Introduction
Neurodevelopmental disorders (NDDs) are a group of conditions that affect the development of the brain and nervous system, resulting in impairments in cognitive, behavioral, social, and motor skills. NDDs include intellectual disability (ID), autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), and learning disabilities, among others. NDDs affect around 3-4% of the world’s population and have a significant impact on the quality of life of individuals, families, and communities.
One of the main causes of NDDs is genetic variation, such as copy number variations (CNVs), which are deletions or duplications of segments of DNA. CNVs can disrupt the function or expression of genes involved in brain development and function, leading to various phenotypes of NDDs. Chromosomal microarray (CMA) is a molecular technique that can detect CNVs across the whole genome with high resolution and accuracy. CMA is the recommended first-tier test for individuals with NDDs and/or congenital anomalies (CAs), as it can provide a definitive diagnosis, inform prognosis, guide management, and facilitate genetic counseling.
However, CMA is not widely available or accessible in many regions of the world, especially in low- and middle-income countries, where the burden of NDDs is high. Moreover, CMA results can be influenced by various factors, such as the platform used, the reference population, the interpretation criteria, and the clinical information. Therefore, there is a need for more studies on the application and utility of CMA in different settings and populations, especially in underrepresented regions.
In this study, the authors analyzed CMA data from 1012 patients from the south of Brazil, who were mainly children with NDDs and/or CAs. The objectives were to interpret CNVs and assess the frequencies and implications of long contiguous stretches of homozygosity (LCSH), which are regions of the genome that are identical on both chromosomes. LCSH can result from various mechanisms, such as uniparental disomy (UPD), inbreeding, population characteristics, or replicative DNA repair events. LCSH can also be associated with recessive diseases, imprinting disorders, or genomic imprinting anomalies.
The authors also focused on ASD, which is a complex and heterogeneous NDD characterized by impaired social communication and interaction, and restricted and repetitive behaviors and interests. ASD affects around 1% of the global population and has a strong genetic component. However, the genetic etiology of ASD is still not fully understood, and the diagnostic yield of CMA for ASD varies widely across studies. Therefore, the authors aimed to investigate the frequency and characteristics of CNVs and LCSH in patients with ASD, as well as the clinical features and outcomes of these patients.
Methods
The study was a retrospective analysis of CMA data from 1012 patients who were referred for genetic testing by geneticists or neurologists between 2013 and 2019. The patients were from the south of Brazil, mainly from the state of Santa Catarina. The inclusion criteria were: (1) having NDDs and/or CAs; (2) having CMA performed using Affymetrix CytoScan HD or 750K platforms; and (3) having available clinical data. The exclusion criteria were: (1) having a known chromosomal abnormality detected by karyotype or fluorescence in situ hybridization (FISH); or (2) having CMA performed using other platforms.
The CMA data were analyzed using the Chromosome Analysis Suite (ChAS) software, which generated reports of CNVs and LCSH. The CNVs were classified as pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign, based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The LCSH were defined as regions of homozygosity longer than 3 Mb, and were further categorized as short (3-5 Mb), medium (5-10 Mb), or long (>10 Mb). The LCSH were also evaluated for their potential pathogenicity, such as UPD, imprinting disorders, or recessive diseases.
The clinical data were collected from medical records, questionnaires, or interviews, and included information on demographics, family history, clinical features, and outcomes. The clinical features were coded using the Human Phenotype Ontology (HPO) terms, and the outcomes were measured using the Global Assessment of Functioning (GAF) scale. The patients were also classified into subgroups based on their main clinical phenotype, such as ID, ASD, ADHD, epilepsy, or CAs.
The statistical analysis was performed using R software, and included descriptive statistics, chi-square tests, Fisher’s exact tests, t-tests, ANOVA, and logistic regression. The significance level was set at p < 0.05.
Results
The study cohort consisted of 1012 patients, of whom 572 (56.5%) were male and 440 (43.5%) were female. The mean age at CMA testing was 7.8 years (range: 0-65 years). The most common reasons for referral were ID (67.6%), ASD (33.3%), ADHD (18.4%), epilepsy (16.9%), and CAs (15.7%). The majority of the patients (87.4%) had more than one clinical feature, and 12.6% had isolated NDDs or CAs.
The CMA analysis revealed that 17% of the patients had pathogenic or likely pathogenic CNVs, 12% had VUS, and 71% had no clinically relevant CNVs. The pathogenic or likely pathogenic CNVs comprised 132 deletions and 74 duplications, ranging in size from 0.02 to 64.5 Mb, and affecting all chromosomes except Y. The most frequently affected chromosomes were 15, 16, 22, and X. The most common syndromes caused by CNVs were 22q11.2 deletion syndrome (n = 14), 16p11.2 deletion syndrome (n = 13), 15q11.2 deletion syndrome (n = 12), and 16p11.2 duplication syndrome (n = 10).
The LCSH analysis showed that 953 patients had at least one LCSH, and the total number of LCSH was 10,246. The mean number of LCSH per patient was 10.8 (range: 1-36), and the mean length of LCSH per patient was 69.8 Mb (range: 3-392 Mb). The most frequently affected chromosomes were 1, 2, and 3. The majority of the LCSH were short (82.4%), followed by medium (15.5%), and long (2.1%). The mean number and length of LCSH were significantly higher in patients with consanguineous parents (p < 0.001). The LCSH were also significantly associated with certain clinical features, such as microcephaly, macrocephaly, hypotonia, hypertonia, and dysmorphic features (p < 0.05). However, no LCSH were found to be pathogenic or indicative of UPD, imprinting disorders, or recessive diseases.
The ASD subgroup consisted of 337 patients, of whom 281 (83.4%) were male and 56 (16.6%) were female. The mean age at CMA testing was 6.4 years (range: 0-38 years). The most common comorbidities were ID (63.8%), ADHD (40.1%), epilepsy (19.6%), and CAs (14.8%). The majority of the patients (92.6%) had more than one clinical feature, and 7.4% had isolated ASD. The CMA analysis revealed that 10% of the patients with ASD had pathogenic or likely pathogenic CNVs, 11.9% had VUS, and 78.1% had no clinically relevant CNVs. The pathogenic or likely pathogenic CNVs comprised 24 deletions and 10 duplications, ranging in size from 0.02 to 64.5 Mb, and affecting 20 chromosomes. The most frequently affected chromosomes were 15, 16, and 22. The most common syndromes caused by CNVs were 22q11.2 deletion syndrome (n = 4), 16p11.2 deletion syndrome (n = 4), 15q11.2 deletion syndrome (n = 3), and 16p11.2 duplication syndrome (n = 3). The LCSH analysis showed that 329 patients with ASD had at least one LCSH, and the total number of LCSH was 3,566. The mean number of LCSH per patient was 10.8 (range: 1-36), and the mean length of LCSH per patient was 70.1 Mb (range: 3-392 Mb). The most frequently affected chromosomes were 1, 2, and 3. The majority of the LCSH were short (82.5%), followed by medium (15.3%), and long (2.2%). The mean number and length of LCSH were significantly higher in patients with consanguineous parents (p < 0.001). The LCSH were also significantly associated with certain clinical features, such as microcephaly, macrocephaly, hypotonia, hypertonia, and dysmorphic features (p < 0.05). However, no LCSH were found to be pathogenic or indicative of UPD
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What are the main differences between NDDs and CAs?
NDDs and CAs are two broad categories of disorders that affect the development and function of the body, especially the brain. NDDs are disorders that impair the development of the brain and nervous system, resulting in cognitive, behavioral, social, and motor deficits. CAs are disorders that affect the formation and structure of the body, resulting in physical malformations or dysfunctions. NDDs and CAs can occur separately or together, and can have different causes, such as genetic, environmental, or multifactorial factors.
What are the genetic risk factors for ASD?
ASD is a complex and heterogeneous NDD with a strong genetic component. However, the genetic etiology of ASD is still not fully understood, and may involve multiple genetic variations and interactions. Some of the genetic risk factors for ASD are:
- CNVs, which are deletions or duplications of segments of DNA, and can disrupt the function or expression of genes involved in brain development and function, leading to various phenotypes of ASD. CNVs can be inherited from the parents or occur de novo in the offspring, and can affect different chromosomes and regions of the genome. Some of the common syndromes caused by CNVs that are associated with ASD are 22q11.2 deletion syndrome, 16p11.2 deletion syndrome, 15q11.2 deletion syndrome, and 16p11.2 duplication syndrome.
- SNVs and indels, which are single or multiple changes in the DNA sequence, and can affect the function or expression of genes involved in brain development and function, leading to various phenotypes of ASD. SNVs and indels can be inherited from the parents or occur de novo in the offspring, and can affect both the protein-coding and non-coding regions of the genome. Some of the common genes that are affected by SNVs and indels that are associated with ASD are CHD8, SCN2A, ADNP, SYNGAP1, and SHANK3.
- Gene-gene interactions, which are the combined effects of multiple genes on the phenotype of ASD. Gene-gene interactions can be additive, synergistic, or antagonistic, and can modulate the expression or function of genes involved in brain development and function, leading to various phenotypes of ASD. Gene-gene interactions can be influenced by the genetic background and the environmental factors of the individual, and can affect different pathways and networks of the brain. Some of the common pathways and networks that are affected by gene-gene interactions that are associated with ASD are the synaptic transmission, the neuronal migration, the chromatin remodeling, and the immune system.
What are the benefits of genetic diagnosis for patients with NDDs and/or CAs?
Genetic diagnosis can provide several benefits for patients with NDDs and/or CAs, such as:
- Confirming or ruling out a specific genetic syndrome or condition, which can help to explain the cause and mechanism of the disorder, and to predict the natural history and prognosis of the disorder.
- Guiding the medical management and intervention of the disorder, such as choosing the appropriate medication, therapy, or surgery, and monitoring the response and outcome of the treatment.
- Providing genetic counseling and support for the patients and their families, such as explaining the inheritance and recurrence risk of the disorder, and discussing the reproductive options and implications for the patients and their relatives.
- Enabling the participation in research and clinical trials, which can help to advance the scientific knowledge and clinical practice of the disorder, and to access novel and experimental therapies or interventions.
What are the challenges of genetic diagnosis for patients with NDDs and/or CAs?
Genetic diagnosis can also pose several challenges for patients with NDDs and/or CAs, such as:
- The complexity and heterogeneity of the genetic etiology of NDDs and/or CAs, which can make it difficult to identify the causal or contributory genetic variations, and to interpret their clinical significance and impact.
- The variability and overlap of the clinical phenotype of NDDs and/or CAs, which can make it hard to distinguish between different disorders, and to correlate the genetic and clinical data.
- The ethical and social issues of genetic diagnosis, such as the privacy and confidentiality of the genetic information, the informed consent and disclosure of the genetic results, the psychological and emotional impact of the genetic diagnosis, and the potential discrimination and stigma of the genetic diagnosis.
What are the advantages of CMA over traditional karyotyping?
CMA is a molecular technique that can detect CNVs across the whole genome with high resolution and accuracy. CNVs are deletions or duplications of segments of DNA that can disrupt the function or expression of genes involved in brain development and function, leading to various phenotypes of NDDs. CMA can detect CNVs that are too small or too complex to be seen by karyotyping, which is a cytogenetic technique that visualizes the chromosomes under a microscope. CMA can also detect LCSH, which are regions of the genome that are identical on both chromosomes, and can result from various mechanisms, such as uniparental disomy (UPD), inbreeding, population characteristics, or replicative DNA repair events. LCSH can also be associated with recessive diseases, imprinting disorders, or genomic imprinting anomalies.
How does CMA compare to other genomic tests, such as WES or WGS?
CMA, WES, and WGS are different genomic tests that can detect different types of genetic variations. CMA can detect CNVs, which are deletions or duplications of segments of DNA, and LCSH, which are regions of the genome that are identical on both chromosomes. WES can detect SNVs and indels, which are single or multiple changes in the DNA sequence, but only in the protein-coding regions of the genome, which account for about 1-2% of the total DNA. WGS can detect SNVs and indels in both the protein-coding and non-coding regions of the genome, which account for about 98-99% of the total DNA. Therefore, WGS is the most comprehensive genomic test, but also the most expensive and complex to perform and analyze. CMA is the most cost-effective and accessible genomic test, but it cannot detect SNVs and indels, which may also contribute to NDDs and/or CAs.
What are the recommendations for CMA testing in patients with NDDs and/or CAs?
The recommendations for CMA testing in patients with NDDs and/or CAs are based on the guidelines and consensus statements of various professional organizations and expert groups, such as the ACMG, the International Standard Cytogenomic Array (ISCA) Consortium, and the American Academy of Pediatrics (AAP). The general recommendations are:
- CMA should be offered as a first-tier test for patients with unexplained NDDs and/or CAs, such as developmental delay, intellectual disability, autism spectrum disorder, or multiple malformations, as it can provide a definitive diagnosis, inform prognosis, guide management, and facilitate genetic counseling.
- CMA should be performed using high-resolution and validated platforms that cover the whole genome and include probes for known syndromic and submicroscopic regions, as well as SNPs that can detect LCSH and UPD.
- CMA should be interpreted using standardized and evidence-based criteria and databases, such as the ACMG guidelines, the ISCA database, and the Database of Genomic Variants (DGV), and should consider the clinical information and the family history of the patient.
- CMA results should be reported using clear and consistent terminology and nomenclature, such as the International System for Human Cytogenomic Nomenclature (ISCN), and should include the size, location, type, and clinical significance of the CNVs and LCSH detected, as well as the recommendations for follow-up testing and genetic counseling.
What are the limitations of CMA testing in patients with NDDs and/or CAs?
CMA testing can also have some limitations in patients with NDDs and/or CAs, such as:
- CMA cannot detect balanced chromosomal rearrangements, such as translocations or inversions, which can also cause NDDs and/or CAs by disrupting or separating genes or regulatory elements, or by creating fusion genes or novel transcripts. Balanced chromosomal rearrangements can be detected by other cytogenetic techniques, such as karyotyping or FISH.
- CMA cannot detect point mutations or small indels, which can also cause NDDs and/or CAs by altering the function or expression of genes or regulatory elements, or by creating premature stop codons or frameshifts. Point mutations or small indels can be detected by other molecular techniques, such as WES or WGS.
- CMA can detect VUS, which are CNVs or LCSH that have uncertain or unknown clinical significance or impact, and can pose challenges for interpretation and counseling. VUS can be resolved by performing additional tests, such as parental testing, trio analysis, functional studies, or validation experiments, or by consulting with experts or databases.
- CMA can detect incidental findings, which are CNVs or LCSH that are unrelated to the indication for testing, but may have clinical relevance or implications for the patient or the family, such as predisposition to cancer or other diseases, carrier status for recessive diseases, or ancestry information. Incidental findings can raise ethical and social issues, such as the informed consent and disclosure of the results, the psychological and emotional impact of the results, and the potential discrimination and stigma of the results.
How can CMA testing be improved and optimized for patients with NDDs and/or CAs?
CMA testing can be improved and optimized for patients with NDDs and/or CAs by using various strategies and resources, such as:
- Developing and implementing new and advanced platforms and technologies that can increase the resolution, accuracy, and coverage of CMA, and that can integrate CMA with other genomic tests, such as WES or WGS, to provide a comprehensive and holistic view of the genome.
- Updating and expanding the databases and repositories that store and share the CMA data and results, and that provide the evidence and references for the interpretation and classification of the CNVs and LCSH, such as the ISCA database, the DGV, and the ClinGen database.
- Establishing and standardizing the criteria and guidelines for the indication, performance, interpretation, reporting, and counseling of CMA, and for the management and follow-up of the patients and families, such as the ACMG guidelines, the ISCA consensus statements, and the AAP recommendations.
- Enhancing and facilitating the collaboration and communication among the stakeholders and professionals involved in CMA, such as the clinicians, geneticists, cytogeneticists, molecular biologists, bioinformaticians, genetic counselors, and researchers, and among the different centers and institutions that offer and perform CMA, such as the laboratories, hospitals, clinics, and universities.
- Educating and empowering the patients and families who undergo CMA, such as providing them with clear and accurate information and resources about CMA, involving them in the decision-making and consent process, supporting them in coping with the results and implications, and engaging them in research and advocacy.
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