Copy number variations (CNVs) are often a significant cause of neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, developmental delay, epilepsy, etc. Epilepsy is one of the most common neurological disorders characterized by recurrent seizures due to excessive excitation of neurons, with approximately 70% of cases determined by genetic factors (Thomas and Berkovic, 2014). According to reports, 5%–12.7% of epilepsy patients have CNVs (Coppola et al., 2019; Orsini et al., 2018). At present, there are few reports on 9q21.13 microdeletion syndrome, which is characterized by severe developmental delay, epilepsy, neurobehavioral disorders, and recognizable facial features such as hypertelorism, long philtrum, and thin upper lip. There is no unified standard for fragment size in existing reports. Moreover, there is no clear syndrome record in the CNV Syndromes and GeneReviews of DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources) database. Boudry-Labis et al. (Boudry-Labis et al., 2013) described the 750 Kb minimal overlapping deletion region in the 9q21.13 locus, which contains 4 genes (RORB, TRPM6, NMRK1, OSTF1) and 2 open reading frames (C9orf40, C9orf41). Among these genes, RORB is a strong candidate gene for neurological phenotypes.

The RORB (RAR-related orphan receptor B) (OMIM *601972) gene which encodes RORβ, having 2 differentially expressed subtypes, namely, RORβ1 and RORβ2 (André et al., 1998). In humans, RORβ1 is mainly expressed in the cortex, spinal cord, and pituitary gland (Baglietto et al., 2014), while RORβ2 is mainly expressed in the retina and pineal gland (Lal et al., 2015). Jabaudon et al. (Jabaudon et al., 2012) demonstrated that the expression level of RORβ controls the cellular structural pattern of neocortex neurons during development. In addition, it has been reported that RORβ is specifically expressed in cortical samples from patients with temporal lobe epilepsy (Rossini et al., 2011). Rudolf et al. observed that nonsense and missense variants, as well as CNVs of various sizes in the RORB gene, may lead to RORB haploinsufficiency (HI), resulting in common phenotypic features such as intellectual disability and systemic epilepsy (Rudolf et al., 2016).

In this article, we identified a novel 9q21.13 microdeletion containing the RORB gene with a fragment size of approximately 2.35 Mb. The microdeletion is stably inherited in 4 individuals from a 3-generation Chinese family and shares the similar clinical phenotypes as the reported 9q21.13 microdeletion syndrome. The aim of this study is to enrich the phenotypic features of 9q21.13 microdeletion syndrome and expand the possible segments of 9q21.13 microdeletion syndrome.

Here we report on a girl with a 2.35 Mb microdeletion at 9q21.13 inherited from her mother and grandmother, with clinical phenotypes of epilepsy, intellectual disability, speech impairment, delayed motor development, and autism. Unfortunately, the fetus in the mother’s womb also had microdeletion in the same region. Intuitive family history is presented in the pedigree chart (Figure 2).

Although the deletion loci are consistent, the clinical phenotypes of family members are not completely the same. The grandmother has the lightest symptoms, only showing intellectual disability. The mother has slightly lower intelligence, and the electroencephalogram shows epileptic waves, but there are no epileptic seizures. While the proband has the most severe symptoms and an early onset age. Different individuals exhibit significant differences in the same variation. Not only in this study, but other studies have also shown significant differences in clinical phenotype and age of onset among individuals with the same variation.

In 2012, the first case with 9q21.13 microdeletion syndrome was identified by Bartnik et al., which is characterized by epilepsy with eyelid myoclonia, generalised tonic-clonic seizures and autism (Bartnik et al., 2012). By comparing the genotypes and phenotypes of 13 patients, Boudry Labis found that these patients share common major clinical features, including mental retardation, developmental delay, epilepsy, neurobehavioral disorders, and characteristic facial features (Boudry-Labis et al., 2013). 13 cases have a minimum overlapping deletion region of 750 Kb, including 4 genes (RORB, TRPM6, NMRK1, OSTF1) and 2 open reading frames (C9orf40, C9orf41). Notably, RORB is lost in all 13 cases, which further confirms that RORB is a strong candidate for neural phenotypes (Boudry-Labis et al., 2013). From PubMed database, only one case with a microdeletion fully included in our patients’ deletion was selected. The patient has a deletion of 582 Kb on 9q21.13 only encompassing exon 1 of RORB, with symptoms of childhood absence epilepsy, intellectual disability, and behavioral problems with aggressive features (Lal et al., 2015; Rudolf et al., 2016). In 2015, Genesio et al. reported a case of 9q21.13 microdeletion syndrome caused by chromothripsis characterized by severe intellectual disability, epilepsy, global developmental delay, dysregulation of platelet aggregation, dysmorphisms, genitalia malformations and hypothyroidism (Genesio et al., 2015). In another case, Tuğ et al. identified a new interstitial deletion within 9q21.11q21.32 confirmed by array comparative genomic hybridization (aCGH) (Tuğ et al., 2018). The patient has many typical features, including but not limited to intellectual disability, development delay, speech disorder, epilepsy, autistic behaviour/behavioral problems as well as craniofacial dysmorphic features (Tuğ et al., 2018). In 2023, a 7-year-old boy with a de novo deletion on 9q21.13 was described, presenting global developmental delay, intellectual disability, autistic behaviour, seizures and facial dysmorphism (De et al., 2023).

Apart from the PubMed database, we also searched 31 patients with the deletion in 9q21.13 locus overlapping with our patients’ microdeletion from DECIPHER database. Among them, Boudry-Labis’ 13 patients are included. Meanwhile, we excluded 4 cases without clinical information (291952, 374605, 385645, 501796). The detailed information of the remaining 14 cases is shown in Table 1.

The location of deletion region in the patients we detected is different from before, and it does not cover the minimum overlapping region of 750 Kb as previously reported (Boudry-Labis et al., 2013). However, there are similar clinical phenotypes to previous reports, indicating that 9q21.13 microdeletion syndrome is not only caused by a specific gene or genomic region. We should analyze the specific deletion specifically. The deletion region in our study contains 6 protein coding genes, namely, ALDH1A1, ANXA1, GDA, RORB, TMC1 and ZFAND5. Of which, RORB and TMC1 are OMIM Morbid genes.

RORB, as a nuclear orphan receptor, can regulates neuronal patterns during cortical development and may be involved in neuronal cell differentiation (Jabaudon et al., 2012; Rossini et al., 2011). Rorb−/− mice exhibited damage in several neural reflexes, retinal degeneration, altered circadian activity, increased exploratory activity, less depressive-like behavior, reduced anxiety behavior, and ataxia, indicating RORB may be involved in sensory processing and emotional regulation (Masana et al., 2007). In addition, recent studies from SNP tagging and genotyping manifest that there is a correlation between RORB and bipolar disorder (McGrath et al., 2009; Partonen, 2012). Sadleir et al. reported 11 affected individuals from 4 families with inherited RORB variants with an overlap of occipital epilepsy and photosensitive genetic generalised epilepsy (GGE) (Sadleir et al., 2020). Each family has one novel variant, 3 families with missense variants and one with exon 3 deletion of RORB, which located in key evolutionary conserved domains and were all predicted to be pathogenic by in silico tools, and not previously seen in population databases (Sadleir et al., 2020). What’s more, the intragenic microdeletion was predicted to give rise to loss-of-function (Sadleir et al., 2020).

In 2013, a report revealed that RORB has been deleted in all 13 cases with 9q21.13 microdeletion syndrome characterised by epilepsy, intellectual disability, speech delay, autism, and moderate facial deformities (Boudry-Labis et al., 2013). In this family, no obvious facial deformities were observed, which may be due to atypical facial features or inconsistent fragments with previous reports, which needs further observation and in-depth research. Gokce-Samar et al. (2024) reported 32 different heterozygous variants in RORB carried by 35 patients, including 28 single nucleotide variants or small insertions/deletions and 4 microdeletions; among them, 89% of patients had epileptic seizures, and 85% of patients had varying degrees of intellectual disability. A large-scale European case-control cohort study on GGE identified a microdeletion only containing the exon 1 of RORB gene from a male patient with childhood absence epilepsy (Lal et al., 2015), overlapping with the critical region of 9q21.13 microdeletion syndrome (Boudry-Labis et al., 2013). The deleted region we detected contains ALDH1A1, ANXA1, GDA, TMC1, ZFAND5 and the first exon of RORB and caused epilepsy and intellectual disabilities, which is consistent with the phenotypes of patient in the case-control study mentioned above. Therefore, we assume that the RORB gene plays a crucial role in the 9q21.13 microdeletion. Other evidence suggests that nonsense and missense variations involving the RORB gene, as well as various sizes of CNVs, may lead to HI of RORB, resulting in common phenotypic features involving intellectual disability and generalised epilepsy (Rudolf et al., 2016).

The above numerous pieces of evidence have shown that the RORB is a strong candidate gene for complex neurodevelopmental disorders and also has a strong association with 9q21.13 microdeletion syndrome. As is known, the RORB gene is associated with susceptibility to idiopathic generalized epilepsy-15 (EIG15; OMIM #618357), an autosomal dominant disease with possible incomplete penetrance which is characterized by the occurrence of different types of epileptic seizures within the first decade (Rudolf et al., 2016; Dong et al., 2021). In 2016, in a 4-generation French family with EIG15, 4 affected individuals carried a heterozygous nonsense variation in the RORB gene which is detected by WES, and confirmed by Sanger sequencing (Rudolf et al., 2016). And this variation segregated with the disease phenotype in this family, which may be a loss-of-function variation (Rudolf et al., 2016). The evidence from Rudolf et al. (2016) strongly indicates that both nonsense and missense variants, and various CNVs in RORB may lead to the RORB HI, which is almost absent in the control population, generating some related neurodevelopmental phenotypes such as intellectual disability and epilepsy.

TMC1, another OMIM Morbid gene, whose variation associated with autosomal dominant deafness-36 (DFNA36; OMIM #606705) and autosomal recessive deafness-7 (DFNB7), also known as DFNB11 (OMIM #600974) (Kurima et al., 2002). None of our patients have a phenotype of hearing loss. In a study of autism rat models, it was found that the retinoic acid synthase ALDH1A1 was downregulated at both mRNA and protein levels in the valproic acid (VPA)-treated offspring (Yuan et al., 2023). Supplementing with retinoic acid improves motor coordination in autism model rats, indicating the role of ADH1A1 in the synthesis of retinoic acid (Yuan et al., 2023). However, There is no relevant OMIM disease information for ALDH1A1, as well as ANXA1, GDA and ZFAND5. It require extensive research and case analysis to determine their association with diseases.

In summary, we detected microdeletions at the 9q21.13 in the samples of the proband and her mother using WES technology. Due to the detection limitations of WES in detecting CNVs, we then validated the microdeletions via CMA technology and found that the locations of the microdeletion of the proband, her mother and her grandmother were identical, which was generally consistent with the regions detected by WES. The final assessment of CNV pathogenicity was based on CMA result, i.e., pathogenic. Moreover, CMA testing of amniotic fluid DNA revealed the same variation in the fetus with no clinical phenotypes. Nevertheless, it cannot be guaranteed that it will not develop symptoms after birth, and the severity of its phenotypes cannot be predicted. Given the symptoms of other family members and other reported cases of 9q21.13 microdeletion syndrome, the classification of this CNV is pathogenic. Therefore, it deserves the attention of prenatal diagnostic physicians and genetic counselors. Intellectual disability is a common feature among the affected members in this 3-generation Chinese family, and other clinical phenotypes include epilepsy, speech impairment, delayed motor development, and autism. After reviewing relevant literatures, we speculate that RORB has a significant contribution to the clinical phenotypes caused by this microdeletion.