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Application of array comparative genomic hybridization in Korean children under 6 years old with global developmental delay

Purpose Recent advancements in molecular techniques have greatly contributed to the discovery of genetic causes of unexplained developmental delay. Here, we describe the results of array comparative genomic hybridization (CGH) and the clinical features of 27 patients with global developmental delay. Methods We included 27 children who fulfilled the following criteria: Korean children under 6 years with global developmental delay; children who had at least one or more physical or neurological problem other than global developmental delay; and patients in whom both array CGH and G-banded karyotyping tests were performed. Results Fifteen male and 12 female patients with a mean age of 29.3±17.6 months were included. The most common physical and neurological abnormalities were facial dysmorphism (n=16), epilepsy (n=7), and hypotonia (n=7). Pathogenic copy number variation results were observed in 4 patients (14.8%): 18.73 Mb dup(2)(p24.2p25.3) and 1.62 Mb del(20p13) (patient 1); 22.31 Mb dup(2) (p22.3p25.1) and 4.01 Mb dup(2)(p21p22.1) (patient 2); 12.08 Mb del(4)(q22.1q24) (patient 3); and 1.19 Mb del(1)(q21.1) (patient 4). One patient (3.7%) displayed a variant of uncertain significance. Four patients (14.8%) displayed discordance between G-banded karyotyping and array CGH results. Among patients with normal array CGH results, 4 (16%) revealed brain anomalies such as schizencephaly and hydranencephaly. One patient was diagnosed with Rett syndrome and one with Möbius syndrome. Conclusion As chromosomal microarray can elucidate the cause of previously unexplained developmental delay, it should be considered as a first-tier cytogenetic diagnostic test for children with unexplained developmental delay.


Introduction
Global developmental delay is defined as significant delay (2 or more standard deviations) in two or more developmental domains: gross or fine motor skills, speech and language, cognition, personal and social interactions, and activities of daily living 1,2) . The term 'global de velopmental delay' is usually reserved for younger children (i.e., typically less than 5 years of age) while the term 'intellectual disability' is usually applied to older children when intelligence quotient testing is valid and reliable 3) . Global developmental delay is estimated to affect 1%-3% of children, many of whom will display intellectual disability with older age 1,2) .
It is difficult to make an etiologic diagnosis for patients with global developmental delay because they do not reveal sufficient specific medical history or clinical features. Recent introduction of chromosomal microarray analysis such as array comparative genomic Korean J Pediatr 2017;60(9):282-289 hybridization (CGH) and single nucleotide polymorphism array has greatly contributed to the discovery of genetic causes of unex plained developmental delay 4,5) . CGH enables more sensitive detec tion of DNA copynumber changes on a genomewide scale with at least 10x higher resolution compared to that of conventional karyo typing 4) . Chromosomal microarray has been recently recommended as a firsttier cytogenetic diagnostic test for patients with develop mental delay or congenital anomalies 5) . However, although chromo somal microarray analysis is widely used for research purposes, its use is still not permitted in the field of medical practice in Korea.
Here we aimed to describe the results of array CGH and clinical features in 27 Korean children with global developmental delay accompanied by physical or neurological problems. In addition, we summarized the phenotypes of five cases showing abnormal or uncertain significant results of array CGH in conjunction with the genetic testing results.

Patients
We included 27 patients who fulfilled the following conditions for this study: (1) Korean children under 6 years with global develop mental delay who were admitted to the Department of Pediatrics at Ulsan University Hospital between April 2011 and February 2014; (2) children who had at least one more physical or neurological problems other than global developmental delay, such as facial dy smorphism, polydactyly, epilepsy, and hypotonia; and (3) patients in whom both array CGH and Gband karyotyping tests were per formed. We retrospectively reviewed the medical records of those patients to obtain their clinical information. Written informed con sent was obtained from all patients' parents or guardians. This study was approved by the Institutional Review Board of Ulsan University Hospital (IRB No. 201104038).

Array CGH and confirmatory tests
Oligonucleotidebased microarray analysis was performed us ing a 135K CGX3 wholegenome microarray platform (Roche NimbleGen Inc., Madison, WI, USA). Genomic DNA was extract ed from the patients' peripheral blood samples. Patients' DNA and normal control DNA (Human Genomic DNA: Male/Female, Pro mega, Madison, WI, USA) were then labeled with Cy3 and Cy5 dyes via NimbleGen DualColor DNA Labeling Kit (Roche NimbleGen Inc.). Array hybridization and washing were performed according to the manufacturer's instruction. The array was scanned at a re solution of 2.5 µm using NimbleGen MS 200 Microarray Scanner. Results were then analyzed using NimbleScan v2.6 software (Roche NimbleGen Inc.) and signal intensity ratios were analyzed using custom oligo nucleotide array CGH analysis software (Genoglyphix, Signature Genomics, Spokane, WA, USA) with the use of aberration filter of 3 contiguous probes. The criteria for quality control me trics were determined using standard deviation less than 0.14 and median absolute deviation less than 0.23 as major parameters for highqu ality array reference values. Log2 values less than 0.3 were excluded from the analysis, and the patient to control ratio of 2:3 or greater were considered to be copy number variations (CNVs). Public databases, such as the American College of Medical Genetics Prac tice Guidelines, University of California Santa Cruz Genome Browser (http://genome.ucsc.edu), Decipher (http://decipher.sanger. ac.uk), the Database of Genomic Variants (http://projects.tcag.ca/ variation/), the International Standards for Cytogenomic Arrays (http://iscaconsortium.org), the National Center for Biotechnology Information, and the Genoglyphix Chromosome Aberration Data base, a genoglyphixuser database provided by genolgyphix, were used to classify and interpret the identified CNVs into benign CNVs, pathogenic CNVs or variants of uncertain significance. All pathogenic CNVs and variants of uncertain significance detected by microarray analysis were confirmed by fluorescence in situ hybri dization (FISH) or realtime quantitative polymerase chain reaction (qPCR). FISH was performed using bacterial artificial chromosome probes located in the regions of gain or loss (target region) and other regions (internal control). DNA of bacterial artificial chromosome clones, RP11480N14 (2p24. StepOne RealTime PCR System apparatus (Applied Biosystem, Fos ter City, CA, USA). Each sample was analyzed in triplicate for target gene and reference gene. In each assay, a normal control and no template control were included. The relative gene copy number was quantified by the comparative CT method 6) . The costs of array CGH and confirmatory tests, such as FISH and qPCR were not charged to the patients.

Other diagnostic tests
To make etiologic diagnosis of global developmental delay accom panied with physical or neurological abnormality, the follow ing tests were also selectively performed before, after or simulta neously with array CGH and Gbanded karyotyping: (1) magnetic resonance imaging (MRI) of the brain; (2) genetic testing for the following known genetic disorders: fragile X syndrome, PraderWilli/Angel man syndrome, Rett syndrome, spinal muscular atro phy, myotonic dystrophy, DiGeorge syndrome, Williams syn drome, and infantile neuronal ceroid lipofuscinosis; (3) workup for metabolic disorders, such as plasma amino acid analysis, urine organic acid analysis, tandem mass spectrometry, electron microscopic study on skin for neuronal ceroid lipofuscinosis, and Berry spot test and cetylpyridi nium chloride precipitation test for mucopolysaccharidosis, and (4) neurophysiologic studies such as electroencephalography, elec tromyography and nerve conduction study, brainstem auditory evoked potential, and visual evoked potential.

Result of array CGH and G-banded karyotyping
Four patients (14.8%) revealed 6 pathogenic CNVs in array CGH. Three CNVs were deletions and the remaining three were du plications. The size of the 6 pathogenic CNVs ranged from 1.19 Mb to 22.31 Mb. There were no CNVs associated with specific known syndromes. One patient (3.7%) showed a CNV of uncertain signifi cance (461.17kb deletion). The clinical phenotypes and gene tic testing results of the 5 patients who showed pathogenic CNVs and variants of uncertain significance in array CGH are described in Table 1.
Three patients (11.1%, patients 1, 2, and 3) revealed abnormalities (1) 102 (2) 17 (1) same as the (1) 1 and 2p22.32p25.1) containing genome of normal copy number between them, which was also verified by FISH (Fig. 2)   development of neuroblastoma 7,8) . However, careful workup for neuroblastoma at 32 months of age revealed no sign of the disease. Among the 5 patients with pathogenic CNVs and variants of un certain significance in array CGH, only one patient's parents (patient 2) underwent the Gbanded karyotype test, the results of which were normal. No patients' parents underwent the other genetic testing including array CGH. The parental phenotypes of all 5 patients who showed pathogenic CNVs and variants of uncertain significance were discordant to the probands. All 27 patients had benign CNV loci, which were composed of losses of 42 loci in 23 patients and gains of 32 loci in 19 patients.

Results of other diagnostic tests
Diagnostic tests other than array CGH and Gbanded karyotyping performed in the 27 patients are described in Table 2. Twentyfive patients (92.6%) underwent MRI of the brain, which was the most commonly performed diagnostic examination other than array CGH and Gbanded karyotyping. Four patients (16%) revealed brain anomalies that were recognized as the causes of developmental delay such as schizencephaly, hydranencephaly, pachygyria in both frontal and temporal lobes, and subependymal heterotopia. All 4 patients with brain anomalies displayed normal result in array CGH and Gbanded karyotyping tests despite accompanying physical problems such as facial dysmorphism, polydactyly, or hypotonia. Genetic testing for 8 known genetic disorders was performed in 17 patients (63.0%). Only 1 patient (5.9%) was diagnosed with Rett syndrome from the result of the MECP2 gene test. Diagnostic tests for metabolic disorders were performed in 14 patients (51.9%). No patient was diagnosed with specific metabolic disorder. Neurophy siologic studies were performed in 16 patients (59.2%). Ten of 11 pa tients (90.9%) who underwent electroencephalography displayed abnor mal findings, including 2 patients with focal epileptiform dis charges, 2 patients with background abnormality, and 6 patients with both abnormalities. Seven of these patients were also diagnosed with epilepsy. Nerve conduction study of the face revealed abnormal findings in 1 patient with facial asymmetry. Two of 10 patients (20 %) who underwent brainstem auditory evoked potential displayed abnormal results. No etiologic diagnosis of global de velopmental delay was made by the neurophysiologic study despite abnormal re sults. Finally, one patient was also diagnosed with Möbius syn drome by the clinical features such as facial paralysis and strabismus.

Discussion
In the present study, four of 27 patients (14.8%) revealed 6 patho genic CNVs, and 1 patient (3.7%) revealed a variant of uncertain sig nificance in array CGH analysis, whereas 3 patients (11.1%) revealed abnormalities in Gbanded karyotyping. Based on the number of patients with pathogenic CNVs divided by the total num ber of patients tested, there was no significant difference of diagnostic yield between array CGH and Gbanded karyotyping in this study (14.8% vs. 11.1%). However, four of 5 patients showing pathogenic CNVs and variants of uncertain significance in array CGH demons trated discordant results compared to those of Gbanded karyotyp ing. Whereas Gbanded karyotyping identified only largesized genomic imbalances, array CGH analysis was able to detect addi tional genomic imbalances smaller than 5 Mb. Moreover, array CGH analysis could provide the exact location and size of the genomic imbalance, unlike Gbanded karyotyping, which also enabled analysis of whether specific known genes were involved in the abnormalities.
Before microarray technology was available, tests such as G banded karyotyping, FMR1 gene testing, subtelomeric FISH, MeCP2 gene testing, MRI of the brain, and visual and hearing assessments were recommended for evaluation of unexplained global develop mental delay based on a diagnostic yield of greater than 1% 9) . Diag nostic yield of chromosomal microarray analysis in children with de velopmental delay/intellectual disability has been reported between 7% and 20% 1018) . One literature review regarding the use of chromo somal microarray as a diagnostic test for patients with developmen tal disabilities, autism, or congenital abnormalities re viewed 33 original reports, including 21,698 patients tested by chromo somal microarray, showed that the average detection rate of pathogenic CNVs was 12.2%, whereas the average diagnostic yield of G band ed karyotyping was less than 3% if Down syndrome was excluded 5) . Genomewide array (~100 kb) showed diagnostic yield of 11%-15%, whereas targeted chromosomal microarray showed diagnostic yields of 7%-11% 5) . In another literature review, in which 367 research articles were selected through literature searches for the years 1980-2009, revealed that the average diagnostic yield of chromosomal microarray in subjects with unexplained global de velopmental delay/intellectual disability was 7.8% with a range of 0%-50%, whereas those of karyotype studies and FISH were 4% and 3.5%, respectively 19) . As patients with multiple CNVs usually present with a more severe phenotype than a single CNV 20) , identification of additional CNVs even in patients who had chromosomal aberrations already detected in karyotyping is useful for not only genetic coun seling, but also in understanding the phenotype of the patients.
Diagnostic yield has increased as the resolution of cytogenetic tests for patients with developmental delay has improved. Although conventional karyotyping can detect chromosomal abnormalities larger than 5 Mb, genomic imbalances in the range of 5-10 Mb are often undetected. However, the development of array CGH techno logy has enabled the detection of genomic imbalances of less than 500 kb 4,5) . Resolution of array CGH is determined by the density of coverage across the genome (spacing) and size of nucleic acid tar gets (length of DNA probe) 21) . Uniform coverage throughout the genome with resolution less than 400 kb is usually recommended as a chromosomal microarray platform, although chromosomal microarray analysis with much higher resolution is commercially available nowadays 5) . This study also showed that four CNVs less than 5 Mb which could not be detected in conventional karyotyping were detected by array CGH.
In this study, we included children who had not only global de velopmental delay but also physical or neurological abnormalities with the expectation of higher diagnostic yield of array CGH com pared to that in children with developmental delay only. Above mentioned largescale literature review demonstrated higher aver age diagnostic yield of chromosomal microarray in subjects with syndromic features than in those with global developmental delay/ intellectual disability (10.6% vs. 7.8%) 19) . Diagnostic yield of the pathogenic CNVs in the present study was 14.8%. However, since this is a small case series study, it could not be assessed whether the results indicate that array CGH analysis in patients with global developmental delay accompanied with physical or neurological abnormalities offers higher yield than in patients with develop mental delay only.
In regard to association of severity of developmental delay/in tellectual disability with diagnostic yield of array CGH, a recent report, which included 329 children with developmental delay/ intellectual disability, demonstrated 16% diagnostic yield of array CGH without difference of diagnostic yield according to the degree of developmental delay/intellectual disability. Based on their results, the authors emphasized the need to conduct array CGH on all pa tients with developmental delay/intellectual disability, regardless of the severity 22) .
Although array CGH analysis can identify aneuploidies, microde letion or microduplication, and unbalanced chromosomal rear range ment on a genomewide scale with high resolution, it may not detect balanced translocation or inversion, polyploidy, and mosaicism 4,5) . To identify chromosomal aberrations that may go undetected in array CGH, Gbanded karyotyping should be conduct ed together with array CGH analysis in cytogenetic testing. However, only a small portion (<1%) of all cases of unexplained developmental delay/intellectual disability and/or congenital ano malies reveal balanced rearrangement and lowlevel mosaicism 5,23) . In the present study, lowlevel mosaicism (11.5%) in 1 patient was detected by karyotyping, but not by array CGH. This suggests that Gbanded karyotyping can play a role in the genetic diagnosis of developmental delay despite advances in chromosomal microarray testing.
Clinical interpretation of CNVs detected in array CGH is critical and often very complicated. CNVs are classified into pathogenic, benign, and variants of uncertain significance. CNVs present in databases of healthy individuals, CNVs associated with welldefined clinical phenotype/syndrome, identical CNVs inherited from an affected parent, expanded CNVs, especially deletions containing many genes, or morbid genes reported in the Online Mendelian Inheritance in Man can be easily categorized into benign and patho genic 5) . However, since enormous genetic variations are present among healthy individuals and populations, categorization of CNVs is often a tough process. Recent data on CNVs detected in healthy individuals using high density arrays demonstrated that overall median CNV length is less than 2.9 kb, 95% of CNVs are less than 100 kb, and 1%-2% of CNVs in normal individuals are larger than 1 Mb 2427) . Another recent study, which reconstructed the genome of 2,504 individuals from 26 populations using a combination of lowcoverage wholegenome sequencing, deep exome sequencing, and dense microarray genotyping revealed in total over 88 million variants including 84.7 million single nucleotide polymorphisms, 3.6 million short insertion/deletion, and 60,000 structural variants 28,29) . Despite higher diagnostic yield, chromosomal microarray testing has not yet been routinely applied as a screening test in patients with developmental delay owing to the perception that it is expensive. However, when compared to the combined cost of a Gbanded karyotype, a subtelomeric FISH analysis, and additional other work ups, chromosomal microarray testing is not expensive 30) . The present study also showed that positive yield of genetic tests such as FISH for known genetic disorders and testing for metabolic disorders, was very low (5.9% and 0%, respectively), although positive yield of brain MRI (16%) was similar to that of array CGH. In addition, the abnormal results of neurophysiologic studies did not reveal the etiology of developmental delay despite high positive yield. These results suggest that array CGH is useful in diagnosing patients with developmental delay with respect to costeffectiveness, because the higher diagnostic yield of array CGH compared to previous conven tional diagnostic tests could reduce necessity of additional tests.
The present study has several limitations. First, the number of patients was small. The diagnostic yield of the Gbanded karyotyp ing (11.1%) in this study was significantly higher than those in previous studies (approximately 3%) such that the diagnostic yield of the Gbanded karyotyping was not significantly different with those from the array CGH (14.8%). Nonetheless, the results ap pear to be affected by a sampling bias originating from the small number of the patients in this study, where three patients with chromosomal abnormalities larger than 5 Mb were incidentally in cluded in the study cohort. Second, parental genetic tests for deter mining whether the CNVs were de novo or inherited could not be performed. Third, diagnostic tests for developmental delay, such as the Bayley Scales of Infant and Toddler Development test, were not performed for each patient, although we included patients with obvious global developmental delay to obtain a higher diagnostic yield from the array CGH. Nevertheless, this study showed the usefulness of applying array CGH in children under 6 years with both unexplained global developmental delay and physical or neurological problems to detect genomic imbalance of less than 5 Mb, which could not be detected in conventional karyotyping. In addition, this study provided summaries of phenotypes from the 5 patients displaying pathogenic and uncertain significant CNVs as detected by array CGH, in conjunction with the locations and sizes of the CNVs. In conclusion, as array CGH can elucidate the cause of developmental delay which was unidentified by past conventional testing, it should also be actively considered as a firsttier diagnostic tool for children with unexplained developmental delay.

Conflicts of interest
No potential conflict of interest relevant to this article was re ported.