Prevalence of CNVs on the X chromosome in patients with neurodevelopmental disorders

Abstract

Background

The X chromosome is enriched with genes related to brain development, and the hemizygous state of these genes in men causes some difficulties in the clinical interpretation of copy number variations (CNVs). In this study, we present data on the frequency and spectrum of CNVs on the X chromosome in a cohort of patients with neurodevelopmental disorders (NDDs).

Methods

Chromosomal microarray analysis was performed for 1175 patients with NDDs. CNVs were confirmed by real-time quantitative PCR. X chromosome inactivation was analysed by methyl-sensitive PCR. To determine the pathogenic significance of the CNVs, several criteria, including the origin (inherited or de novo), variant type (microdeletion or microduplication), and X chromosome inactivation pattern in asymptomatic and symptomatic carriers, were considered. Additionally, the spectrum, size and molecular bases of copy number changes in genes or gene regions involved in the development of the pathological phenotype in each patient were considered.

Results

CNVs on the X chromosome were identified in 33 patients (2.8%). Duplications and triplications (27 cases) were four times more common than deletions (6 cases). In 74% of patients, CNVs were of maternal origin; in 10% they were of paternal origin; and in 16% they arose de novo. The frequency of skewed X inactivation among family members who were healthy carriers of pathogenic and likely pathogenic CNVs and variants of uncertain significance (VUSs) on the X chromosome was 23%. For the first time, we reported several CNVs, including a pathogenic microdeletion at Xq26.1q26.2 involving the ARHGAP36 gene and a microduplication at Xp22.2 involving the OFD1 gene,

Conclusions

This study expands on the frequency and spectrum of CNVs in patients with NDDs. Pathogenic variants on the X chromosome were present in 15% of cases, LP in 12%, VUS in 57%, and LB in 16% of cases. Previously unreported CNVs aid in the identification of new structural variants and genes associated with X-linked intellectual disability. We propose to consider the X-chromosome inactivation status when assessing the pathogenetic significance of CNVs using the ACMG algorithm (American College of Medical Genetics).

Background

X-linked intellectual disability (XLID) is a group of hereditary diseases caused by mutations in various genes located on the X chromosome. There are 867 protein-coding X-linked genes, 500 of which are expressed in the brain [1]. To date, 162 genes associated with XLID have been identified, with 21 new genes identified since 2017 [2]. Although the discovery rate of new XLID genes has somewhat slowed in recent years, the density of known genes associated with intellectual disability (ID) on the X chromosome is still twice as high as that on any autosome [2]. XLID can be associated with either gene variants or copy number variations (CNVs), such as microdeletions and microduplications.

Pathogenic CNVs associated with microdeletion/microduplication syndromes do not have 100% penetrance and can be inherited by affected children from apparently unaffected parents [3]. CNVs containing known XLID genes are not always pathogenically significant, especially CNVs involving duplications. To correctly interpret the pathogenic significance of a CNV on the X chromosome in a patient with a neurodevelopmental disorder (NDD), several factors should be considered. The first factor is the segregation pattern of the CNV on the X chromosome. In male patients, the CNV is always inherited from the mother, while in female patients, the variant can be inherited from either the mother or the father. Second, male patients are more likely to have more severe clinical manifestations of CNVs on the X chromosome than females, in whom the same variant may either manifest in a milder form or not manifest at all. The manifestation in female CNV carriers depends on several factors, including the size and type of CNV (deletion or duplication) and gene content [4], as well as the pattern of X chromosome inactivation (XCI) [5]. Although there are numerous studies that describe CNVs on the X chromosome in patients with NDDs [4, 6, 7], new variants are often identified that do not always correspond to the previously reported ones and require careful analysis to determine their clinical significance. Therefore, we evaluated several factors (gene content, CNV origin, and XCI in CNV carriers) when analysing the pathogenic significance of CNVs on the X chromosome in a large cohort of patients with NDDs and propose to use the X chromosome inactivation status in CNV carriers among other factors to assess the pathogenetic significance of X chromosome CNVs using the ACMG algorithm (American College of Medical Genetics).

Methods

This study was approved by the Scientific Ethics Committee of the Research Institute of Medical Genetics of the Tomsk National Research Medical Center of the Russian Academy of Sciences (TNRMC) (# 15 from February 28, 2023). Written informed consent was obtained from all parents for themselves and their children.

The age of the patients ranged from 1 to 18 years. All patients underwent an initial examination by a geneticist to obtain clinical data and family histories. Venous blood was subsequently collected. Standard metaphase analysis, molecular genetics and biochemical studies were performed if a particular disease was suspected. Patients with numerical and large structural chromosomal abnormalities, hereditary metabolic disorders or monogenic disorders manifesting with intellectual disability (Martin-Bell, Prader-Willi, Angelman, and others) were excluded. Patients were selected for array comparative genomic hybridization (aCGH) when none of the examination results indicated the cause of the disease. aCGH analysis was carried out for 1175 individuals (740 males and 435 females).

According to the International Classification of Diseases 11th Revision (ICD-11), neurodevelopmental disorders are behavioural and cognitive disorders that occur during development. Patients with the following phenotypes were included: 6A00, disorders of intellectual development; 6A01, developmental speech or language disorders; 6A01.0, developmental speech sound disorder; 6A01.1, developmental speech fluency disorder; 6A01.2, developmental language disorder; 6A02, autism spectrum disorder; 6A03, developmental learning disorder; 6A04, developmental motor coordination disorder; 6A05, attention-deficit/hyperactivity disorder; 6A06, stereotyped movement disorder; and 6A0Y, other specified neurodevelopmental disorders.

The peripheral blood of patients and their relatives was collected in tubes containing EDTA for molecular genetic analyses. Genomic DNA was isolated from blood using phenol‒chloroform extraction.

aCGH analysis was performed using SurePrint G3 Human CGH 8 × 60 K microarrays (Agilent Technologies, Santa Clara, CA, USA) with a 41-kb overall median probe according to the manufacturer’s recommendations. Data analysis was performed using Cytogenomics Software (v. 5.3.0.14) (Agilent Technologies, USA). Interpretation of the clinical significance of CNVs was carried out in accordance with professional international standards [8, 9]. Since CNVs were localised to the X chromosome, the XCI pattern was also determined in obligate and affected carriers of the variant. If a CNV in the Database of Genomic Variants (DGV) occurred in three or more patients, we recalculated the scores of these CNVs according to the generally accepted ones, and these scores were − 0.99 and less. In these cases, the CNV was classified as benign and excluded from further analysis [10,11,12]. Next, variants were identified in the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER) [13]. The functions of genes located in regions of genomic imbalance were retrieved from the NCBI Gene Database and OMIM [14, 15].

In our study, CNVs were classified into four groups: pathogenic (P), likely pathogenic (LP), variants of uncertain significance (VUSs), and likely benign (LB). CNVs were classified according to a semi–quantitative scoring system defined in 2020 by the American College of Medical Genetics and Genomics (ACMG), the Clinical Genome Resource project (ClinGen) [12] and the CNV-ClinViewer Internet resource [11]. CNV classification also considered the origin, gene content, and XCI status in heterozygous carriers. Information about CNVs on the X chromosome in our sample was uploaded into the ClinVar database.

Real-time quantitative PCR was performed using the AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, United States). Specific target sequences were selected for real-time quantitative PCR (qPCR) using Primer 3 software (v. 0.4.0). Two primer pairs were created for each genomic region. A list of primers is shown in Supplementary Table 1. Control genomic DNA was obtained from the peripheral blood lymphocytes of a healthy donor. HEXB, which encodes the beta subunit of hexosaminidase and is located at 5q13.3, was chosen as the control gene.

XCI status was determined based on the amplification of a highly polymorphic CAG repeat in the first exon of the androgen receptor (AR) gene after DNA hydrolysis with the methyl-sensitive restriction endonuclease HpaII according to standard protocol [16]. A degree of inactivation < 80% was considered a random pattern, and a degree of inactivation > 80% was considered a skewed X chromosome inactivation (sXCI).

Results

Cytogenomic testing using aCGH on Agilent 60 K microarrays was performed for 1175 patients (740 males and 435 females) younger than 18 years with NDDs. Overall, CNVs were detected in 36% of patients (422 individuals), including 235 microdeletions and 295 amplifications. CNVs on the X chromosome were detected in 33 patients (Fig. 1a), including 26 boys and 7 girls (sex ratio, 3.7) (Table 1). The identified CNVs in patients were classified as pathogenic (4 males and one female), likely pathogenic (4 male patients), of uncertain significance (19 patients—14 males and 5 females), or likely benign (5 patients—4 males and 1 female) (Table 1). Overall, pathogenic and likely pathogenic CNVs accounted for 0.6%, and VUS and likely benign CNVs accounted for 2.2% of all included patients. For males, the frequency of P and LP variants was 1% and the frequency of VUS and LB variants was 2.4%, and for females, these values were 0.2% and 1.4%, respectively.

Fig. 1

Study design (a); classification of CNVs and status of XCI in healthy mothers (b); and origins of CNVs and distribution of clinical types of CNVs according to sex (c)

Table 1 List of patients with X-linked CNVs

The total frequency of P, LP, VUS and LB variants on the X chromosome in patients with NDDs was calculated as 2.8% (3.5% in males and 1.6% in females). Twenty-two patients had microduplications (82 kb to 1.6 Mb in size), five patients had microtriplications (192 kb to 888 kb in size), and six patients had deletions (81 kb to 10.2 Mb in size). The average size of microduplications and microtriplications was 802 kb, and the average size of microdeletions was 551 kb, excluding the largest deletion of 10.2 Mb in Patient #1 (Table 1).

CNV origin was determined for 18 patients: 13 patients had maternal CNVs (10 males and 3 females), 2 had paternal CNVs (both females), and 3 had de novo CNVs (2 males and 1 female) (Fig. 1b). An analysis of the variant origin was not possible for 15 patients (Fig. 1). Analysis of XCI status was performed in 13 mothers who were healthy carriers of CNVs on the X chromosome and four patients. sXCI was found in three mothers (23%), and random XCI was observed in 10 mothers and 2 patients (Table 1); the other 2 patients were homozygous for the first exon of the AR gene. Of the healthy mothers with sXCI, two had pathogenic CNVs (Patient #2 and Patient #3); for the third mother, since the XCI status of her daughter could not be determined because she was homozygous for the first exon of the AR gene, her CNVs were classified as VUSs (Patient #19).

Patients with X-linked CNVs exhibited multiple phenotypes as follows: disorders of intellectual development (6A00) in 27 patients (81%), developmental speech or language disorders (6A01) in 25 patients (75%), autism spectrum disorder (6A02) in 13 patients (39%), and developmental motor coordination disorder (6A04) in 11 patients (33%). In addition, 10 patients had craniofacial dysmorphism (8A03.1Y) (30%); 9 individuals had behavioural disorders (6C5Z) (9%); 7 patients had metabolic disorders (5D2Z) (21%); and 11 patients had congenital malformations (33%), including motor disorders, seizures, cerebral palsy and other nervous system disorders (Fig. 2). Below, we provide details of the patients with CNVs on the X chromosome from different groups.

Fig. 2

Phenotype distribution in patients with CNVs on the X chromosome

Pathogenic CNVs

CNVs were classified as pathogenic if they had a total score of ≥ 0.99 according to the CNV-ClinViewer calculator (Table 1). Pathogenic CNVs were either large deletions or known microduplication/microdeletion syndromes.

Patient #1. A 9-year-old girl (Table 1) had ID, speech delay, behavioural disorders, short stature, shortening of the tubular bones of the arms and legs, dysmorphic features, and partial atrophy of the optic nerves. The patient had a de novo Xp22.33-p22.2 deletion of 10.2 Mb. The patient had random XCI (73%). The deleted region contained genes that are associated with the development of Langer mesomelic dysplasia (OMIM 249700); Leri-Weill dyschondrosteosis (LWD) (OMIM 127300); short stature, idiopathic familial (OMIM 300582); chondrodysplasia punctata, X-linked recessive (OMIM 302950); intellectual developmental disorder, X-linked (OMIM 300495); nystagmus 6, congenital, X-linked (OMIM 300814); ocular albinism, type I, Nettleship-Falls type (OMIM 300500); hypogonadotropic hypogonadism 1 with or without anosmia (Kallmann syndrome 1) (OMIM 308700); and ichthyosis, X-linked (OMIM 308100).

LWD is a pseudoautosomal dominant disease characterized by short stature, mesomelic limb shortening, and characteristic Madelung’s deformity of the forearms (deformity of the radius and limited pronation/supination of the forearm). LWD is caused by deletions or point mutations of the SHOX gene, which are believed to disrupt regulatory elements. The LWD phenotype is highly variable, with short stature being the only distinguishable manifestation in some patients. Hypoplasia of the lower jaw has also been observed. Although LWD occurs in both sexes, it is usually more severe in females, possibly due to sex differences in oestrogen levels. In our patient, in addition to short stature, shortening of the tubular bones of the extremities was observed, which is consistent with the phenotype of LWD. The ID in this patient may be due to a deletion of neuroligin 4 (NLGN4, OMIM 300427.0002). NLGN4 belongs to the family of neuroligins, which are neuronal cell surface proteins located in synaptic structures. In vitro experiments showed that the neuroligin-neurexin intercellular adhesion complex can trigger the formation of functional presynaptic elements and lead to axonal specialization [17]. Variants in NLGN4 are implicated in a wide range of phenotypes, ranging from mild isolated ID without communication deficits to Asperger’s syndrome with normal or abnormal intelligence. X-linked recessive chondrodysplasia punctata is associated with variants and deletions of the ARSL gene (OMIM 300180), and X-linked ichthyosis is associated with variants in the STS gene (OMIM 300747). Both syndromes manifest in a recessive manner, but the ARSL and STS genes are haploinsufficient; therefore, the patient’s facial dysmorphic features and behavioural disorders may be the result of the deletion of these genes.

Patient #2. A 2.5-year-old boy who presented with X-linked intellectual disability, Nascimento type, caused by a 168 kb microdeletion at Xq24 involving 5 genes (CXorf56, UBE2A, NKRF, SEPT6, and MIR766) inherited from a healthy mother and grandmother with extremely skewed XCI (100%) has been described previously [18].

Patient #3. A 7-year-old boy had ID and speech and motor development delay. The patient is a carrier of a maternal Xq24q25 duplication of 4.8 Mb. The duplication included 51 genes, 11 of which (CXorf56, UBE2A, UPF3B, NDUFA1, RNF113A, LAMP2, CUL4B, C1GALT1C1, GLUD2, GRIA3, THOC2, and XIAP) were annotated in OMIM (Fig. 3). The CXorf56 and UBE2A genes are associated with X-linked ID. Mutations and deletions in the UBE2A gene result in intellectual developmental disorder, X-linked syndromic, Nascimento type (OMIM 300860) or UBE2A deficiency syndrome [19]. The CXorf56 gene is a candidate for XLID (OMIM 301013) and is expressed in the brain. This patient’s unaffected mother had an extremely skewed XCI (100%). The patient’s sister and grandmother were healthy carriers of the duplication, and both also had an extremely skewed XCI (100%). Therefore, we classified this duplication as P variant. An overlapping duplication was annotated in the DECIPHER database with the coordinates arr[GRCh38] Xq24-q25(118564854_123376081) (patient: 279051). The carrier of the duplication had a specific learning disability, but the clinical significance of the duplication was uncertain.

Fig. 3

Results of the CMA (a) and qPCR (b) analyses of Xq24q25 duplication and the XCI status (c) in the family of Patient #3. I—native DNA; II—DNA digested with the restriction endonuclease HpaII

Patients #4 and #5. Two male siblings (2 and 8 years old) had mental and speech development delays, aggressive behaviour, emotional instability, obesity, mild congenital hypothyroidism, enlarged testicles, and hepatomegaly. Both brothers had a 1.16 Mb deletion in the Xq26.1-q26.2 region (Fig. 4), which was not previously reported in the literature. The deletion included seven genes: ENOX2, ARHGAP36, IGSF1, OR13H1, FIRRE, LOC105373338, and LINC01201. The mother of the patients was not available for testing, but since the aberration was the same in both brothers, the deletion was most likely of maternal origin. Three of the deleted genes were annotated in OMIM: ENOX2 (OMIM 300282), ARHGAP36 (OMIM 300937), and IGSF1 (OMIM 300137). The ARHGAP36 gene is highly expressed in the spinal cord and brain during specific periods of mouse embryonic development [19]. A deletion of the IGSF1 gene has been identified in males with central hypothyroidism and testicular enlargement [21]. Of the six patients with overlapping deletions from DECIPHER (ranging in size from 60 to 979 kb; patients: 314664, 385774, 265814, 376920, and 412324), four had speech and language development delay, two had bone abnormalities, and one had autism spectrum disorder (ASD).

Fig. 4

Results of the CMA (a) and qPCR (b) analyses of the Xq26.1-q26.2 deletion in Patients #4 and #5

Patient #6. A 3-year-old boy had global developmental delay, brachycephaly, Dandy-Walker malformation, divergent strabismus, diffuse muscle hypotonia, palmar erythema, and cutis marmorata. He had an Xq28 microtriplication of 246 kb. The microtriplication overlapped with the region involved in chromosome Xq28 duplication syndrome (OMIM 300815). Duplications in the Xq28 region are the most common CNVs in males with XLID, as this region is enriched in segmental duplications predisposed to variants through a nonallelic homologous recombination mechanism [6, 22, 23]. The patient’s microtriplication included 22 genes, three of which (RPL10, EMD, and GDI1) are associated with ID. Variants in the RPL10 gene (OMIM 312173) cause intellectual developmental disorder, X-linked syndromic 35 (MRXS35, OMIM 300998). A variant in the EMD gene has been found to cause Emery-Dreifuss muscular dystrophy (EDMD; OMIM 310300). Copy number gains involving the GDI1 gene (OMIM 300104) are among the most common variants in patients with XLID and epilepsy [23]. Increased expression of GDI1 mRNA is positively correlated with the severity of ID and the probability of epilepsy development [24]. Gains of the gene may be associated with brain defects, such as Dandy-Walker malformation with cerebellar hypoplasia and agenesis of the corpus callosum. The patient’s main clinical features were consistent with Xq28 duplication syndrome. It was impossible to determine the origin of the microtriplication.

Likely pathogenic CNVs

In our cohort, CNVs in the genes responsible for the development of known microdeletion/microduplication syndromes were identified in three patients, but the size of these CNVs was smaller than those described in the literature; however, they affected a critical part of a gene or a causal gene. In all cases, the scores calculated using the CNV-ClinViewer calculator did not reach the cutoff for LP classification (between 0.90 and 0.98 points); however, we still found it possible to classify these CNVs as LP.

Patient #7. A boy, 2 years and 10 months old, had developmental delay, speech delay, difficulties in understanding addressed speech, difficulty in making eye contact, and stereotyped movements. A 191 kb de novo deletion of exon 1 of the MID1 gene (Xp22.22) was detected (Fig. 5). MID1 gene deletions are associated with X-linked Opitz GBBB syndrome (X-OS, OMIM 300000), which is characterized by midline defects, including hypertelorism, cleft lip and/or palate, laryngotracheoesophageal anomalies, cardiac malformations, and hypospadias. Patients with X-OS also have ID and anatomical abnormalities of the brain. Clinical features vary widely, even within the same family. The MID1 gene encodes an E3 ubiquitin ligase. The gene is approximately 400 kb; it has nine coding exons and is transcribed from several promoters in various combinations. The deletion in our patient affected the first exon of the gene, from which the two longest transcripts (MID-205 and MID-206) are synthesized [25, 26]. The phenotype of this patient was not specific to X-OS. The boy did not have dysmorphic features and demonstrated developmental delay and some signs of ASD. The loss of the first exon of this gene appears to not be critical for the development of a complete X-OS phenotype.

Fig. 5

Results of the CMA (a) and qPCR (b) analyses of the Xp22.2 deletion and XCI status (c) of the family of Patient #7. I—native DNA; II—DNA digested with the restriction endonuclease HpaII

Patient #8. A 7-year-old boy had ID, an absence of speech and self-care skills, hypertension syndrome, and movement disorders. Prolonged conjugative jaundice was observed. An Xp11.22 microduplication of 312 kb, which overlaps with the region associated with chromosome Xp11.23-p11.22 duplication syndrome (OMIM 300801), was detected (Table 1). This condition is characterized by borderline to severe ID, speech delay, and electroencephalography (EEG) abnormalities in both males and females. Early puberty was described in 77% of examined patients, and 50% were significantly overweight. Approximately 66% of patients had lower extremity abnormalities, pes planus and pes cavus, fifth toe hypoplasia, and syndactyly. The size of duplications usually varied between 0.8 and 9.2 Mb. In most affected females, the duplicated X chromosome was predominantly active. The phenotypes of all subjects with recurrent 4.5 Mb duplications and nonrecurrent larger duplications were remarkably similar despite the vastly different number of genes involved, including XLID-related genes, indicating a key role of the gene(s) in the overlapping duplicated region in defining the clinical features of the syndrome [27]. In our patient, the duplication included only two genes, SHROOM4 (OMIM 300579) and BMP15 (OMIM 300247). Variants in the BMP15 gene are responsible for ovarian dysgenesis (OMIM 300510) and premature ovarian failure (OMIM 300510). Variants of genes in the Xp11 region are responsible for 30% of all non-syndromic XLID cases. Patient #7 had a smaller microduplication (320 kb) than those previously reported in the literature. The patient’s microduplication overlapped with a critical region described in the literature for this region. The microduplication included the promoter region, exon 1, and intron 1 of the SHROOM4 gene, which is an XLID-related gene [2]. Duplications of this gene are common to almost all patients with Xp11.23-p11.22 duplication syndrome [27]. SHROOM4 is a member of the APX/Shroom protein family. Members of this family are localized in the cytoskeleton and play a role in neurulation, cell architecture, actin remodelling, and ion channel function [27,28,29]. Patient #8 had ID and a lack of speech typical of this syndrome. We did not determine the origin of the microduplication because the family was not available for additional analysis.

Patient #9. A 3-month-old boy had global psychomotor delay and the absence of electrical and mechanical atrial activity. He had a 90 kb Xq25 microduplication, which included exons 14–35 of the STAG2 gene (OMIM 300826). The STAG2 gene encodes one of the main proteins of the cohesin complex, a conserved functional unit involved in DNA replication, gene expression, heterochromatin formation, DNA repair, and sister chromatid adhesion. STAG2 is a dose-sensitive gene, and heterozygous loss-of-function duplication variants of this gene result in cohesinopathy [30]. Heterozygous or hemizygous mutations in the STAG2 gene can cause holoprosencephaly 13, X-linked (OMIM 301043) or Mullegama-Klein-Martinez syndrome (OMIM 301022). X-linked holoprosencephaly 13 is a neurological disorder characterized by midline developmental defects that mainly affect the brain and craniofacial structures. The severity and manifestations of this condition may vary; some patients may have complete alobar holoprosencephaly with cyclopia, while others may have semilobar holoprosencephaly or septooptic dysplasia. Dysmorphic features include microcephaly, hypotelorism, low-set ears, micrognathia, and cleft lip/palate. Patients with a more severe phenotype may die during the neonatal period, while patients with a milder phenotype experience general developmental delay. Mullegama-Klein-Martinez syndrome is a less severe disease with some overlapping features, such as developmental delay with ID, speech delay, hypotonia, microcephaly, and short stature. An increase in the number of copies of the STAG2 gene is associated with chromosome Xq25 duplication syndrome (OMIM 300979), a neurodevelopmental disorder characterized by developmental delay, mild to moderate ID, abnormal behaviour, and dysmorphic features [30, 31]. We did not find any CNVs similar to those detected in Patient #9 in the databases, but since the duplication in the patient was intragenic, it should have affected the mRNA sequence. Patient #9 died shortly after birth, but since he did not show clinical signs of the syndrome, we classified these CNVs as LP. We were unable to determine the origin of the CNVs because the patient’s mother was unavailable.

Variants of uncertain significance

VUSs contained ID-related genes and included those CNVs for which the score according to calculations using CNV-ClinViewer is between − 0.89 and 0.89 points (Table 1).

Patient #10. A 3-year-old boy had global developmental delay, CNS lesions, and neurological symptoms. He had an Xp22.33 microduplication of 192 kb. The duplication included three genes, none of which were associated with ID, but CNV-ClinViewer identified this variant as VUS (Table 1).

Patients #11–17. Overlapping microduplications of the Xp22.31 region were found in seven patients (Table 1). Three females and four males had microduplications ranging in size from 358 kb, containing the VCX, PNPLA4, MIR651, and VCX2 genes, to 1.5 Mb, including the PUDP, STS, and MIR4767 genes. The affected females had random XCI. The age of the patients ranged from seven months to 10 years. Phenotypic features varied widely: three patients had clinical features of ASD, two had speech development delay, and two patients had brain anomalies (perinatal CNS damage of mixed genesis and magnetic resonance [MR] signs of a lesion in the white matter of the left frontal lobe). In addition, six patients had single or multiple developmental anomalies: four had clubfoot (flat valgus foot), one had congenital kidney disease, and two had neurogenic torticollis. The pathogenic role of Xp22.31 duplications affecting the STS and VCX genes is still controversial [32]. In our patients, in addition to developmental delay and clinical features of ASD, disorders such as clubfoot and torticollis were observed, which is consistent with the literature (Table 2).

Table 2 Frequency of clinical features in carriers of Xp22.31 duplications

The frequency of this duplication was 0.15% in an unaffected control population and approximately 0.41% in a cohort of individuals with abnormal phenotypes, including ID (Fisher’s exact test p = 0.1295) [33, 34]. The size of the duplication ranged from 149 kb to 1.74 Mb. The male-to-female ratio was 0.7. However, a more recent study that assessed multiple measures of physical and mental health, cognitive function, and neuroanatomy in duplication carriers questioned the impact of this CNV on cognitive function and learning ability [35]. Most of the affected carriers of the duplication had random XCI. However, notably, the STS, HDHD1A, and PNPLA4 genes were shown to escape X inactivation, although with less than 100% efficiency, with STS activity varying from 1.3 to 1.7 in normal females [36]. Determination of the clinical significance of the Xp22.31 duplication is important since this duplication is frequently found in patients with ID (1/470 males and 1/240 females) [35].

The second duplication in the Xq26.3 region in Patient #17 was 312 kb (Table 1). The duplication included three genes, one of which is annotated in OMIM (FHL1 gene 300163). The product of this gene is the SLIM1 protein, which is expressed exclusively in human skeletal muscles. Mutations in the FHL1 gene lead to the development of X-linked myopathies and muscular dystrophy (OMIM 300280, 300696, 300695, 30018).

Patient #18. An 11-year-old boy had cerebral palsy (Gross Motor Function Classification System [GMFCS] 2, Manual Ability Classification System [MACS] 1); focal gliosis changes in the cerebral hemispheres; cognitive and behavioural disorders; central tetraparesis; parasomnia; subclinical hypothyroidism; bilateral ptosis; left-sided radioulnar synostosis; hypotrophy of the left forearm; shortening of the right lower limb; scoliosis; flexion-adduction functional contracture of the hip, knee and ankle joints; and equino-plano-valgus foot deformity. The boy had a 165 kb maternal Xp22.22 microduplication (Fig. 6). The patient’s mother exhibited random XCI (52%). The microduplication involved three genes: TRAPPC2, OFD1, and GPM6B. Variants in the TRAPPC2 gene are responsible for spondyloepiphyseal dysplasia tarda (OMIM 313400), an X-linked recessive disorder characterized by short stature and bone abnormalities. Loss-of-function variants (missense mutations, splice site mutations, and some deletions) in the OFD1 gene cause several X-linked recessive and dominant diseases, including orofaciodigital syndrome I (OFD1, OMIM 311200). OFD1 is usually fatal in male foetuses and predominantly affects women. OFD1 is characterized by malformations of the face and oral cavity, finger anomalies, polycystic kidney disease, brain anomalies, and ID. Duplications in the Xp22.2 region, including the TRAPPC2 and OFD1 genes, in chorionic villi in spontaneous abortions lead to an increase in gene expression at both the RNA and protein levels [37]. TRAPPC2 plays a role in procollagen transport, while OFD1 regulates the function of cilia [37]. Dysfunction of OFD1 due to overexpression can lead to abnormal ciliogenesis and ultimately to defects in the sonic hedgehog (Shh) and canonical Wnt signalling pathways responsible for regulating cell proliferation, cell migration, and apoptosis [38], which are associated with the differentiation of the neuronal lineage. Our patient had musculoskeletal system abnormalities, mainly of the lower extremities; neurological anomalies; glial changes in the cerebral hemispheres; and ID. These clinical features do not all fit into the classical picture of syndromes associated with mutations in the TRAPPC2 and OFD1 genes; however, the systems of the affected organs are the same.

Fig. 6

Results of the CMA (a) and qPCR (b) analyses of the Xq28 duplication and XCI status (c) of the family of Patient #18. I—native DNA; II—DNA digested with restriction endonuclease HpaII

Patient #19. A 10-year-old girl had ID, speech development delay, anxiety-phobia disorder, and myotonic syndrome. She had an Xp22.13-p22.12 microduplication. The microduplication included three genes, one of which, PDHA, was annotated in the OMIM database (OMIM 300502). The CNV was inherited from an unaffected mother who had skewed XCI (83%). We could not analyse X inactivation in the girl since she was homozygous for the AR gene. Variants in the PDHA gene (OMIM 300502) are associated with a deficiency in pyruvate dehydrogenase E1-alpha. The severity of deficiency in affected females depends largely on the XCI pattern in the brain. Another CNV was detected in the patient: a microdeletion at 9q34.3 (Table 1).

Fifteen genes are located in region 9q34.3, and mutations in 3 genes (NSMF, EHMT1, and CACNA1B) are pathogenetically significant according to the OMIM database. Heterozygous point mutations and deletions affecting the NSMF gene, which is highly expressed in tissues of the central and peripheral nervous system, as well as the kidneys, are associated with the development of hypogonadotropic hypogonadism with/without anosmia (hypogonadotropic hypogonadism 9 with or without anosmia; OMIM 614838). Heterozygous point mutations and deletions affecting the EHMT1 gene are associated with the development of subtelomeric deletion syndrome 9q (9q subtelomeric deletion syndrome or Kleefstra syndrome 1; OMIM 610253). Homozygous and compound heterozygous point mutations and deletions affecting the CACNA1B gene may be associated with impaired neuropsychiatric development accompanied by seizures and nonepileptic hyperkinetic disorders (neurodevelopmental disorder with seizures and nonepileptic hyperkinetic movements; OMIM 618497).

Patient #20. A boy, 6 years and 11 months old, had mental and speech development delays and signs of ASD (autoaggression, partial understanding of addressed speech and simple instructions, stereotyped movements, food selectivity, and partial self-care skills). At birth, diffuse muscle hypotonia was diagnosed. The patient had a 262 kb Xp21.2 microduplication. The microduplication included the Table 3 and FTHL17 genes and exon 79 of the DMD gene. Mutations in the DMD gene (OMIM 300377) cause Duchenne muscular dystrophy (DMD, OMIM 310200) or a milder form of Becker muscular dystrophy (BMD, OMIM 300376). DMD is a progressive neuromuscular disease characterized by severe cognitive and learning impairments, as well as neurobehavioural disorders, some of which are associated with the destruction of dystrophin isoforms. ASD is diagnosed in 15% of DMD patients, full-scale IQ < 70% in 27%, learning disability in 44%, ID in 19%, and attention-deficit/hyperactivity disorder in 32% [39]. BMD is similar to DMD in the distribution of muscle wasting and weakness; however, it is mostly proximal and more benign. Onset occurs at approximately age 12, although some patients do not experience symptoms until much later in life. Loss of ambulation also varies but starts in adolescence, with death usually occurring in the fourth or fifth decade of life. In some cases, ID also occurs [40]. In the DECIPHER database, we found a male patient with an overlapping microduplication and autism (patient 323448). Since the pathogenic role of this microduplication was not shown, we interpreted it as a VUS.

Patient #21. A 7-year-old boy had ASD, dysarthria, and obesity. The child did not jump well, could not ride a bicycle, and tired quickly when walking. Muscle tone was diffusely symmetrically reduced. His creatine kinase and lactate dehydrogenase levels were normal. aCGH revealed an 81 kb Xp21.1 microdeletion in intron 44 of the DMD gene (Fig. 7). Intron 44 is the longest intron in this gene. 25% of deletions in the DMD gene include all or part of intron 44 and usually include adjacent exons [41]. In our patient, the small deletion did not extend beyond the boundaries of the intron, and the biochemical parameters did not correspond to those observed in DMD patients. Therefore, the pathogenic role of this aberration is unclear.

Fig. 7

Results of the CMA (a) and qPCR (b) analyses of the Xp21.1 deletion in Patient #21

Patients #22 and #23. Two unrelated male patients, 8 and 9 years old, had Xq11.2 microduplications. The microduplications were 56.41 kb in size and affected exons 2–8 of the TSPAN7 gene. In Patient #23, the microduplication was inherited from an unaffected mother. In Patient #24, the origin of the CNV was unknown. The mother of Patient #23 had random XCI (54%). Both patients had a delay in mental and speech development and signs of ASD (lack of contact with others, behavioural disorders, aggressiveness, stereotyped movements, and ritual actions), as well as periodic signs of anxiety. An intragenic microduplication in the TSPAN7 gene (OMIM 300096) is considered a VUS. The TSPAN7 gene is highly expressed in the brain, and several TSPAN7 gene variants have been associated with XLID (XLID58; OMIM 300210). The association between TSPAN7 gene duplication and the pathogenesis of ID is still unclear. Previously, Noor et al. [42] showed that TSPAN7 intragene duplication in a patient with ASD did not change the cDNA sequence and, accordingly, could not be the cause of ASD. In addition, duplications of this gene have been found in some healthy individuals [43].

Patient #24. A 1.5-year-old boy had turricephaly, an arachnoid cyst in the pole of the right temporal lobe, anomalies in the development of the substance of the brain and sagittal sinus, a single convulsion, and hypothyroidism. He had an Xp11.3-p11.23 microduplication of 1.3 Mb. The microduplication was inherited from his unaffected mother who had sXCI (80%). The microduplication involved 22 genes; variants in four of these genes, ZNF674 (OMIM 300573), RBM10 (OMIM 300080), ZNF41 (OMIM 314995), and SYN1 (OMIM 313440), are associated with either brain anomalies or XLID. The SYN1 gene is of particular interest, as variants in this gene are associated with X-linked epilepsy-1 with various learning and behavioural disorders (EPILX1, OMIM 300491) and intellectual developmental disorders, such as X-linked 50 (OMIM 300115). This gene is also involved in Xp11.23-p11.22 duplication syndrome (OMIM 300801). In Patient #24, only the last exon of the gene was included in the duplication, and therefore, we cannot interpret this CNV as pathogenic. In the DECIPHER database, we found only one overlapping duplication of uncertain significance (patient 399231). The patient had hypoplastic male external genitalia, lissencephaly, and polymicrogyria.

Patient #25. A 4-year-old girl had speech delay and ASD. A 437 kb Xq21.32 microduplication of maternal origin involving the first exon of the NAP1L3 gene was detected. The proband and her mother had random XCI. The NAP1L3 gene (Gene ID: 4675) encodes a member of the nucleosome assembly protein (NAP) family. This gene is closely related to the genes responsible for several X-linked cognitive disability syndromes [12], but we did not find evidence of the pathogenic significance of this duplication.

Patient #26. A 7-year-old boy had mental and speech development delay, ASD and 2nd -degree obesity. A maternally inherited 82 kb Xq22.1 microduplication was detected. The mother had random XCI. The microduplication included three genes of the ARMCX family (ARMCX4, ARMCX1, and ARMCX6). These genes encode proteins in the mitochondria that control energy supply processes in neurons. Deletions of these genes are associated with ID [44], but there are no data on the pathogenic role of duplications. The DECIPHER database contains one overlapping microduplication of uncertain significance of a larger size (194 kb). The male carrier (Patient: 503427) of the microduplication had ID and speech and language development delays.

Patients #27 and #28. Two unrelated boys (4.5 and 3.5 years old) had speech and motor development delays and a lack of self-care skills. Patient #27 also had stereotypical movements, frequent mood swings, food selectivity, and atopic dermatitis. These patients had overlapping Xq25q26 microduplications of 1.6 Mb and 249 kb, respectively. The larger duplication region included nine genes, three of which are present in OMIM: OCRL, XPNPEP2, and ZDHHC9. Variants in the OCRL (OMIM 300535) and ZDHHC9 (OMIM 300646) genes are associated with XLID. Certain variants of the XPNPEP2 gene (OMIM 300145) predispose patients to angioedema induced by angiotensin-converting enzyme (ACE) inhibitors (OMIM 300909). In Patient #28, only two genes were included in the microduplication: SMARCA1 and OCRL. The OCRL gene was disrupted, because exons 1–14 were duplicated. In DECIPHER, among patients with overlapping duplications (Patients 458073, 301418, 385454, 410054, 434854, and 350034), features such as ID, atypical behaviour, obesity, global developmental delay, and seizures were described.

Likely benign

LB variants are CNVs with a score from − 0.90 to -0.98 points. The LB CNVs were those that were not found in DGV and did not contain genes associated with the disease.

Patients #29 and #30. Two siblings, 6 and 11 years old, had mental and speech development delays and maternally inherited Xp22.33 microduplications that were 888 kb in size. The duplication region included genes that were not associated with XLID.

Patient #31. A 2.5-year-old girl had Crouzon-type acrocephalodysostosis and developmental delay. She had a paternally inherited Xq13.3q21.1 microduplication of 1.28 Mb. The duplication included four genes (PBDC1, MAGEE1, MIR384, and FGF16), three of which were not associated with the disease; mutations in the FGF16 gene were associated with metacarpal 4–5 fusion (MF4; OMIM #309630).

In Patients #29–31, the regions of the variants were enriched in segmental duplications.

Patients #32 and #33. Five-year-old male monochorionic twins had global developmental delay and ASD. The boys had an Xq21.31 microduplication of 457 kb. The duplication included exon 1 and part of intron 1 of the KLHL4 gene. The family had an older son (8 years old) with the same disorders, but no duplication was detected. In addition, the father of the boys also suffered from ID. Paternal transmission in this family ruled out X-linked inheritance.

Discussion

The interpretation of the clinical significance of CNVs on the X chromosome remains highly relevant at present. Phenotypic manifestations of variants and structural aberrations on the X chromosome have a number of characteristics that are different from those caused by autosomal variants, primarily because the male X chromosome is hemizygous. In addition, to compensate for the dose of X-linked genes in females, only one X chromosome remains active. There are more duplications on the X chromosome than deletions, because the deleted region in a male becomes nullisomic, and the complete loss of a segment of a single X chromosome can be lethal. The size of duplications on the X chromosome is larger than the size of deletions due to the potential nonviability of hemizygous individuals with large deletions. In our study, the number of X-linked duplications and triplications was four times greater than the number of deletions (27 gains and 6 losses). The mean size of duplications and triplications was also larger than the mean size of deletions (802 kb and 551 kb, respectively), except for Patient #1, who had a large heterozygous deletion (10.2 Mb). This deletion partially included the pseudoautosomal region, and the female patient had random XCI.

When classifying CNVs on the X chromosome, we used as many criteria as possible: the type and gene content of the CNV and the patient’s phenotype, origin and XCI pattern when parental material was available for analysis, as well as a score using the CNV- ClinViewer calculator. An important criterion when classifying CNVs is the XCI pattern of heterozygous carriers. Current calculators do not take into account the X chromosome inactivation status of heterozygous CNV carriers. For a more correct classification of CNVs on the X chromosome, we propose to introduce a semi-quantitative assessment of the XCI status in heterozygous CNV carriers. In the case of random XCI, the score will be 0. If a CNV carrier has a skewed XCI (from 80 to 100%), the scores can range from 0.45 to 0.9. A skewed XCI equal to 80% is estimated at 0.45 points. A skewed XCI equal to 90% is estimated at 0.7 points and 95–100% (one of the X chromosomes is expressed exclusively) is estimated at 0.9 points. For example, in the case of the large duplication of Xq24q25 in a boy with ID and learning disabilities (Patient #3), all healthy heterozygous carriers of CNVs in this family had an extremely skewed XCI. According to the CNV calculator, this patient’s CNV score is 0.45. We added 0.90 points to this score and obtained 1.35 points. This score corresponds to pathogenic, and this CNV was classified as pathogenic (Table 1). Most X-linked diseases (from 30 to 80%) are inherited from a phenotypically healthy mother. The majority of CNVs on the X chromosome (from 30 to 80% in various pathologies) are inherited from a phenotypically healthy mother [6, 45, 46].The mothers of all of our patients were unaffected. In most women, X-linked diseases are not manifested, either because they are not homozygous for the pathogenic variant or because the cells that express the mutant allele derive from cells that express the gene product of the normal allele in sufficient quantities to perform the essential function. A skewed XCI is often associated with pathological phenotypic variants in humans and can occur for a variety of reasons, but one of the most likely causes is lethal mutations or microstructural aberrations of the X chromosome [5]. Plenge et al. [47] analysed the inactivation patterns of 20 different forms of XLID and reported that sXCI was present in ∼ 50% of the families. Unfortunately, we were not able to analyse the XCI pattern of all asymptomatic CNV carriers in our study. An sXCI (≥ 80%) was detected in three of 13 families (23%). The frequency of sXCI in mothers with P CNVs was 40%, and in mothers with VUSs, it was 16%. However, in one family with an Xq24 microdeletion (Patient #2), the aberration was associated with a known syndrome. Heterozygous carriers of this aberration are healthy but have extreme sXCI. We were unable to assess the XCI inactivation status of Patient #19 with a VUS. Her mother had sXCI (83%), but her affected daughter was homozygous for the CAG repeat in the AR gene. Therefore, we cannot associate the status of X inactivation with the clinical phenotype.

The frequency of CNVs on the X chromosome in our cohort of patients with NDDs was 2.8%. VUSs accounted for 57%, and P and LP variants accounted for 15% and 12%, respectively. The frequency of CNVs on the X chromosome varies widely across different studies (from 1.3 to 20%) depending on sample size and patient selection criteria (Table 3).

Table 3 Frequency of CNVs on the X chromosome in different patient cohorts

Submicroscopic aberrations of the X chromosome can be intragenic or can affect an entire gene or several genes. It is believed that deletions of several exons of a gene on the X chromosome in males will lead to the same lack of a functional product as the loss of the entire gene. However, in our study, small intragenic deletions did not lead to clinical symptoms of a particular syndrome in some patients. For example, Patients #6 and #21, with intragenic deletions of the MID1 (Fig. 1) and DMD genes (Fig. 7), respectively, manifested only some clinical signs of X-linked Opitz GBBB syndrome and Duchenne syndrome or Becker syndrome, respectively.

Intragenic duplications can lead to the formation of incorrect transcripts and elongated proteins. When the breakpoint of a duplication occurs within a gene, the outcome depends on whether the duplication is tandem or inverted or whether the duplication is inserted elsewhere in the genome. For example, duplications of exons 2–8 of the TSPAN7 gene are classified in the literature as VUSs since it was previously shown that intragenic duplication of TSPAN7 in a patient with autism did not lead to a change in the cDNA sequence [42]. However, there are data indicating that this duplication may be pathogenically significant. We analysed the clinical phenotypes of 29 patients in the DECIPHER database [13], two patients from our study, and two brothers with the same X-chromosome duplication previously described in the literature. ID, speech delay and ASD were observed in almost 100% of patients, and muscle hypotonia was observed in 15% of patients. Brain lesions (heterotopic grey matter, cerebellar atrophy, and encephalopathies), hearing loss, skin anomalies, joint hypermobility, and obesity have also been reported. In addition, 20% of hemizygous duplication carriers had dysmorphic features such as abnormal skull shapes (microcephaly, brachycephaly, and dolichocephaly), attached earlobes, hypertelorism, underdeveloped alia nasi, a short smoothed filter, a high palate, and widely spaced teeth.

The majority of P and LP variants and VUSs involved two or more genes. CNVs that overlapped with known microdeletion/microduplication syndromes but had a smaller size than described in the literature were classified as LP. In addition, when classifying CNVs, we considered the score calculated using the CNV-ClinViewer Internet resource. When CNVs did not overlap with known deletions/duplications but contained genes associated with development or normal brain function, we classified them as LP or of uncertain significance. CNVs were classified as P if the clinical picture of a patient was similar to the phenotype of individuals with previously established variants. In the DECIPHER database, Xq26.1-q26.2 deletions are classified as P, LP or VUS. In our cohort of patients, deletion of this region was found in two siblings (Patients #4 and #5) with intellectual and speech development delays, aggressive behaviour, emotional instability, obesity, mild congenital hypothyroidism, enlarged testicles, and hepatomegaly. This large deletion of 1.16 Mb contains 7 genes, three of which are listed in OMIM. The product of one of these genes, ARHGAP36 (OMIM 300937), is required for the development of the mouse spinal cord and brain [19, 20]. Therefore, we classified this variant as pathogenic. The duplication involving the OFD1 gene in Patient #18, who had severe neurological, cognitive, and behavioural disorders and multiple congenital developmental anomalies, is most likely pathogenically significant. However, this aberration was previously found only in the placenta of women with spontaneous abortions and was not described postnatally; therefore, we classified it as a VUS. Mutations and deletions of the OFD1 gene are associated with XLID [2].

In conclusion, the frequency of clinically significant CNVs, which include P and LP variants, was 1% in our cohort of patients with NDDs. The frequency of VUSs and LB variants was 1.8%, but it should be noted that novel CNVs may be a reservoir for the identification of new structural variants and genes associated with XLID.