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DNA copy number variations and craniofacial abnormalities in 1,457 children with neurodevelopmental disorders

Italian Journal of Pediatrics volume 51, Article number: 9 (2025) Cite this article

Abstract

Background

This study aimed to investigate deoxyribonucleic acid (DNA) copy number variations (CNVs) in children with neurodevelopmental disorders and their association with craniofacial abnormalities.

Methods

A total of 1,457 children who visited the Child Health Department of our hospital for unexplained Neurodevelopmental disorders (NDDs) between November 2019 and December 2022 were enrolled. Peripheral venous blood samples (2 mL) were collected from the children and their parents for whole-exome sequencing. Positive results were verified through Sanger sequencing for locus and pedigree validation. Simultaneously, a specific sign-scoring scale was created to evaluate characteristics related to the developments of eyes, nose, ears, eyebrows, head, mouth, face, trunk, limbs, and reproductive, urinary, and cardiovascular systems.

Results

A total of 536 children (36.78%, 536/1,457) were found to have genetic variations, with 379 (70.71%, 379/536) exhibiting pathogenic monogenic mutations. Furthermore, 157 children (29.29%, 157/536) harbored DNA copy number variants, encompassing microdeletions (68.15%, 107/157) and microduplications (31.85%, 50/157). Regarding the pathogenicity of CNVs, 91 (57.96%, 91/157) were identified as pathogenic, 28 (17.83%, 28/157) as variants of uncertain clinical significance (VOUS), and 38 (24.20%, 38/157) as benign according to the American College of Medical Genetics and Genomics (ACMG).Using a specific sign-scoring scale, the proportion of pathogenic CNVs in children graded 1 point or higher (64%, 58/91) was significantly higher than that of non-pathogenic CNVs (43%, 29/66) (P < 0.05). Furthermore, the proportion of microdeletions in children graded 1 point or higher (60.75%, 65/107) was significantly higher than those carrying microduplications (44%, 22/50) (P < 0.05). The proportion of pathogenic microdeletions in children graded 1 point or higher (73.43%,47/64) was significantly higher than those carrying pathogenic microduplications (40.74%, 11/27) (P < 0.05).

Conclusion

The positive rate of whole-exome sequencing for children with combined craniofacial abnormalities and NDDs exceeds the international average in our study cohort. Thus, whole-exome sequencing may be recommended for precise diagnosis of neurogenetic diseases in such cases.

Background

Neurodevelopmental disorders (NDDs) encompass a group of conditions that originate during the developmental stage. NDDs typically emerge in early life (often before school age), manifesting as developmental abnormalities that impair social, academic, or occupational functioning. NDDs include comprehensive developmental delay/intellectual disability (DD/ID) and autism spectrum disorders (ASD). Globally, DD/ID and ASD are closely associated with childhood disability. DD/ID, a group of neurodevelopmental disorders of high clinical and genetic heterogeneity, are characterized by ASD and attention deficit hyperactivity disorder. The prevalence of ID is around 1% worldwide, and that of severe ID is about 0.6% [1,2,3]. ASD has shown an escalating incidence since its initial recognition in 1943. The global prevalence of ASD is approximately 1%, according to the World Health Organization (WHO) data from 2012 [4].

The etiology of NDDs involves exogenous and genetic factors. Exogenous factors, like infections, toxins, trauma and malnutrition, can be well controlled [5]. Genetic factors pose an increasing weight on the pathogenesis of NDDs. For example, genetic factors are responsible for two-thirds of ID cases, including chromosomal abnormalities, single/multi-gene mutations, and congenital metabolic defects, which may result in developmental delay, mental disorders, distinctive facial features, endocrine abnormalities, and behavioral changes. Health education and rehabilitation can relieve clinical symptoms of ID patients, but pose a heavy financial cost [6,7,8,9].

DNA copy number variations (CNVs) are defined as alterations in deoxyribonucleic acid (DNA) stretches in contrast to a reference genome, with a size ranging from a kilobase to an entire chromosome (monosomy/trisomy). CNVs, which may appear in multiple, single or intergenic regions, are pathogenic or benign [10]. Chromosomal microdeletions and microduplications constitute a portion of CNVs, but either microdeletions or microduplications, though emerging in the same region, may lead to inconsistent phenotypes, bringing patients with clinical and genetic heterogeneities. New genetic technology has made possible to deepen into the etiology in NDDs, including molecular analysis (a-CGH) and NGS (panels, whole-exome sequencing, whole-genome sequencing).

Children with NDDs may display craniofacial and organ malformations alongside brain dysfunction. Craniofacial malformations are related to the abnormal growth of cranial neural crest cells (CNCCs) caused by genetic variants, such as choromosomal deletions or duplications [11]. Unique facial features are common in numerous syndromes, and seldom change with time. Some genetic diseases are often recognized by distinctive facial features; however, their rarity remains a challenge for clinicians, who have to identify an ever-growing number of different genetic disorders. 3D imaging offers preliminary facial screening, but its application is limited also due to the high costs. For NDDs children displaying craniofacial malformations, a new tool method may be useful for clinical diagnosis and analysis.

This study aimed to identify DNA CNVs though whole-exome sequencingin a group of children with DD/ID or ASD. We also designed a new diagnostic scale to assess craniofacial features and analyze children’s clinical phenotypes. We examined the correlation between DNA CNVs and clinical phenotypes. Therefore, an assessment of special facial characteristics using the sign-scoring scale favored the early screening and recognition of high-risk genetic diseases, as well as whole-exome or whole-genome sequencing for a pedigree study. A timely intervention by family members and professional institutions would greatly benefit children with NDDs, thus reducing family and social burdens and improving the quality of life of the affected subjects.

Methods

Participants

A total of 1,457 children diagnosed with unexplained intellectual disability (ID) were included. These children were treated at the Child Health Department of the Children’s Hospital of Nanjing Medical University (Nanjing, China) from November 2019 to December 2022.

Inclusion criteria: (1) Children under 4 years old were assessed using Gesell and Griffiths diagnostic scales. If two or more developmental areas (including adaptability, large movement, fine movement, language, and personal social/behavior) differed by more than two standard deviations, comprehensive developmental delay was considered. Children scoring below 50 points in five developmental areas were included. (2) Intelligence Quotient (IQ) was measured using Wechsler scales in children aged 4–14. Based on IQ value and social adaptability, ID was categorized as mild (IQ: 50–69), moderate (IQ: 35–49), severe (IQ: 20–34), or extremely severe (IQ: < 20). All enrolled children had moderate, severe, or extremely severe ID. (3) Autism spectrum disorders (ASD) were diagnosed according to the criteria outlined in the US Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).

Excluded were those with (1) known specific genetic diseases or genetic metabolic diseases indicated by clinical manifestations and laboratory examinations; (2) DD/ID or ASD caused by perinatal ischemia, hypoxic brain injury, kernicterus, central nervous system infection, poisoning, or a history of trauma [12].

Procedures

With the informed consent from children’s guardians, 2 ml of peripheral venous blood was collected from each of the child and his or her parents for whole-exome sequencing and CNV analysis. For those with positive results, mutation site and pedigree were confirmed through the Sanger sequencing.

Target Region Capture Sequencing: the DNA fragments within the target region were enriched and subsequently sequenced using a high-throughputsecond-generation sequencing platform. In this study, a total of 23 exonic regions in 23,000 genes were captured using MyGenostics’ GenCap kit through procedures of randomly fragmenting genomic DNA, ligating it to the Illumina (NovaSeq6000, USA) PE adaptor oligonucleotide mixture, and performing link-mediated polymerase chain reaction (ligation-mediated PCR) amplification and purification to create the DNA library. Quality testing was conducted, and PCR products were hybridized into a target region capture chip to enrich the sequences of interest. Subsequently, the enriched sequences were sequenced using the Illumina Nova 6000 sequencer, followed by preliminary raw data processing, including image recognition and sample differentiation.

Bioinformatic Analysis: contaminated and overlapped data were removed. Then, utilizing BWA software (http://bio-bwa.sourceforge.net/) [13, 14], the filtered sequences were aligned against the human genome reference sequence (hg19) from the NCBI database. GATK software (https://software.broadinstitute.org/gatk/) [15] was employed to analyze and identify single nucleotide variants (SNVs) and insertion-deletion mutations (INDELs). A further annotation of all single nucleotide polymorphisms (SNPs) and INDELs were annotated using ANNOVAR software (http://annovar.openbioinformatics.org/en/latest/) [16]. Mutation sites at a frequency lower than 0.05 were selected from the normal human databases, including the Thousand Genomes Project (http://www.1000genomes.org), Exome Variant Server (http://evs.gs.washington.edu/EVS) and EXAC (http://exac.broadinstitute.org/). Pathogenicity and conservative predictions were performed on SIFT (http://sift.jcvi.org/) [17,18,19], PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) [20,21,22], MutationTaster (http://www.mutationtaster.org/) [23], and GERP + + (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html) [24]. Splicing sites were analyzed with SPIDEX (http://www.deepgenomics.com/spidex) software.

PCR Amplification and Sanger Sequencing: variant sites were further verified via PCR amplification and Sanger sequencing. The PCR primer pairs were designed using the online Primer 3.0 software [25,26,27]. Subsequently, the PCR products were subjected to Sanger sequencing and analyzed on an ABI 3130 Genetic Analyzer (Applied Biosystems Genetic Analyzer, 3130, USA). Family members were assessed for co-segregation.

We have designed a rating scale for craniofacial anomalies in children with neurodevelopmental disorders in the Procedures section (Table 1). Based on the assessment of craniofacial anomalies specifically in the head, skin/hair, face, eyebrows, eyes, ears, nose, oral cavity and other parts, any anomaly (single or multiple anomalies) in one craniofacial region was graded as 1 point, otherwise it was graded as 0. For example, a 4-year-old boy with positive characteristics of wide eye fissure, bilateral bulge, too long eyelash, low nasal bridge and asymmetric nostrils was graded as 2 points, including 1 point for three anomalies in the eyes and 1 for two anomalies in the nose.

Table 1 A self-designed rating scale for craniofacial anomalies in children with neurodevelopmental disorders
Full size table

Statistical analysis

Data were recorded using EpiData 3.0, and SPSS 22.0 software was used for statistical analysis. Numeration data were expressed in percentage or frequency, and compared using the Chi-square test. P < 0.05 indicated statistical significance.

Results

General situation analysis

Out of the 1,457 children, 536 (36.78%) were identified to carry genetic variations. Among them, 379 (70.71%) exhibited pathogenic single-gene mutations. Additionally, 157 children (29.29%) presented DNA CNVs, including 107 (68.15%) with microdeletion and 50 (31.85%) with microduplication. Among the 157 children with CNVs, 85 were male and 72 were female. Their ages ranged from 1 month to 13 years, with a mean age of 3.98 ± 2.68 years (Table 2).

Table 2 Basic characteristics of NDD patients
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Pathogenicity classification of CNVs

Of the 157 CNVs, 91 (57.96%, 91/157) were pathogenic, 28 (17.83%, 28/157) benign, and 38 (24.20%, 38/157) VOUS [28, 29] (Table S1).

Among the 91 pathogenic CNVs, 20 (21.98%, 20/91) were located at chromosome 7, 13 (14.29%, 13/91) at chromosome X, 11 (12.09%, 11/91) at chromosome 15. Williams syndrome, observed in 16 cases (17.58%, 16/91), was the most common (Fig. 1).

Fig. 1
figure 1

Distribution of pathogenic CNVs on chromosomes (1. red means male and green means female; 2. Pathogenic/ Likely pathogenic/ Uncertain significance from outside to inside; 3. A direction toward the center of the circle indicates deletion, and outward indicates duplication.)

Full size image

Analysis of clinical special signs of DNA CNVs

CNVs were divided into six groups for analysis of clinical special signs: microdeletion group, microduplication group, pathogenic group, non-pathogenic group (benign CNVs and VOUS), pathogenic microdeletion group and pathogenic microduplication group.

In the microdeletion group, 65 cases (60.75%) were scored by ≥ 1 point, significantly more than 22 cases (44%) in the microduplication group (P < 0.05). In the pathogenic CNVs group, 58 cases (64%) were scored by ≥ 1 point, significantly more than the 29 cases (43%) in the non-pathogenic CNVs group (P < 0.05). In the pathogenic microdeletion group, 47 cases (73.43%) were scored by ≥ 1 point, significantly more than the 11 cases (40.74%) in the pathogenic microduplication group (Table 3). The probabilities of eye deformity in 32 cases (35%) and oral deformity in 38 cases (42%) were higher in the pathogenic case group, and oral deformity was more likely to occur in either the microdeletion group and the microduplication group or the pathogenic microdeletion group and the pathogenic microduplication group (P < 0.05) (Table 4).

Table 3 Special sign scale scores in six groups of different DNA CNVs
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Table 4 Comparison of cranial and facial signs among six groups of CNVs
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Discussion

NDDs, presenting different manifestations, often bring a high rate of disability, but a lack of effective treatment options. Genetic factors play a pivotal role in their etiology, spanning chromosomal abnormalities, single or multigene mutations, and congenital metabolic defects. Notably, children with the same NDD exhibit similar craniofacial and other organ malformations, implying that common genetic factors may underpin craniofacial malformations.

Craniofacial development occurs during the gastrula stage, and needs the orchestration of diverse signaling pathways and germ layers to ensure a normal morphogenesis. Cranial neural crest cells (CNCCs) are key contributors to craniofacial development. Although craniofacial traits exhibit a strong heritability, however, a high heterogeneity exists in different population and individuals [30]. Genome-wide association analysis have identified over 300 loci linked to facial morphogenesis, predominantly within the enhancer regions of neural crest cells or fetal maxillofacial tissues [31].

Genetic mutations are the culprit of congenital craniofacial malformations [32], such as head shape anomalies, facial irregularities (e.g., low nasal bridge, asymmetrical eyes, sparse eyelashes, ear dysplasia), hair aberrations, and other features. [33,34,35]. Numerous studies have associated specific genes with various phenotypic facial features, such as teeth, ears, and hair [36, 37]. Growing evidence within the past decade has supported the enhanced accuracy of next-generation sequencing (NGS) in diagnosing NDDs [38]. NGS is the preferred examination in children with NDDs, although a systematic guideline for its clinical application is scant [39,40,41,42,43,44,45,46]. Whole-exome sequencing offers a higher diagnostic rate of NDDs than chromosomal microarray analysis (CMA) and array comparative genomic hybridization (aCGH) [45, 46], and is recommended as a first-tier diagnostic test. In our center, we also performed whole-exome sequencing to identify children with NDDs. Besides, according to these associations and our clinical experience, we established a special sign scale to evaluate these deformities.

In the present study, we carried out genetic testing for NDD children exhibiting a special sign score of ≥ 1. Among the 1,457 children, 536 cases were diagnosed with genetic variations, achieving a diagnostic rate of 36.78%. The rate of single pathogenic CNV was 25.88%, and that of DNA copy number variation was 10.78%, significantly higher than earlier reported 15%−20% [47,48,49]. The scale improved the positive diagnostic rate in outpatients, demonstrating its discriminative value.

CNVs constitute approximately 13% of the genome [50], and a considerable portion of them are benign [51]. However, pathogenic CNVs may lead to psychiatric and neurodevelopmental disorders, somatic diseases, neurological conditions, and cancers [52]. However, there lack specific tools in diagnosing pathogenic CNVs. The present study revealed that the likelihood of pathogenic CNVs was notably higher in children with a score of ≥ 1 by the special sign scale, suggesting that the scale holds huge potential to predict the pathogenicity of CNVs.

DNA alterations, including CNVs, result from the natural evolution and adaptation of organisms [52]. In humans, deletions and duplications are more balanced than in other mammals [53]. Some repeats play a crucial role in driving human evolution, leading to brain enlargement and the emergence of novel human-specific genes [54]. In theory, deletion and duplication should happen simultaneously with the same frequencies, but research has shown that CNVs are more likely to occur during early gamete formation. Clinical observations have also highlighted that microduplications are less deleterious and may lack clinical phenotypes. Moreover, at the chromosome level, the tolerance of an individual to triploidy is higher than that to haploidy, implying that triploids have a higher survival rate than haploids [55,56,57]. Our study further revealed that children in the deletion group displayed more craniofacial abnormalities, particularly oral malformations, in the special sign scale. These findings underscore that gene deletions tend to be more detrimental than duplications [58, 59]. Interestingly, our research identified 7q11.23 deletions as the most frequent cause of Williams syndrome, a finding that contrasts with previous studies on microdeletions and microduplications. As such, additional clinical evidence is required to ascertain the effect of deletions and duplications on human development. We identified that certain genes, such as GTF2I, LAT 2, LIMK 1, ARSA, and SHANK3, were associated with higher scores on the assessment scale. However, the extent to which these genes correlate with CNCC development warrants further investigation.

In the present study, 157 children harbored DNA CNVs, including 107 (68%) with microdeletions and 50 (32%) with microduplications. Among these, 91 cases (58%) were unequivocally pathogenic. Notably, most of the pathogenic CNVs were located on chromosome 7 (20%), followed by X chromosome (14%) and chromosome 15 (12%). These findings deviate from prior studies that reported chromosomes 18 and 22 as the most common sources of pathogenic CNVs [60]. These differences may be attributed to sample size and age. Thus, further exploration is warranted through studies involving a larger sample of children with neurodevelopmental disorders.

Overall, we created a specific sign-scoring scale to evaluate craniofacial abnormalities associated with NDDs and provided more references for illustrating the phenotype-genotype relationship of NDDs. In addition,craniofacial abnormalities indicated by the sign-scoring scale, especially anomalies in the eyes and oral cavity, significantly improve the positive rate of whole-exome sequencing. The use of the sign-scoring scale is conductive to the increased etiological diagnosis rate of genetic diseases and rare diseases, allowing for a more effective allocation of medical resources.We think broadly about the future use of the sign-scoring scale to neonatal screening, especially in parents with abnormal family and gestational history. The sign-scoring scale was able to provide an alarm for high-risk populations to rapidly receive genetic counseling, whole-exome sequencing and interventions.

Limitations

Several limitations warrant consideration in this study. Firstly, the sample was mainly pooled from Nanjing and surrounding areas. Secondly, all subjects are Han Chinese, which may bring with ethnicity-related bias. Consequently, the study results may not be representative for the overall Chinese population.

Conclusions

Craniofacial abnormalities represent significant clinical phenotypes associated with DNA copy number variations in children. The special sign scale, primarily focusing on cranial and facial anomalies, can be used to guide primary screening and genetic testing for children with NDDs.

Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

Abbreviations

DNA:

Deoxyribonucleic acid

CNVs:

Copy number variations

NDDs:

Neurodevelopmental disorders

DD/ID:

Developmental delay/intellectual disability

ASD:

Autism spectrum disorders

WHO:

World Health Organization

CNCCs:

Cranial neural crest cells

IQ:

Intelligence Quotient

PCR:

Polymerase chain reaction

SNVs:

Single nucleotide variants

INDELs:

Insertion-deletion mutations

SNPs:

Single nucleotide polymorphisms

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Acknowledgements

We are very grateful for the support we received from the psychology technicians in the Children’s Health Care Department of the Children’s Hospital of Nanjing Medical University. We also thank the children and parents for their participation.

Funding

This research is supported by the Pediatric medical research special fund project of Jiangsu Medical Association (SYH-32034–0070) and Jiangsu Medical Association Scientific Research Special Fund (SYH-32034–0097).

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Author notes

  1. Dandan Wu and Ran Chen contributed equally to this work.

Authors and Affiliations

  1. Child Mental Health Deparment, Children’s Hospital of Nanjing Medical University, NanjingJiangsu, 210008, China

    Dandan Wu

  2. Child Healthcare Department, Children’s Hospital of Nanjing Medical University, Jiangdong South No.8 Road, Nanjing, Jiangsu, 210008, China

    Ran Chen, Wu Yan, Mengyin Chen, Dongqing Xia, Xiaonan Li, Yanyan Dai, Yinhua Chen & Rong Li

  3. Basis International School Nanjing, Nanjing, Jiangsu, 210008, China

    Jerry Zhang

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Contributions

DDW and RC participated in the study design, data analysis, and data interpretation and prepared the manuscript. JZ, WY, MYC, DQX, XNL, YYD participated in data collection. YHC, RL were responsible for the study design and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yinhua Chen or Rong Li.

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Ethics approval and consent to participate

We confirmed that all the written informed consents were obtained from the participants or if participants are under 16, from their legal guardians. The study protocol was approved by the Medical Ethics Committee of the Children’s Hospital of Nanjing Medical University (NMUB202110083-1), and all methods were performed in accordance with the ethical standards as laid down in the Declaration of Helsinki.

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The authors declare no conflicts of interest.

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Wu, D., Chen, R., Zhang, J. et al. DNA copy number variations and craniofacial abnormalities in 1,457 children with neurodevelopmental disorders. Ital J Pediatr 51, 9 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13052-025-01839-6

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Italian Journal of Pediatrics

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