl11b plays a critical role in the development of corticospinal motor neurons axonal projections to the spinal cord in vivo by loss-of-function experiments in null mutant mice for Bcl11b (Arlotta et al., 2005); on the other hand, Bcl11b was expressed in all T cell compartments and was indispensable for T lineage development and commitment in mice (Li L. et al., 2010; Li P. et al., 2010). Those patients carrying the pathogenic variations of the BCL11B gene tend to have immune deficiency or intellectual developmental disorder, the major clinical manifestions are speech delay, dysmorphic facies, and T-Cell abnormalities. Meanwhile, it is accompanied by various phenotypes, such as autistic symptoms, attention deficit-hyperactivity disorder, anxiety, dental anomalies, and other behavioral abnormalities (Punwani et al., 2016; Lessel et al., 2018). So far, there have been merely19 cases reported worldwide (Punwani et al., 2016; Lessel et al., 2018; Qiao et al., 2019; Prasad et al., 2020; Yan et al., 2020; Yang et al., 2020).

Located at chromosome 14q32.1, the human BCL11B gene encodes a C2H2-type zinc finger protein. The isoform 1 (NM_138576.2) of BCL11B consists of 894 amino acids and 4 exons (Lennon et al., 2016). All of these six zinc finger domains are located in the last exon (exon 4), in which the ZnF2 and ZnF3 domains are responsible for DNA binding. Besides, the N-terminal CCHC zinc finger motif plays a vital role in the formation of the BCL11B dimer (Kominami, 2012; Grabarczyk et al., 2018). Currently, there are 17 variants of the BCL11B gene identified in 19 patients through different studies. The major types of BCL11B genetic variation include frameshift, non-sense, missense, and complex chromosome rearrangement. However, there is still no report on splicing variation.

In this study, 4 patients with neurodevelopmental disorders and extended phenotypes were reported. Notably, a novel splicing variant and a de novo frameshift variant were detected through whole exome sequencing from the two studied families separately. Furthermore, the function of the two gene variants was verified by an in vitro minigene assay and an NMD assay.

This study identified a new splice variant and a novel frameshift variant of the BCL11B gene in two unrelated pedigrees, including 3 patients in Family 1 and 1 patient in Family 2 with neurodevelopmental disorders. According to the in vitro minigene assay, the splice variant could cause the abnormal splicing of the BCL11B gene. Meanwhile, the extended phenotype with developmental retardation was observed in the proband of Family 1, suggesting the clinical heterogeneity and complex biological mechanisms of the disease.

It is widely known that BCL11B-related disorder can lead to two phenotypes with common features, namely, type 49 immunodeficiency and intellectual development disorders with malformation, speech delay, and T-cell abnormalities. There were 19 patients reported to show delayed psychomotor development, of whom only one showed the severe combined immune deficiency caused by missense variants (Punwani et al., 2016). In addition to the common features shown by all patients, such as facial dysmorphism, it was also found in this study that the proband in Family 1 also showed regression in intellectual and language development, which has been unreported previously. Moreover, the clinical manifestations of the 3 patients in Family 1 varied significantly. The proband showed more typical features of the disorder than her mother and brother did, whereas the younger brother exhibited milder symptoms. This finding suggested phenotypic heterogeneity of the splice site variant in BCL11B-related disorder from this family-based study. There are various reasons for this phenotypic heterogeneity. It is suspected that the detected splice variation may lead to various sets of abnormal transcripts in different patients, which is associated with the significant variation in gene expression and function. Additionally, the other variants might be excluded from WES analysis, which is suspected to be a pathogenic factor located in non-coding regions or associated with complex structural rearrangements. For the proband’s younger brother, the clinical phenotype showed changes due to aging. A follow-up survey or gene investigation could be conducted to clarify this.

At present, the diverse roles of BCL11B in T cell biology and Group 2 innate lymphoid cell (ILC2) development have been studied by many researchers (Walker et al., 2015; Yu et al., 2015, 2016; Xu et al., 2019). It was in 2016 that the first patient manifesting severe combined immunodeficiency (SCID) and multisystem anomalies was reported (Punwani et al., 2016), with nearly half of the reported patients displaying persistent infections, eosinophilia, allergy, and asthma. Consistently, it was discovered in our study that the proband and her younger brother in Family 1 exhibited persistent infections as well, and that the proband in Family 2 showed only allergy. Moreover, flow cytometric analysis was conducted to reveal that the ratio of CD4 + /CD8 + was the lowest in the proband and her younger brother of Family 1, but normal in the proband in Family 2. According to the study of Lessel et al. (2018) this is linked to the impairment of T cell immunity. Given a lack of definitive immune deficiency diagnosis, it is necessary to reveal the impact of BCL11B gene variation on the immune system through more clinical cases and pathogenic mechanism studies.

De novo cryptic splice variants contribute significantly to rare genetic disorders, especially intellectual disability and autism spectrum disorders (2015; Jaganathan et al., 2019). In this study, a minigene expression assay was performed to figure out the in vitro effects of the c.427 + 1G > A variant on gene function of BLC11B. By causing the absence of the splice donor on Intron 2 and the incidence of a novel cryptic donor splice site on Exon 2, the splice variant shortened Exon 2 and led to the generation of an abnormal pre-mRNA (c.142_427del). On the translation level, the abnormal pre-mRNA produced a prematurely truncated protein p.Val48Glyfs*14 but with no ZnF_C2H2 domains of the BCL11B protein. Given the involvement of ZnF2 and ZnF3 domains in DNA binding (Kominami, 2012), it is speculated that the c.427 + 1G > A gene variant could interfere with the recognition of BLC11B protein and DNA and the interaction between them. Besides, the abnormal truncated variant p.Val48Glyfs*14 is located in Exon 2, and it is thus predicted to trigger NMD and cause haploinsufficiency as a result. In summary, the genetic etiology of patients in Family 1 is closely associated with the splice variant c.427 + 1G > A of the BCL11B gene.

It is also known that de novo frameshift variants can result in severe intellectual disability and developmental delay (Kosmicki et al., 2017). For all the BCL11B variants associated with developmental delay and intellectual disability, frameshift variants account for 76% (13/17), with nearly all of them being de novo. However, there was only one frameshift variant p.Asp534Thrfs*29 reported as hereditary (Lessel et al., 2018). In our study, the proband in Family 2 carried a de novo frameshift variant p.Glu821Glyfs*28 in the BCL11B gene. As confirmed by the overexpression experiments, this variant could escape NMD to form a truncated protein, despite no significant difference observed in protein and RNA expression between the mutant plasmid and the wild-type plasmid. So far, the variant p. (Gly820Alafs*27) has been confirmed to result in a functional null allele and suppress progenitor cell proliferation in hippocampal of Bcl11b mutant mice. Also, it has been found to interfere with the expression of the BCL11B in dentate neurons (Lessel et al., 2018). As a neighboring variant, the p.Glu821Glyfs*28 truncated variant as identified in this study has a potential to produce a functional null allele, thus causing the disorder. By summarizing the phenotypes of all patients with a frameshift/non-sense variant (Supplementary Table 3), it can be found out that all the frameshift variants may cause the loss of the last two or three C-terminal ZnF_C2H2 domains, except for p.Ala891Profs *106 and p.Cys81Leufs*76 variants. As a result, NMD can be activated (Hamby et al., 2011). However, there was no significant correlation observed between the number of retained Zn finger domains and the severity of symptoms. Since the zinc finger domain, usually, must be connected in series for binding DNA (Avram et al., 2002), the loss of the zinc finger domain may offset the physiological impact of BCL11B protein by affecting its transcription-regulatory activity.

In conclusion, a novel splicing variant of the BCL11B gene and a novel frameshift variant were identified in this study from two different families separately. The findings of this study expand the spectrum of gene variation associated with the disease. The genetic and clinical data used in the study provide evidence for a study to better understand the pathogenic mechanism and genetic etiology of the disease. BCL11B-related disorder is an intellectual development disorders characterized by malformation, speech delay, and T-cell abnormalities or immune deficiency. Genetic testing is conducive to improving the accuracy of a clinical diagnosis and providing firm evidence for gene counseling.

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Xi’an Children’s Hospital. Written informed consent to participate in this study was provided by the participants; legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s); legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

FC and YY contributed to conception and design of the study. XT and LZ collected the clinical data. FC and XT wrote the first draft of the manuscript. HL revised the manuscript, while XZ and MD coordinated and supervised data collection. All authors contributed to revising the manuscript and read through and approved the submitted version.