Chromosomal Aberrations in Pediatric Patients with Developmental Delay/Intellectual Disability: A Single-Center Clinical Investigation

Introduction Chromosomal microarray analysis (CMA) has currently been considered as the first-tier genetic test for patients with developmental delay/intellectual disability (DD/ID) in many countries. In this study, we performed an extensive assessment of the value of CMA for the diagnosis of children with ID/DD in China. Methods A total of 633 patients diagnosed with DD/ID in West China Second University Hospital, Sichuan University, were recruited from January 2014 to March 2019. The patients were classified into 4 subgroups: isolated DD/ID, DD/ID with multiple congenital anomalies (MCA), isolated autism spectrum disorders (ASDs), and DD/ID with epilepsy. CMA was performed on Affymetrix 750K platform. Results Among the 633 patients, 127 cases were identified as having pathogenic copy number variations (pCNVs) with an overall positive rate of 20.06%. Of the 127 cases with abnormal results, 76 cases had 35 types of microdeletion/microduplication syndromes (59.84%) including 5 cases caused by uniparental disomy (UPD), and 18 cases had unbalanced rearrangements (14.17%) including 10 cases inherited from parental balanced translocations or pericentric inversions. The diagnostic yields of pCNVs for the subgroups of isolated DD/ID, DD/ID with MCA, isolated ASD, and DD/ID with epilepsy were 18.07% (60/332), 34.90% (52/149), 3.70% (3/81), and 16.90% (12/71), respectively. The diagnostic yield of pCNVs in DD/ID patients with MCA was significantly higher than that of the other three subgroups, and the diagnostic yield of pCNVs in isolated ASD patients was significantly lower than that of the other three subgroups (p < 0.05). Conclusion Microdeletion/microduplication syndromes and unbalanced rearrangements are probably the main genetic etiological factors for DD/ID. DD/ID patients with MCA have a higher rate of chromosomal aberrations. Parents of DD/ID children with submicroscopic unbalance rearrangements are more likely to have chromosome balanced translocations or pericentric inversions, which might have been missed by karyotyping. CMA can significantly improve the diagnostic rate for patients with DD/ID, which is of great value for medical management and clinical guidance for genetic counseling.


Introduction
Developmental delay/intellectual disability (DD/ID) affects approximately 3% of the general population [1]. In China, 11,820,000 people were diagnosed with DD/ID, of whom 954,000 were younger than 6 years of age [2]. Taking care of a patient with DD/ID exerts a substantial financial and emotional burden on his/her family and society. Approximately more than half of DD/ID cases resulted from genetic etiologies, including chromosomal abnormalities, microduplication or microdeletion syndromes, and monogenic disorders [3]. Other etiologies include teratogenic exposures, perinatal asphyxia, infections, etc. [4].
Submicroscopic chromosomal aberrations (copy number variants, CNVs) play a significant role in the pathogenesis of DD/ID, and the diagnostic yield of chromosomal microarray analysis-(CMA-) detected CNVs associated with these disorders ranges from 12% to 29% [5][6][7][8]. Currently, 2.2. Chromosomal Microarray Analysis. Whole genomic DNA was extracted from peripheral blood cells of each patient and his or her parents using QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) and subjected to CMA-single nucleotide polymorphism (SNP) array analysis by using the Affymetrix ® CytoScan ™ 750K Array (Affymetrix, Santa Clara, CA, USA). e procedure was described in our previous publication [13].
When the fragment size of absence of heterozygosity (AOH) was larger than one-third of the chromosome, analysis software UPD tool_0.2 was used to separate the AOH into uniparental disomy (UPD) or consanguinity by comparison with the parental results. e detected CNVs were systematically evaluated for clinical significance. e procedure was also described in our previous publication [13].

Chromosomal Karyotyping.
When a gain and a loss of more than 5 Mb were simultaneously detected at one end of two different chromosomes or at the both ends of a single chromosome in one sample, peripheral blood samples of the normal parents were karyotyped to confirm whether the parents had chromosomal balanced translocations or inversions.

Statistical
Analysis. Statistical analysis was performed by using SPSS software, version 24. e frequency of pCNVs was compared among subgroups of isolated DD/ID, DD/ID with MCA, DD/ID with ASD, and DD/ID with ASD by using the chi-square test. A value of p < 0.05 was considered to indicate statistical significance.

Submicroscopic Unbalance Rearrangements.
Of the 127 cases with abnormal results, 18 cases were detected with submicroscopic unbalance rearrangements (14.17%), including 10 cases inherited from parental balanced translocations or pericentric inversions (Figure 1(b)). Fifteen cases had subtelomeric aberrations at the end of two different chromosomes, of which 8 cases were inherited from normal parents with balanced translocations confirmed by karyotyping. ree cases had subtelomeric aberrations at both ends of the same chromosome, of which 2 cases were inherited from normal parents with pericentric inversions confirmed by karyotyping.

Discussion
e establishment of genetic etiological diagnoses for DD/ ID children is usually challenging due to the high frequency of relatively nonspecific symptoms shared by numerous potential syndromes. We identified pCNVs in 20.06% of cases, which was comparable to other reported series [8,[14][15][16][17]. Interestingly, our study revealed some new findings with certain clinical significance.

More Deletions than Duplications in pCNVs.
In our study, the proportion of deletions was extremely higher than duplications in pCNVs. is finding is consistent with the notion of Ruderfer et al. [18] that many duplications present in the human genome are benign, and most phenotypically normal individuals possess a higher number of duplications than deletions. e dosage-sensitive genes have the ability to cause phenotypes [9]. In our study, 32 genes were confirmed with "sufficient evidence for haploinsufficiency" in the pathogenic deletions, while only 2 genes were confirmed with "sufficient evidence for triplosensitivity" in the pathogenic duplications (https://www.clinicalgenome.org/), which influenced the phenotypes of these patients.
us, deletions contributed more pathogenic interpretations than duplications.

Diagnostic Yields Associated with the Phenotypes.
e diagnostic yield of pCNVs (including microdeletion/ microduplication syndromes) in the MCA subgroup was significantly higher than that in the other 3 subgroups, which implied that severe and complex phenotypes, such as dysmorphology or congenital anomalies, tend to have a higher likelihood of identifying a genetic etiology [4]. Case 92 is a 13-year-old female who has mild ID, specifically a learning disability with a cleft palate. CMA revealed a 5242-kb duplication in the 15q11.2q13.1 (15q11-q13 duplication syndrome) inherited from her normal mother and a 748-kb deletion in 16p11.2 (16p11.2 recurrent microdeletion) inherited from her normal father. Evidence suggests that maternally derived 15q11.2q13.1 duplications are more frequently associated with abnormal phenotypes [19]. Weiss et al. [20] reported that the phenotype of 16p11.2 recurrent microdeletion is characterized by DD, ID, and/or ASD. It is rare that one patient suffers from two different microdeletion/microduplication syndromes. We hypothesized that both the duplication and deletion contributed to the phenotype of the patient. e probability of her parents having another baby with one of the pCNVs or for both is extremely as high as 75%. Wolfe et al. [21] identified that 16p11.2 deletions and 15q11.2q13.1 duplications had incomplete penetrance with high frequencies in neurodevelopmental disorders; however, they sometimes can be observed in healthy controls. So, the phenotype of the baby with pCNV(s) could not be confirmed before birth.
In the isolated DD/ID subgroup and DD/ID with epilepsy subgroup, the diagnostic yields of pCNVs were significantly lower than those of the MCA subgroup but significantly higher than those of the isolated ASD subgroup. e more phenotypes the patients had, such as epilepsy, the higher the likelihood of finding a genetic etiology [9]. However, the diagnostic yields of pCNVs between these two subgroups were not statistically significant. Next-generation sequencing (NGS) also contributes to the identification of epilepsy caused by monogenic mutations [22], which might be omitted by CMA. e diagnostic yield of pCNVs was significantly lower in the patients with isolated ASD than in the other 3 subgroups, which was consistent with the results of Ho et al. [16]. We assumed that some other genetic etiologies, such as singlegene disorders, may contribute to the pathogenesis of ASD,           which requires further investigation. We detected 3 microdeletion/microduplication syndromes in this subgroup, including Smith-Magenis syndrome, Potocki-Lupski syndrome, and 2q37 monosomy, which were reported in the previous studies [23,24]. us, we believe that the correct genetic diagnosis confirmed by CMA is imperative to medical management and prognostic evaluation of patients with DD/ID.

Assessment of Recurrence
Risks. In our study, microdeletion/microduplication syndromes were detected in 76 patients. As most of the syndromes are de novo (63/77), the recurrence risk of these sporadic syndromes is extremely low. However, the parents of the DD/ID patients with maternally derived 15q11-q13 duplication (Cases 23, 24, and 92) or some parentally derived recurrent CNVs such as 16p11.2 microdeletion (Case 92) or 16p13.11 microduplication/microdeletion (Cases 22 and 86) have a recurrence risk of 50%. In addition, the parents of male patients with maternally derived X-chromosomal aberrations including Xp22.31 deletion, Xq28 duplication, or Xp11.22 duplication have a recurrence risk of 25%. Hence, the CMA results of these parents are more vital to evaluate the recurrence risk in reproduction.

Syndromes
Isolated DD/  ID  DD/ID with MCA DD/ID with ASD DD/ID with epilepsy Total   Williams-Beuren syndrome  7  6  0  0  13  Angelman syndrome  4  1  0  2  7  Silver-Russell syndrome  0  3  0  0  3  15q11-q13 duplication syndrome  2  1  0  0  3  results of Cheng et al. [25]. In the 18 cases, 8 families were confirmed with parental balanced translocations and 2 families were confirmed with pericentric inversions by karyotyping. ese families have an extremely high risk of having another child with submicroscopic subtelomeric aberrations induced DD/ID (10/18). Conventional cytogenetics can only recognize chromosomal rearrangements with a limited resolution of 5∼10 Mb [9]. ere were still 8 cases diagnosed as de novo submicroscopic subtelomeric aberrations by comparing with the karyotypes of their parents. ese parents should be further tested whether they have balanced translocations or pericentric inversions by locus specific FISH probes according to the results of CMA. Fortunately, all the 18 families may possibly have a healthy child if effective genetic counseling was given based on reasonable techniques of prenatal or preimplantational diagnosis.

Limitations of CMA.
Parental study is usually indispensable because it not only helps with the interpretation of the clinical significance of CNVs but also contributes to genetic counseling and the evaluation of recurrence risk of genetic abnormalities [26]. However, even though the results of normal parents were compared with their children, there was still 1.11% VUS in our study. In general, the rate of VUS will decrease as more CMA results are obtained from the normal parents. e establishment of a normal individual CMA database might be helpful to address this issue. CMA has been confirmed as a vital technology to offer extremely higher diagnostic yield compared with chromosomal karyotype analysis in DD/ID. However, the genetic etiology of approximately 80% of patients remains unknown. Development of NGS offers another option for the genetic diagnosis of DD/ID. Currently, with an increased number of pathogenic mutations of genes associated with DD/ID detected by NGS, the diagnostic yield could be further improved by 20∼30% [27,28]. A combination of CMA and NGS could be a comprehensive strategy, but the cost-effectiveness should be considered.

Data Availability
e CMA data used to support the findings of this study may be released upon application to Prenatal Diagnosis Center, West China Second University Hospital, Sichuan University, who can be contacted at e-mail of the director of Prenatal Diagnosis Center.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.