Microdeletion 3q13.33-3q21.2: A Rare Cause of Neurodevelopmental Disorder

Abstract

Chromosomal sub-microscopic imbalances, such as microdeletions and microduplications, are associated with multiple genetic disorders. Here, we illustrate microdeletion 3q13.33q21.2 might be responsible for neurodevelopmental disorder in two patients.

There are two patients with neurodevelopmental disorder in a family of seven. We used chromosomal microarray analysis to identify the microdeletion 3q13.33q21.2. Next-generation sequencing was utilized to exclude the presence of allelic mutations within the microdeletion region 3q13.33q21.2, which may have a potential role in the development of disease in patients affected with secondary genetic alterations.

Patient 4 was diagnosed with dilated left third ventricle, neurodevelopmental disorder, and mild abnormalities in electroencephalogram through a series of clinical examinations. Patient 6 was diagnosed with attention deficit hyperactivity disorder, short stature, intellectual disability, and concurrent epilepsy. By investigating the 3q13.33q21.2 band of the University of California, Santa Cruz database, we screened out the genes related to developmental delay and intellectual disability, including ADCY5 SEMA5B andKPNA1, which were highly suspected to be related to intelligence. This region also involves CASR, a gene that has been reported to be associated with epilepsy.

The ADCY5 and SEMA5B genes may be key genes to cause neurodevelopmental disorder. Abnormal expression of the CASR gene may lead to the occurrence of epilepsy.

Introduction

Chromosomal sub-microscopic imbalances at the microscopic level, such as microdeletions and microduplications, are linked to a variety of genetic disorders, including intellectual disability (ID), developmental delay (DD), autistic spectrum disorders (ASD), and congenital abnormalities. ¹ ID and multiple congenital anomalies impact 1 to 3% of the population ² and encompass a significant and diverse range of disorders. The molecular mechanisms underlying these conditions remain unresolved in a substantial number of cases due to the significant heterogeneity, presenting diagnostic challenges. More recently, they were identified with next-generation sequencing (NGS), which gave a diagnostic yield of approximately 28.8%. ³ NGS and targeted re-sequencing would play a very important role in the diagnosis of neurodevelopmental disorders (NDDs) and epilepsy. ^{4 5} The genetics of epilepsy is complex and several genetic tests are available. 3q deletion syndrome is a genomic disorder characterized by significant variability in phenotypes correlated with the size of the deletion. The clinical manifestations of 3q deletion syndrome include NDD, DD, congenital heart diseases, renal and gastrointestinal malformations, attention deficit hyperactivity disorder (ADHD), and epilepsy. ^{6 7} The researchers conducted a review of 27 patients with deletions spanning different sizes within the 3q12.3–3q21.3 region. They determined that patients with deletions encompassing the critical region 3q13.31 exhibited common characteristics, including DD, abnormal male genitalia, postnatal overgrowth, and dysmorphic features. Recently, Libotte et al ⁸ reported a case of a fetus with a newly identified interstitial deletion of 12.87 Mb in the chromosome region 3q13q21.2, along with a paternally inherited microdeletion of 1.2 Mb in the chromosome region 12p13.3. The fetus presented with corpus callosum dysplasia and a mild ventriculomegaly as observed in the fetal ultrasound scan.

In this study, we present a comprehensive clinical and molecular analysis of a family with a 2.74 Mb deletion on chromosome 3q13.33-3q21.2. The affected individuals consistently exhibit clinical symptoms, including NDD, speech delay, and ADHD. Additionally, we conduct a literature review on the candidate genes that have been suggested to be associated with these phenotypes.

Materials and Methods

Clinical Data Collection and Analysis

Patient no. 6, who experienced their first seizure around the age of 8 years while attending school, was admitted to the hospital. Subsequently, other six patients were referred to specialized brain hospitals for a comprehensive battery of tests. Following the referral for clinical genetic evaluation, six members of the family underwent assessment to ascertain their participation in the study except one child (out of the six) declined to undergo whole-exome sequencing (WES) due to needle phobia. This decision was duly respected and meticulously documented as part of the clinical data collection process. Inclusion of family members facilitated a more comprehensive understanding of potential genetic factors. . The clinical investigations and genetic analyses adhered to the guidelines outlined in the Declaration of Helsinki. Moreover, ethical approval was obtained from the ethics committee at the Sixth Affiliated Hospital of Sun Yat-Sen University. Clinicians furnished the identified clinical information for the six patients, ensuring its accuracy and reliability. Informed consent was obtained from the patients or their legal guardians for the utilization and publication of pertinent clinical information.

Chromosomal Microarray Analysis and Classification

Genomic DNA was isolated from the peripheral blood samples using the Maxwell RSC Blood DNA kit (Magen, China) according to the manufacturer's instructions. Comparative genomic hybridization assays were conducted with the Illumina Infinium OmniZhongHua-8 v1.4 BeadChip (Illumina, San Diego, California, United States). Using the Illumina Infinium OmniZhongHua-8 v1 BeadChip, 407,040 ± 2,635 single nucleotide polymorphisms (SNPs) were obtained, and the concordance rate was 99.94% ± 0.02% (false discovery rate = 0.06% ± 0.02%). The manufacturer's guidelines were followed for whole-genome SNP array tests. Specifically, the Illumina chip contained nearly 850,000 empirically selected SNPs spanning the whole genome. The average interprobe distance was approximately 1.8 kb, and the overall effective resolution was approximately 18 kb. The processed chip was scanned on the NextSeq550 system (Illumina). The data were analyzed using GenomeStudio 2.0 software (Illumina).

Each copy number variation (CNV) was classified according to the American College of Medical Genetics (ACMG)/Clingen recommendations. International databases such as Database of Chromosome Imbalance and Phenotype in Human using Ensembl Resources (DECIPHER), PubMed, Online Mendelian Inheritance in Man (OMIM), and Genome Aggregation Database (gnomAD) were also consulted for evaluating genotype–phenotype association.

Whole-Exome Sequencing

DNA was extracted from the patient and patient's family members using the Maxwell RSC Blood DNA kit (Magen, China). The gDNA was sent to the Yunkang gene company for library construction. Exome sequencing was performed using the IDT xGen Exome Research Panel v2 (IDT, United States). DNA samples were prepared according to the platform recommendations. Genomic DNA was captured with a biotinylated oligonucleotide probe library (IDT, United States), followed by 150 bp paired-end massively parallel sequencing on an Illumina Novaseq 6000 (Illumina, United States). We used bamdst v.1.0.9 to assess coverage with default parameters. All variants identified in the affected individuals were annotated with databases such as refGene, Avsnp150, gnomAD211, Clinvar, dbnsfp41a, Intervar by snpeff 5.0d, and annovar 2020 Jun. Candidate mutational events were then inspected with the integrative genomics viewer. The resulting variants were excluded when the frequency was over 1/1,00 in the gnomAD. The candidate variants were then assessed under the protocol issued by ACMG.

Results

Clinical Testing Result

A family from Guangdong, South China, consisting of seven members spanning across three generations has been included in the present study (see Fig. 1 ). Table 1 presents a comprehensive comparison of the clinical characteristics between patient 4 and patient 6. Patient 4 was born through an uncomplicated pregnancy and delivered without any complications. Patient 4, with a height of 145 cm, encountered academic difficulties, particularly in mathematics, during her primary school years. Significant delays in language development, attention deficits, immature social-emotional skills, and social barriers were observed. As a result, the challenging decision was made to discontinue education at the junior high school level, leading to subsequent difficulties in finding suitable employment. Currently, she is capable of self-care and can perform simple household tasks. The administration of the Raven Standard Progressive Matrices test revealed a low IQ score of 66, indicating a complete absence of abstract reasoning ability. Neurological evaluation showed mildly abnormal findings on the electroencephalogram (EEG), characterized by suppressed α waves observed both with eyes open and closed. The cranial computed tomography spiral scan revealed bilateral enlargement of the lateral ventricles and third ventricle in Patient 4, shown in Supplementary Fig. S1 in the Supplementary Material.

Fig. 1.

“ ” means male; “ ” means female; “ ” means patient, patient 4 is the mother, and patient 6 is the eldest son.

Table 1. Clinical features of patients with 3q microdeletion.

	Patient 4	Patient 6	Molin et al (case 4)	Molin et al (case 6)	Molin et al (case 15)
Decipher ID	−	−	251017	252522		396427	396167	395642	501032	500983
Sex	F	M	M	F	M	F	M	M	M	M
CNV(hg19)	arr 3q13.33q21.2(121705842-124422005) × 1, arr 4p16.1(8311643-10128355) × 3	arr 3q13.33q21.2(121695622-124435710) × 1, arr 4p16.1(8311643-10238615) × 3	arr 3q13.11q21.3(104580989-126976442) × 1	arr 3q13.13q13.33(108633408-121150880) × 1	arr 3q13.32q21.2(117778747-125103009) × 1	chr3:121217310-142617302	chr3:121217310-142617302	chr3:106017310-129017302	chr3:123896874-124020828	chr3:123896874-124020828
Inheritance	De novo	Inherited from mother	De novo	De novo	De novo	De novo	De novo	De novo	Unknown
Age	35 y	8 y	1 y 6 mo	5 y	8 y	5 y	Less than 1 y	Less than 1 y
Weight	45 kg	18.7 kg	85th–97th	19 kg	40 kg
Height	145 cm	112 cm	85th–97th	112.5 cm at 4 y 10 mo	133 cm					Short stature
Developmental delay	+	+	+	+	Delayed speech	Intellectual disability		Intellectual disability	Global developmental delay	Global developmental delay
Epilepsy	−	+
Neurocognitive	IQ 66 (age = 35), social and emotional immaturity	ADHD, WISC-IV 47 (age = 7)	Autism, repetitive behavior, anxious, IQ?
Skull abnormalities	Ventriculomegaly	−	Brachycephaly, relative microcephaly	Ventriculomegaly		Primary microcephaly		Delayed closure of the anterior fontanelle, hypoplasia of the corpus callosum, dolichocephaly		High forehead
Craniofacial						Short palpebral fissure	Short palpebral fissure
Bital hypertelorism	+	+	+	+	+			+
Epicanthal folds	+	+				+	+	+
Broad/flat nasal	−	+	−				Convex nasal ridge	+		Abnormality of the nose
Ears	−	Large, low set	Small, low set		−	Crumpled ear		Low-set ears, abnormal pinna morphology		Protruding ear
Other malformations				Hypertelorism, antimongoloid slant, myopia	Myopia, small hands with bilateral single palmar crease	Cleft palate	Ptosis, blepharophimosis, congenital diaphragmatic hernia, premature birth	Wide intermamillary distance, cryptorchidism, micropenis, urethral atresia, urethral valve, nystagmus, exaggerated cupid's bow, long philtrum, narrow forehead

Abbreviations: ADHD, attention deficit hyperactivity disorder; IQ, intelligence quotient; WISC-IV, Wechsler Intelligence Scale for Children, 4th edition.

Note: Spaces left blank indicate there were no data available for that feature; “ + ” for clinical manifestations; “ − ” for no clinical manifestations. Case 4,case 6, and case 15 are the cases reported in Molin et al literature. The WHO Child Growth Standards were used to describe the standard height and weight.

Patient 6 was delivered through a cesarean section, with a birth weight of 3.24 kilograms. He initiated independent walking at 18 months and exhibited language development from the age of 3. At the age of 8, his weight reached 20 kilograms, with a height of 112 centimeters. Reports from his family suggested difficulties in expressing coherent sentences. The first epileptic seizure episode occurred during school hours, lasting 5 minutes and accompanied by salivation, convulsions, and loss of consciousness. Subsequent magnetic resonance imaging scans did reveal signs of mild cerebral atrophy. The EEG analysis demonstrated abnormal patterns in EEG III (wake/sleep) with intermittent episodes of epileptiform discharges localized in the cerebral region and the posterior left head area. Consequently, he received a clinical diagnosis of growth retardation, NDD, and short stature at a pediatric hospital. His Webster's intelligence score indicated a value equal to or below 47. The pediatric psychiatrist identified mild impairments in vestibular balance function, bilateral differentiation function of the brain, as well as mild sensory integration dysfunction and motor developmental disorders. Moreover, pronounced deficits were observed in visual-spatial and morphological sensory functions, alongside significant impairment in proprioception and body coordination. Learning capabilities and emotional state were deemed within normal range, along with stress management skills and self-image recognition. A subsequent survey conducted at the age of 9 revealed brief communication abilities and adeptness in following instructions.

Chromosomal Microarray Analysis

All seven affected subjects were assessed by neurologist and a clinical geneticist. Chromosomal microarray analysis (CMA) and NGS were performed on all family members, with the exception of patient 6 who did not undergo NGS testing. Patient 4 was detected to carry microdeletion 3q13.33q21.2 and patient 6 inherited the 3q13.33q21.2 microdeletion. The 3q13.33q21.2 microdeletion is shown in Fig. 2 . The CMA assay results indicated that both patient 4 and patient 6 tested positive for deletions at arr 3q13.33q21.2. Specifically, patient 4 showed a result at arr 3q13.33q21.2(121705842-124422005)×1, while patient 6 exhibited a result at arr 3q13.33q21.2(121695622-124435710)×1, which The size and position of the deletion was almost identical for, suggesting maternal inheritance of the deletion in patient 6. The 3q13.33q21.2 microdeletion encompasses a total of 23 RefSeq protein-coding genes, out of which five are classified as Morbid genes. No similar reports of polymorphisms have been identified in population databases such as Database of Genomic Variants (DGV) and gnomAD. Moreover, it is noteworthy that this specific deletion region does not exhibit any overlap with known pathogenic CNV regions, as documented in databases like ClinGen and Decipher. In the Decipher database, there are four cases of patients classified as potentially pathogenic whose deletions overlap with the ones reported here. These patients are identified as Patient: 396427, 396167, 395642, and 501032.

Fig. 2.

( A ) It is shown that there is a deletion in the long arm of chromosome 3 from 13.33-21.2. The red part is the deleted region. The B allele has only two lines, and a LogR value of 1 means that the copy number is equal to 1. ( B ) it is shown that there is duplication in the short arm of chromosome 4 from p16.1. The blue region has a copy number of 3, and some positions in the middle are not covered by probes. The B allele has four lines, and a LogR value of 3 means that the copy number is equal to 3.

At the same time, a microduplication in the 4p16.1 region, approximately 1.8 mb, was detected in patient 2, patient 4, patient 3, patient 6, and patient 7. The 4p16.1 microduplication is shown in Fig. 2 . The genetic microarray analysis unveiled a 1.81 Mb duplication in the p16.1 region of chromosome 4 in the individual under investigation, impacting multiple OMIM pathogenic genes. This particular region does not encompass any genes with a triplosensitivity score of 3. Only two comparable duplications have been documented in ClinGen/ClinVar, with one categorized as a potentially benign variant and the other classified as a variant of uncertain clinical significance. Furthermore, there is currently a lack of literature reports pertaining to this CNV. However, it is noteworthy that this duplication does not segregate as a causative variant for disease within the reported pedigree. Healthy individuals within the family likewise carry this segment. Thus, in accordance with the ACMG/ClinGen recommendations, ⁹ the clinical significance of this segment remains uncertain.

Whole-Exome Sequencing

Clinically nonpathogenic, probable pathogenic, or unclear variants were identified that could explain the subject's phenotype. We focused on screening-related genes of NDD, schizophrenia, abnormal behavior, convulsions, epilepsy/convulsions, etc. that doctors are more concerned about, and found no potentially clinically significant variants matching the phenotype of the subjects (including SNV and Indel).

In the WES, according to the WES CNV prediction analysis software, the subject is prompted to Seq arr 4p16.1(8311643-10128355) × 3 position repeat copy. The copy number is 3 (consistent with the CMA results; due to the limitations of NGS, WES CNV prediction analysis software cannot accurately determine the specific position of CMA).

The sensitivity of these three genes to trisomy dose is unknown, the population database DGV is included (phenotype unknown), and there are only two similar duplications in ClinGen/ClinVar; one as a probable benign variant, and another one is of an unknown clinical significance variation. This CNV has not been reported in the literature. It was observed through the joint analysis of the second-generation data of the family that the CNV of patient 3 was inherited from patient 2. According to the clinical phenotype of patient 3 we collected, we believe that the clinical phenotype of this CNV-related disease is not highly consistent with the subject's current clinical symptoms, and the 4p16.1 microduplication is inherited from patient 2. However, patient 2 has no clinical symptoms, so this CNV may not be the main cause of the current clinical symptoms. Based on the above evidence, we evaluate this CNV as a variant of unknown clinical significance (consistent with the CMA judgment result).

Our attention was also directed towards the 3q13.33q21.2 microdeletion, and we did not detect any pathogenic, probable pathogenic, or uncertain clinically significant variants within this genomic region. However, it is a pity that patient 6 failed to cooperate with the blood draw for WES to verify the mutation site information.

Discussion

The molecular investigation revealed deletions within the 3q13q21.2 region, characterized by heterogeneous boundaries and variable sizes. It included the smallest documented deletion of 1.2 Mb located at 3q13.33q21.2. Genotype–phenotype correlation analysis in a cohort of five patients with this minimal overlapping deletion region uncovered notable shared characteristics, such as DD and orbital hypertelorism. ⁷ Clinical data from a total of 10 patients, including both new cases and previously published ones, were collected and are summarized in Table 1 . These features encompassed typical gestation and full-term delivery, with few exceptions. Among them, 9 out of 10 cases exhibited DD or ID, while one case (patient: 396167) did not present DD. Additionally, low-set ears were observed in 3 out of 10 cases, with one instance of crumpled ear (patient: 396427) and one instance of protruding ear (patient: 500983). Furthermore, 6 out of 10 cases displayed various phenotypic abnormalities in the skull. Epicanthal folds were present in 5 out of 10 patients. Notably, patients 501032 and 500983 listed in Table 1 had very small deletions involving a partial loss of the KALRN gene, which may result in limited clinical manifestations. The listed facial features in the table demonstrate epicanthal folds, hypertelorism, and antimongoloid slant. Due to the limited number of cases and variations in breakpoints among these patients, synthesizing a comprehensive phenotype profile for proximal 3q deletions is challenging.

We conducted a meticulous mapping analysis of partially overlapping deletions in patients exhibiting loss of pathogenicity ( Fig. 3 ). The analysis was performed under the assumption of incomplete penetrance, thus only the cases with reported pathogenicity are included in the mapping. All of these patients had NDDs or developmental problems of various levels of delay. This highly similar phenotype suggests evidence of haploinsufficiency (HI) in this region of genes associated with nervous system development. It can also be seen in Fig. 3 that our case is the smallest reported pathogenic deletion in this region reported so far.

Fig. 3.

( A ) The genes contained in the 3q13.33q21.2 microdeletion, gnomAD pLI (probability of loss intolerance): genes with higher numbers are more likely to be dosage-sensitive. The copy number variable region shows some case reports overlapping the 3q13.33q21.2 microdeletion. This figure is referenced from the Decipher databases. ( B ) A physical map of the chromosomal region 3q13.3 to 3q21.2, illustrating the deletions. The deletions identified in novel patients are shown in orange, previously reported deletions that have been molecularly characterized are shown in blue. Molin et al (case 15) present in last column. The author's information and the specific genomic location (hg19) are marked later.

Individuals with various large genomic rearrangements often exhibit NDDs and delays, indicating the crucial role of proper gene dosage in the development of the central nervous system. We next performed comprehensive annotation of this region using public databases, including University of California, Santa Cruz (UCSC) Genome Browser, Gene Reviews, OMIM, and PubMed. According to the UCSC Genome Browser (build 37.2), the region of the 3q13.33q21.2 deletion contains 21 known named genes ( Fig. 3 ).

Based on the presence of a deletion at 3q13.33-3q21.2 spanning a size of 1.2 Mb and encompassing 23 protein coding genes, we can potentially infer the involvement of HI gene in the development of this NDD. The %HI value is about 10%, o/e value is ≤0.35, and the pLI (probability of loss intolerance) ≥0.9. ¹⁰ The genes that meet the above conditions are: ADCY5, CASR, MYLK, and KPNA1, suggesting its strong HI status. Investigation for aforementioned 1.2 Mb deletion of 3q chromosome by DECIPHER database reveals seven OMIM genes ( Table 2 ). We propose HI of one or more genes, such as plasma membrane G protein-coupled receptor ( CASR ; OMIM#601199), members of the semaphorin protein family ( SEMA5B ; OMIM#609298), adenylate cyclase 5 ( ADCY5 OMIM#600293), and karyopherin alpha-1 ( KPNA1 OMIM#60068) included in the deletion region could influence the phenotype.

Table 2. Morbid genes located in the 3q13.33q21.2 microdeletion.

Name	Location	pLI	LOEUF	o/e	%HI	OMIM
ADCY5	chr3:123282296-123449090	1	0.25	0.135	11.41	600293
CASR	chr3:122183668-122291629	0.05	0.45	0.266	3.37	601199
CSTA	chr3:122325248-122341969	0	1.92	1.351	76.56	184600
ILDR1	chr3:121987323-122022247	0	1.06	0.746	57.36	609739
MYLK	chr3:123610049-123884332	0	0.53	0.398	15.69	600922
KALRN	chr3:124033369-124726325	1	0.13	0.084	10.98	604605
KPNA1	chr3:122421902-122514945	1	0.24	0.095	11.62	600686

Note: gnomAD pLI (probability of loss intolerance): genes with higher numbers are more likely to be dosage-sensitive. gnomAD LOEUF: genes with lower numbers are more likely to be dosage-sensitive. o/e: genes with lower numbers are more likely to be dosage intolerant; o/e values provided by the gnomAD database. %HI (haploinsufficiency rank): genes with lower numbers are more likely to be dosage-sensitive.

For each of the observed phenotypes in our case, we performed a detailed analysis of candidate genes. MYLK is not associated with neurological disease development in the OMIM database. It mainly causes aortic aneurysms, familial thoracic aneurysms, macrocystis-microcolon-intestinal hypoperfusion syndrome, and smooth muscle disorders. ^{11 12 13} Moreover, we performed detailed mapping of partially overlapping deletions in patients with these phenotypes ( Fig. 3 ). The analysis was performed under the assumption of incomplete penetrance; therefore, only cases with reported genetic variations in the KALRN and SEMA5B genes, which have been linked to specific phenotypic characteristics, were included in the mapping.

The patient 6 had epilepsy, while patient 4 had no symptoms of epilepsy. Kapoor et al reported a novel epilepsy genetic locus on chromosome 3q13.3-q21. ¹⁴ This finding may be related to the CASR gene. The measurement of peripheral blood samples in patient 6 indeed showed elevated plasma calcium levels of 2.59 mmol/L, with a normal reference range of 2.11 to 2.25 mmol/L. It is likely that patient 6 has a pathogenic mutation in the CASR allele that causes epilepsy, which may also be due to the heterogeneity of CASR onset. Unfortunately, we have no way to verify it because the family refused to confirm with WES. Adenylyl cyclase 5 (ADCY5) mutations are associated with heterogenous syndromes, including familial dyskinesia and facial myokymia, paroxysmal chorea and dystonia, autosomal-dominant chorea and dystonia, and benign hereditary chorea. ADCY5-related diseases have considerably expanded in recent years. To date, from over 70 ADCY5-related disease reports, only 3 have been associated with an autosomal recessive inheritance. The rest of the reports were linked to an autosomal dominant inheritance. ^{15 16 17 18}

All patients in the family who carry the 3q13.31-22.1 microdeletion segment have mental retardation suggesting that semaphorin 5B (Sema5B) regulates the elimination of synaptic connections in cultured hippocampal neurons. Overexpression of full-length Sema5B in hippocampal neurons reduces synapse number, while expression of a Sema5B construct lacking the semaphorin domain has no effect. ¹⁹

Importin α1/KPNA1, a member of the Importin α family is widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. A study by Sakurai et al ²⁰ revealed the Kpna1 KO mice showed anxiety-like behaviors, impaired short-term memory and sensorimotor gating, and increased depressive-like behaviors. Interestingly, in humans, examinations of the exomes of schizophrenia patients have identified a de novo nonsense mutation in KPNA1 (human importin α5 gene), ²¹ suggesting a possible link between KPNA1 and schizophrenia; however, no studies have reported whether it is associated with NDD .

Conclusion

One of the most regrettable aspects of this study is the lack of a blood sample from patient 6 for WES examination due to the refusal of his family. The occurrence of epilepsy may be related to CASR gene mutations, but this could not be confirmed. However, for the phenotype of NDD, we can determine that it is related to the 3q13.33q21.2 microdeletion. This report is expected to provide a reference for clinicians facing with prenatal diagnosis and genetic counseling in pregnant women with diagnosis of 3q13.33q21.22 deletions. The effect of the ADCY5 gene on brain development may be eventually explained in future research. In conclusion, our study not only expands the phenotypic spectrum of NDD, but also the genetic spectrum of NDD.

Funding Statement

Funding The study was supported by the National Natural Science Foundation of China (grant no.: 81801454) and the Natural Science Foundation of Guangdong Province (grant no.: 2017A030310556).