Journal of Biomedical Sciences

Keywords

Neurodevelopmental disorders; Global developmental delay; Chromosomal microarray

Abbreviations

ASD: Autism Spectrum Disorder; CMA: Chromosomal Microarray; CNV: Copy Number Variation; ID: Intellectual Disability; GDD: Global Developmental Delay; KR: Mental Retardation

Introduction

The human brain is a highly complex structure, and its normal development and functioning are tightly orchestrated by a network of genes. The Neurodevelopmental Disorders (NDDs) are characterized by complex constellations of symptoms that include intellectual disability, emotional dysregulation and aberrant behaviors [1]. Sub microscopic chromosomal rearrangements have formed the foundation for neurodevelopmental disorders and are an important source of genetic and phenotypic variations. The prevalence rates of Neurodevelopmental Disorders (NDDs) from different regions of the country ranges from 1%-3% of the general population [2]. The various clinical entities such as Intellectual Disability (ID), Mental Retardation (MR), Global Developmental Delay (GDD), schizophrenia and Autism Spectrum Disorder (ASD), show considerable comorbidity and may be associated with a variety of neurological features within complex syndromes. Even with recent advances in genetic testing, there are still patients without an etiologic diagnosis, bearing the long diagnostic odyssey in search of accurate etiology. Some of these imbalances can be explained by gross chromosomal abnormalities, detected by conventional cytogenetic techniques such as GTG-banding. Although banding allows for the detection of numerical and structural chromosomal abnormalities present in the entire genome but have a limitation of resolution of 5-10 Mb and a detection rate of only 3%-5% [3]. Therefore, the etiology of congenital anomalies in 40%-60% of the cases remains unclear. In the past two decades, traditional banding has been combined with targeted molecular technologies to improve the resolution at which one can detect genomic changes.

The International Collaboration for Clinical Genomics (ICCG), also known as International Standard for Cytogenomic Array (ISCA) Consortium, has recommended CMA as the first-tier clinical diagnostic test for patients with DD, MR, and ASD of unknown causes [4]. Also, the American College of Medical Genetics and the American Academy of Paediatrics has endorsed, CNV analysis using array-based comparative genomic hybridization (Array-CGH) or CMA is now routinely performed in clinical genetics laboratories [5]. CMA detects Copy Number Variations (CNVs) in the entire genome with a much higher resolution than conventional cytogenetic. Recent studies using such genome-wide arrays to investigate patients with MR with and without dysmorphic features have suggested a diagnostic yield of 10%-25%, of which de novo findings count for approximately 10% [6-8]. The diagnostic yield of clinically significant CNVs varies between 12%-20%, depending on the clinical preselection and resolution of the array [9]. The CNVs can be divided into benign, pathogenic, likely pathogenic and CNVs of unknown clinical significance and can be polymorphic with a frequency greater than 1.0% or rare, less than 1.0%. The genomic disorders result from DNA rearrangements caused by de novo anomalies or are inherited, multiallelic or biallelic, Non- Allelic Homologous Recombination (NAHR) between regionspecific low copy repeats (LCRs) or non-homologous end joining, leading to interstitial deletions, duplications, and inversions as well as unbalanced translocations [10]. Our study is an attempt to unravel the application of microarray in patients with neurodevelopmental disorders in Indian Scenario. In the present study, we have used the CytoScan_750k array platform provided by the Affymetrix to analyze 102 patients with unexplained neurodevelopmental disorders and congenital anomalies to identify chromosomal rearrangements in these patients.

Methods

Clinical evaluation

A total of 102 patients with unexplained neurodevelopmental disorders and congenital anomalies were referred for chromosomal microarray analysis by physicians from pediatrics, rehabilitation, neurology, and psychiatric departments of different hospitals and research institutes. These patients were found to suffer from for unexplained DD, ID, MR, and ASD, microcephaly with or without dysmorphism or seizures. Written informed consent was obtained from patients or guardians for genetic analysis. The average age of patients ranged from 50 days to 32 years.

DNA preparation and chromosomal microarray

DNA was extracted from peripheral blood using DNAse blood and tissue kit (Qiagen) according to manufacturer's instructions and was stored at -200?C until further use. Genomic DNA concentration was measured by Nano drop spectrophotometer (Thermo Fisher). Approximately, 250 mg of high-quality genomic DNA was digested with Nsp1 restriction enzyme and digested DNA was then ligated to Nsp1 adapters. The ligation product was then amplified via Polymerase Chain Reaction (PCR) to produce amplicons in the range of 200 to 1100 bp. The amplicons are then purified and digested with DNAse I to produce 25 to 125 bp fragments. The fragments are end-labeled with a modified biotinylated base [11]. Samples were hybridized to Cytoscan 750k array in an Affymetrix Hybridization Oven at 600?C for 16 hours. Washes and staining of the arrays were performed with an Affymetrix Fluidics Station 450, and images were obtained using CytoScan 750K (Affymetrix, Santa Clara, CA, USA). The array is characterized with 750,000 CNV markers, including 200,000 genotype-able SNP probes and >550000 nonpolymorphism probes. The overall average marker space is 4125 base pairs. All data were visualized and analyzed with the Chromosome Analysis Suite (ChAS) software package (Affymetrix, ChAS V3.3) using Human Genome build hg19 and reporting threshold of the CNVs was set at 100 kb with a marker count of >50. The total number of autosomal, pseudo autosomal, intragenic and intragenic markers are 702,346; 811; 532,850 and 217,586, respectively.

Cytogenetic studies using G-banding standard karyotype at an average resolution of 750-800 bands and FISH analysis was also performed on the patients showing duplication of chromosome 22 at band q11.2. In order to perform FISH analysis, DiGeorge TUPLE1 (TUPLE1 [MIM 600237) (HIRA) region probe at 22q11.2 and a control probe, arylsulfatase-A (ARSA [MIM 607574]), at 22q13.3 (Vysis), were used on leukocytes in metaphase [12]. This is a direct-labeled dual-color probe mixture with TUPLE1 (HIRA) probe labeled in orange and ARSA probe labeled in green. Slide preparation, denaturation, hybridization, and posthybridization washes were all performed according to already established procedures and the manufacturer's recommendations, with minor modifications [12]. For each patient, at least 100 interphase and 10 metaphase cells were scored for both the TUPLE1 and ARSA signals.

Interpretation

Detected CNVs (gains/duplications or losses/deletions) were classified as pathogenic and likely pathogenic (possible clinical relevance), in accordance with the recommended guidelines from the International Standard Cytogenomic Array and the American College of Medical Genetics [13,14]. In this study, the pathogenic abnormalities has been classified as the ones where the detection of CNVs is in the known pathogenic regions, deletion/duplication are >3 Mb in size and deletions/ duplications <3 Mb and previously reported as pathogenic. Confidence is determined on a marker by marker basis by evaluating the concordance of the log ratio at each marker with the copy number state assigned by the hidden Markov model (HMM). The average confidence score of markers in gain and loss segments determines the confidence score of that segment.

The data were interpreted by using information available in the scientific literature, public databases and other general information about pathogenic CNVs (size, the content of Online Mendelian Inheritance in Man (OMIM) morbid genes or dosagesensitive genes, and type of dosage imbalance: duplication or deletion) [9]. Genomic map from the UCSC Genome Browser [15] was used to map the locations of CNVs and gene distribution. The Database of Genomic Variants [16] provided catalogues of structural variations found in normal healthy controls. The dbVar [17] database was also used to get information about CNVs from both normal and diseased populations. We also used the Database of Chromosomal Imbalance and Phenotype in Humans using Ensemble Resources [18] as a reference for known micro deletion and micro duplication syndromes, and the OMIM [19] for disease-causing genes, their functions and inheritance patterns.

Results

A total of 102 patients with GDD, ID, dysmorphism with or without multiple congenital anomalies were considered in the study. The genomic DNA was isolated, followed by digestion, ligation, PCR, fragmentation, and labeling (Figure 1). In this communication, only pathogenic or likely pathogenic CNVs have been reported. Out of 102 patients studied, 48 patients were found to have a normal karyotype of 46XX/XY. The pathogenic CNVs were found in 53 (57.6%) patients and an interesting case 199, showed the exclusive occurrence of high levels of homozygosity. The ratio of affected males to females was found to be (2.1:1). The rate of duplications was found to be more than that of deletions in the affected patients accounting to 74.3% and 25.6%, respectively.

Figure 1: Representative agarose gel electrophoresis of cases 66, 79, 99, 118 and 122 (a) Genomic DNA isolated from the peripheral blood samples of the patients corresponding to cases 66, 79, 99, 118 and 122, where M denotes the 1Kb ladder. (b) PCR amplification of the isolated, digested and ligated DNA products corresponding to the cases mentioned in the top panel. Each sample was amplified in quadruplicates (c) A 4% agarose gel depicting fragmentation of the purified amplified PCR products, where M denotes the 50 bp ladder.

Patients with pathogenic CNVs

In 53 patients with pathogenic CNVs, 67.3% of patients had CNV involving single chromosome while 33.9% of patients showed co-occurrence of CNVs on two chromosomes.

Description of patients with pathogenic CNVs

Of the total 53 patients, 18 (34.6%) showed duplication of 22q11.2 region detected by Cytoscan 750K microarray Figure 2. These duplications were later confirmed by karyotyping at a band resolution of 750-800 and by Fluorescence In Situ Hybridization (FISH) (Figure 3A and 3B). FISH analysis of interphase/metaphase cells revealed a duplication of the TUPLE1 probe on one chromosome 22q, reflecting increased gene dosage in the VCFS critical region (Figure 3B). A total of 100 cell nuclei were analyzed, and all cells had three red signals for TUPLE1 of similar intensity and two green signals of Locus- Specific Identifier (LSI) ARSA. The ARSA is the most commonly used commercial probe for internal control to determine the presence, deletion, or duplication of TUPLE1. The duplication was present in all 100 interphase cells screened and, duplication on chromosome 22 was visualized as a more intense signal of the TUPLE1 probe on one chromosome 22 as compared to its homolog in all cells (Figure 3B).

Figure 2: A Microarray profile depicting duplication of the long arm of chromosome 22 at band 11.2. (a) Representative microarray profile of the patient depicting duplication of 22q11.2, each dot represents a single probe spotted on the array. The log ratio of the chromosome is plotted as a function of chromosomal position. The increase in the copy number of chromosome 22q11.2 has been encircled. (b) (i) The whole genome view showing the duplication of chromosome 22 (encircled). (ii) The red arrow in the karyogram depicts the region of gain in chromosome 22.

Figure 3: Karyotyping and Fluorescence In Situ Hybridization (FISH) depicting duplication of the long arm of chromosome 22 at band 11.2. (a) G-banded karyotype of a patient, at band resolution of 750-800 showing a gain in the copy number chromosome 22 at band q11.2 (arrow). (b) (i) Metaphase, showing microduplication of TUPLE1 (red) on one chromosome 22 (arrow) and normal control probe, ARSA (green). The microduplication is seen as a larger-sized signal compared with the normal. (ii) Interphase cell showing duplication of TUPLE1 (three red signals) and two control probe (ARSA) signals (green).

Patients with pathogenic CNVs and known syndromes

Five out of 53 sporadic patients (Case 66, 79, 99, 118 and 122) had known micro deletion syndromes (Figure 4). The Case 66 showed 17p11.2 deletion; Smith-Magenis syndrome coupled with the mosaic loss of chromosome 17p13.2, case 79 depicted loss of Xp22.13 region overlapping with Rett syndrome, case 99 had 1q21.1 micro deletion syndrome along with Turner Syndrome, case 118 showed 4p16.3 terminal deletion; Wolf- Hirschhorn syndrome and case 122 had 7q11.23 deletion; William syndrome. Four patients presented with previously reported but rare CNVs case 49 (22q13.33 and 22q11.2 duplication), case 56 (2q13, 4q35.1 and 15q11.2 duplication), case 115 (7p22.3 deletion and 10q25.3 duplication) and case 129 (15q13.3 and 17p13.3 duplication).

Figure 4: Pathogenic genomic imbalances CytoScan 750K microarray profile. (a to i) The copy number gain or loss shifts the log 2 ratio. The lower panel corresponds to each case. (j) Exceptionally, the karyoview of case 199 represents a high degree of LOH regions across the whole genome.

Micro duplication of 15q13.3 and 17p13.3

The case 129 showed micro duplication of both 15q13.3 and 17p13.3 regions (Figure 4). Individuals with duplication in 15q13.3 region showed variable symptoms like intellectual disability, communication difficulties, and behavioural problems including autism spectrum disorders [20]. Only one gene name Cholinergic receptor, Neuronal nicotinic, Alpha polypeptide 7 (CHRNA7) was found to be affected in the patient which is a critical gene associated with autism and behavioural disorders [21]. Also, micro duplication of 17p13.3 encompasses the same region that is deleted in Miller-Dieker syndrome [22]. Patients with 17p13.3 duplication exhibit neurobehavioral disorders, including delayed development, mental retardation, and attention deficit-hyperactivity disorder.

Mosaic loss of chromosome 17p13.2 and loss of 17p11.2

There was a loss of the short arm of the chromosome 17 at bands p13.2 and p11.2, respectively in case 66 (Figure 4). These regions were found to be 100% overlapping with the critical region of Smith-Magenis syndrome [23-25]. Smith-Magenis syndrome is associated with a deletion of the proximal short arm of chromosome 17, including the critical RAI1 gene region. Although the phenotype is variable, the syndrome can be suspected in patients with failure to thrive, brachycephaly (short head), prominent forehead, microcephaly (small head), flat and broad mid face, broad nasal bridge, strabismus, myopia, malformed ears, high and cleft palate, prognathism (protruding mandible), short and broad hands and feet, scoliosis (laterally curved spine), and cryptorchidism (undescended testes). Mental retardation is variable but usually severe with seizures and hyperactivity.

Micro deletion of chromosome Xp22.13

The case 79 had a clinical history of Global developmental delay with epileptic encephalopathy and dysmorphism. Microarray array analysis revealed a micro deletion of chromosome Xp22.13. This micro deletion resulted in a loss of five OMIM relevant genes (Figure 4). These genes included SCML2, CDKL5, RS1, PPEF1, and PHKA2. Out of all these five genes, CDKL5 is a main causative gene for epileptic encephalopathy which is an X-linked dominant severe neurological disorder characterized by the onset of seizures in the first months of life and severe global developmental delay resulting in mental retardation and poor motor control. Other features include lack of speech development, subtle dysmorphic facial features, sleep disturbances, gastrointestinal problems, and stereotypic hand movements. There is some phenotypic overlap with Rett syndrome [26,27].

Micro deletion of 7p22.3 and duplication of 10q25.3

A de novo micro deletion of chromosome 7p22.3 and duplication of chromosome 10q25.3 was identified in a 3-yearold boy (case 115) with loss of 2.5 Mb of chromosome 7p22.3 deletion and gain of 17.8 Mb of chromosome 10q25.3 (Figure 4). This abnormality corresponds to a der(7)t(7;10)(p22;q26) that is an unbalanced version of a t(7;10) translocation, which was paternally inherited. Investigation of the few genes in the region of chromosome 7p also revealed FAM20C and Platelet-Derived Growth Factor Alpha (PDGFA), as genes that have potential roles in patient's developmental delay and bone findings [28]. In case of, 10q25.3 duplication several putative candidate genes on 10q including DPYSL4 (neuronal differentiation), PPP2R2D (embryonic growth and development), INPP5A (intracellular signaling), and GPR123 (G-protein coupled receptor expressed in brain) may contribute to the pathogenesis in this patient [29-31].

Wolf-Hirschhorn syndrome (terminal deletion of 4p16.3)

A patient (case 118) was found to have ~ 6 Mb deletions at 4p16.3 region (Figure 4). The 4p deletion also known as Wolf- Hirschhorn syndrome came to existence in 1965 by Wolf et al, 1965 and Hirschhorn et al, 1965 [32,33]. It is a contiguous gene syndrome caused by partial loss of chromosomal region from the terminal region of chromosome 4p [34]. These patients are reported to have hypertelorism, arched eyebrows, epicanthic folds, short philtrum, micrognathia, hypotonia, seizures, EEG abnormalities and heart defects [35]. Patient 118 has similar phenotype such as Global developmental delay, failure to thrive and atrial septal defect. This genetic disorder is microdeletion syndrome with CNV of a variable size known to be caused by dosage-sensitive genes, and atypical recognized syndromes associated with non-recurrent micro deletions that might have been clinically missed at birth. Furthermore, even in a welldefined syndrome, non-recurrent chromosome deletions can be of different sizes, leading to a broad phenotypic spectrum.

The case with high LOH content

A case 199, corresponding to a female depicting dysmorphism, low set ears, and poor coordination showed extended contiguous regions of allele homozygosity (>8 Mb) in multiple chromosomes that is consistent with common descent (related parents). Several large regions of homozygosity were detected, encompassing approximately 17.5% of the genome. These may be added to provide a measure of identity by descent which in this case is equivalent to brother-half-sister parentage.

Discussion

Several neurodegenerative and neurodevelopmental disorders are now known to be caused by disparate recurrent and nonrecurrent genomic rearrangements that are mediated or stimulated by complex regional genomic architecture occurring throughout the human genome. The clinical utility of microarray technologies used in the post and prenatal diagnosis lies in its ability to detect sub microscopic copy number changes that are associated with clinically significant outcomes. Though the usefulness of CMA in identifying CNVs has been well recognized over the last decade but the data on its application in Indian scenario has not been explored. CMA analysis when conducted on 102 unexplained DD, MR and ASD patients, revealed duplication 22q11.2 region as the most frequent abnormality. The clinical phenotype of patients with this abnormality ranges from a mild learning disability to the presence of severe congenital malformations. The chromosome 22q11.2 region is highly susceptible to both micro deletions and duplications due to its misalignment of eight Low Copy Repeats (LCR) regions, LCR22-A through LCR22-H, which mediate nonallelic homologous recombination. Both micro deletions and micro duplications might be expected to occur at the same frequency. The micro duplication of chromosome 22q11.2 was faintly visible at higher resolution 750-800 in all the patients studied. Since 750-800 band resolution karyotypes are not routinely analyzed, FISH studies on interphase nuclei play a key role in the identification of patients with dup(22)(q11.2). Interphase FISH detected the duplication in all patients, but it was confirmed by metaphase FISH showing three TUPLE1 signals. Micro duplications of 22q11.2 have previously been diagnosed primarily using interphase FISH [36,37].

Pathogenic CNVs were found to be randomly distributed across the genome and were located on 1q21.1, 1p36, 4q13, 4q21, 7q11, 10q11.2, 15q11, 15q15.3, 22q13, Xq11, Xp22.13 and Xq27 regions of chromosomes (Table 1). Kaminsky et al. found that other than the common micro deletion syndromes, recurrent CNVs found were deletions in 16p13.11, 17q21.31, 17q12, 8p23.1 and 3q29 region, while recurrent duplications were found in 1q21.1, 16p13.11, 17q12, 22q11.2 regions [33]. In our study, no such recurrent deletions or duplications were found which could be attributed to a small number of cases. The size of pathogenic CNVs ranged between 36 kb to 15.5 Mb. This is similar to reported data in literature where these have been found to vary from 0.14 Mb to 17.58 Mb [38]. Among all the patients with pathogenic CNVs, thirty-five patients (67.3%) harbored a CNV on single chromosomes; whereas seventeen patients (33.9%) had CNVs on two different chromosomes. Patients in the latter category showed more prominent features for deletion, suggesting haploinsufficiency of genes had a more pronounced effect which leads to decreased protein levels and is less tolerated than duplication. Startlingly, high levels homozygosity in multiple chromosomes corresponding to case 199 validates the phenomenon that multiple generations of consanguinity can increase the levels of allele homozygosity. Although this result is not diagnostic of a specific condition, it raises the possibility of a recessive disorder with a causative gene located within one of these regions. Additionally, these results could indicate a familial relationship (first or second degree) between this individual's parents.

Table 1: Clinical features and cytogenetic findings of patients with pathogenic CNVs.

Thus, high-resolution screening using microarray not only detects sub microscopic chromosomal imbalances but also allows accurate delineation of the duplicated or deleted chromosomal segment. Our data involving detailed phenotype analysis and CNVs can add to databases for future genetic studies for discovering new candidate genes and molecular pathways underlying unexplained neurodevelopmental disorders.

Conclusion

Our study is an attempt towards deconvoluting the effect of copy number variants in genetic diseases and more broadly in the clinical evaluation of patients with unexplained DD/ID, congenital anomalies and dysmorphic features. Further, enhancements in genomic microarray analysis will soon allow the reliable analysis of all copy number variations throughout the chromosome at the kilobase or single exon resolution. Also, clarification of the genetic profile generated by CMA coupled with knowledge-based genetic counseling, rational clinical action and follow up familial studies may aid in directing medical management.

Acknowledgment

Authors are grateful to the patient and their family to support the case study. We would also like to thank the management of Dr. Lal Path Labs for providing excellent facilities.

Conflict of Interest

The author(s) declare they have no conflict of interests.

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