Genomic testing refers to the analysis of human DNA to detect disease-causing variations. These variations could be chromosomal abnormalities or single gene defects (monogenic or Mendelian disorders). Chromo-somal abnormalities can be numerical (aneuploidy) or structural, which include loss or gain of a large part of one or more chromosomes, translocations, inversions and insertions. Loss or gain of smaller regions of a chromosome, known as copy number variations (CNV), usually involve more than one gene and are implicated in many human diseases [1]. While chromosomal aneuploidies are traditionally detected by karyotyping, chromosomal microarray analysis (CMA) is now widely used to detect chromosomal abnormalities. Next gene-ration sequencing (NGS), which includes targeted panel testing, exome sequencing (ES) and whole genome sequencing (WGS), has emerged as the most powerful tool for diagnosis of monogenic disorders, which are mostly caused by sequence variations in the coding portion of the DNA. With technological advances, cost of these tests has decreased drastically and they have become widely available. This review discusses the techniques, clinical utility, advantages and limitations of CMA and NGS.

CHROMOSOMAL MICROARRAY

CMA, otherwise known as genomic microarray, enables the study of chromosomes at a higher resolution as compared to traditional karyotyping. It has replaced karyotyping as the first-tier investigation of children with intellectual disability, multiple malformations and autism [2,3].

Principle

CMA is based on complementary hybridization of nucleotides in the probe and target DNA. Probes are oligonucleotides, varying in length from 25 to 70 bp, which are immobilized on a glass slide or a chip (array) [4-7]. They are spread across the genome at regular intervals (form the ‘backbone’ and defines the resolution of CMA) and are usually enriched for regions of clinical interest. They are designed to detect CNVs or single nucleotide polymorphisms (SNPs) or both. A CNV is a segment of DNA, which is 1kb or more, and has a variable copy number (extra or less) compared to reference genome [8]. SNPs are the most common genetic variations found in a population across the human genome. Genotyping of millions of SNPs across the genome provides information on alleles and their copy numbers, in addition to mosaicism, uniparental disomy, triploidy and regions of homozygosity. The different types of oligo array platforms include comparative genomic hybridization arrays (array CGH) and SNP arrays (Fig. 1a and 1b). Most commercially available platforms are hybrid arrays and contain oligonucleotide probes for detecting both CNVs and SNPs. Array design can be targeted (for specific regions of interest), whole genome (evaluates entire genome) or a combination of whole genome and targeted (most commercially available platforms).

Fig. 1 (a) Comparative genomic hybridization array, and (b) Single nucleotide polymorphism array.

Interpretation

The variants identified are critically evaluated based on their size, gene content and published reports in literature [9,10]. Penetrance (how many of individuals with this variant have a phenotypic effect) and variable expressivity (varying severity of disease in individuals with a particular genotype) are considered. The databases used for CNV interpretation are given in Web Table I. The CNVs are classified into pathogenic, benign or variant of uncertain significance (VOUS) based on American College of Medical Genetics and Genomics (ACMG) criteria given in Table I. VOUS are variants, which are not directly linked to the patient's phenotype but have some evidence for causation. Usually laboratories using SNP arrays report variants above 50 to 100kb in size [11]. Testing of parents may be required to ascertain the significance of the variant.

Table I Classification of Copy Number Variants (CNVs) Based on American College of Medical 
Genetics and Genomics criteria [9]

		Type of CNVs   

		   

		   Criteria

		Pathogenic

		•

		CNVs associated with a known microdeletion/duplication syndrome

		•

		CNVs reported as clinically significant in peer-reviewed journals and public databases

		•

		CNVs that are more than 3-5Mb size and are cytogenetically visible 

		Uncertain clinical significance

		Likely pathogenic

		•

		CNVs reported in a single case report, but with breakpoints and phenotype correlating to the patient's features

		•

		CNV interval has a gene whose function is relevant to the clinical features of the patient

		No sub-classification

		•

		CNVs described in multiple peer-reviewed journals with no conclusive evidence regarding clinical significance.

		•

		CNV interval has genes but it is not known whether the genes are dosage sensitive 

		Likely benign 

		•

		CNVs are seen in small number of people in databases of variations in normal individuals

		•

		No gene in the CNV interval; but it is included because of the size cut off set by the laboratory

		Benign

		•

		CNVs reported as benign variants in multiple peer- reviewed publications or curated databases

		•

		CNVs whose benign nature has been characterized

		•

		CNVs represents a common polymorphism and has a population frequency of more than 1%

CMA has the highest diagnostic yield for any single test in evaluating cognitive impairment, developmental delay, multiple malformations of unknown etiology or autistic spectrum disorder [2,12]. It is the first line investigation for antenatally detected structural abnormalities, stillbirth or intrauterine demise [13], and when a karyotype shows a marker chromosome or extra chromosome material of unknown origin. CMA can identify gain or loss of chromosomal material in up to 20% of individuals with an apparently balanced chromosome translocation [14,15]. Box I enumerates the advantages and disadvantages of CMA as compared to karyotyping.

One should know the design and resolution of the testing platform and the genomic regions covered. Most of the commercial platforms available have probes for known microdeletion/ duplication syndromes along with genome wide probes for other clinically significant CNVs. In a clinical setting, a low-resolution array, covering all well-delineated microdeletion and microduplication syndromes is usually sufficient. High-resolution arrays are more accurate in delineation of CNVs and SNPs, but result in a large number of variants, which are difficult to interpret. Its utility is limited to the research context.  Both pretest counseling (for the yield, specific benefits and limitations) and post-test counseling are also essential.

NEXT-GENERATION SEQUENCING

NGS, also known as massively parallel sequencing or deep sequencing, is a high throughput sequencing technology which allows simultaneous sequencing of millions of DNA base pairs at a comparatively lower cost and higher speed. Exomes comprise only 1% of 6.2 billion base pairs in human DNA, which code for proteins [16]. NGS can analyze the whole genome (whole genomic sequencing, WGS), exome (exome sequencing, ES) or a targeted region of interest in the human genome (targeted gene panel testing). The features of WGS, ES and targeted sequencing are summarized in Table II.  The steps involved are illustrated in Web Fig. I. Depth of sequencing is the number of times a nucleotide is read during sequencing. A depth of 20x implies that a particular variant or nucleotide is sequenced 20 times. Coverage usually refers to the fraction of the target region of interest sequenced satisfactorily (usually at least 20 times or 20x).

Table II Characteristics of NGS Based Tests

Interpretation

The variants are sorted to narrow down to a single variant that is likely to explain the disease or phenotype. As monogenic diseases are rare, it is assumed that the disease-causing variant is usually not seen in genomes of healthy individuals in the population. Disease-causing variants are likely to result in a change in quantity or quality of the protein coded by the gene, thus affecting the function of the protein. They are also likely to be conserved across different species. Several computational tools are now available to predict the effect of a change in the nucleotide sequence of a gene. The sorting (also popularly called filtering) is also aided by published databases of normal variants and disease-causing variants (Web Table II). If in-house databases with frequency of variants in a particular population are available, they can be very powerful tools for variant analysis as we expect unique genetic variations in different ethnicities. In 2015, ACMG published guidelines for interpretation of sequence variants and categorized them into five categories, i.e., pathogenic, likely pathogenic, benign, likely benign and VOUS [17]. The results are then correlated with clinical features and communicated to the patient. For efficient filtering and clinical interpretation of the variants, a patient should be referred to a trained clinical geneticist.

NGS testing generates a large number of variants in an individual's exome or genome. Clues from evaluation of pedigree, clinical examination and routine medical tests are vital to determine the effect of the variant on the phenotype. Often Human Phenotype Ontology [HPO] terms are used for this purpose. NGS should not be considered as an alternative for thorough clinical examination and ancillary laboratory tests.

 Clinical Indications

•    Targeted panel testing can be done when a particular phenotype is caused by variations in more than one gene (locus heterogeneity). For example, variations in about 20 different genes are implicated in osteogenesis imperfecta. A panel, which covers all the genes for osteogenesis imperfecta is more efficient than Sanger sequencing one gene after the other. Other examples are deafness, Noonan syndrome (RASopathies), congenital myopathy and pediatric epilepsy. Large genes like dystrophin can be tested by NGS either singly or in a panel for muscular dystrophy or myopathy when deletion and duplications are ruled out by multiplex ligation dependent probe amplification (MLPA) in a child with Duchenne muscular dystrophy. 

•    ES can be performed in patients with genetically heterogenous monogenic disorders when targeted panel testing fails.

•    WGS may be considered when ES fails to identify a disease-causing variant. It detects variants in coding and non-coding regions of the genome and regions not well captured and sequenced in ES, CNVs and structural chromosomal abnormalities. It has the potential to become a single test replacing most of the current tests.

•    NGS-based tests hold promise in area of carrier testing, pre-symptomatic testing, pharmacogenetic testing, and predictive testing, which are beyond the scope of this review.

Even though genome sequencing and exome sequencing are described as 'whole' genome or 'whole' exome sequencing, they do not evaluate all the genes in the human genome. The word 'whole' distinguishes these tests from panel testing and should not mislead clinicians and patients to believe that these tests would be 100% sensitive to detect all the disease-causing variants. The coverage of known genes by these tests vary from 85%-92% [18]. ‘Clinical exome’ or ‘focused exome’ is a commercial panel test that uses a customized capture kit to interrogate only genes associated with a known clinical phenotype, usually listed in Online Mendelian Inheritance in Man (OMIM). Hence the term 'clinical exome' is better avoided. In strict sense, 'clinical' genome or exome sequencing implies sequencing of exome or genome for clinical applications [19]. Before ordering a test, it is essential to check the coverage of genes of interest. The decision whether to order a targeted panel test or ES or WGS will depend on the clinical features of a patient and the ability of a clinician to arrive at a diagnosis.  An ideal targeted panel test should be able to diagnose disease-causing variants in the genes of interest of the suspected genetic disorder and should also include methods to detect deletion and duplications, which can cause a specific disease phenotype. Analyzing only selected regions or genes of interest may not qualify to be called a targeted panel, unless the laboratory fills the gaps in sequencing by alternate methods like Sanger sequencing and does a deletion/ duplication analysis. For example, in a child with leukodystrophy, before ordering a targeted panel test for leukodystrophy, it is essential to check whether all the genes of interest are covered. Krabbe disease is often caused by deletions in GALC gene and might be missed if an NGS test is ordered without asking for deletion/duplication analysis of GALC gene. If a specific genetic diagnosis cannot be made, ES or WGS may be considered. ES is cheaper and is often preferred to WGS as the first investigation for undiagnosed single gene diseases, which mostly result from variations in exons. A singleton or single exome means exome sequencing of a proband, whereas 'trio' exome means exome sequencing of the proband and parents.

Consent and Counseling in NGS Tests

Informed consent is essential before NGS based testing. Pretest counseling is essential to explain the yield, utility and implications of a ‘negative’ or ‘positive’ report for family. Limitations of science in interpreting VOUS and identification of secondary variants are specific issues in NGS testing.  Secondary variants in genes are associated with diseases unrelated to the proband’s condition and are common in ES and WGS. Secondary findings in genes causing cancer and sudden cardiac death may have implications for the patient and family members.  A genetic diagnosis may not have any direct impact on the treatment of the patient but may aid in long-term management, genetic counseling and prenatal diagnosis. Post-test counseling by a geneticist is thus needed. Sanger sequencing is done to validate the variant in the proband and for segregation analysis. Good quality NGS often obviates the need for Sanger confirmation. Segregation analysis determines segregation of the variants in the other affected or unaffected members in the family and is crucial for causal association in the proband. If a negative test result is obtained, the family should be counseled about the need to re-evaluate the data at a later date.

At present there are no regulations governing clinicians, laboratories and counselors in India. Direct marketing of these tests may result unregulated commercialization.

Variables to Consider in NGS Report

The NGS report mentions the methodology, capture kit, depth and coverage of sequencing. Capture kits may be customized for different panel tests and ES.  It is important to check for depth and coverage of sequencing before conveying the report to the patient. 

Some clinical scenarios where CMA and NGS have aided in diagnosis are described in Web Table III.

CONCLUSIONS

Chromosomal microarray, exome sequencing and whole genome sequencing using NGS techniques are powerful methods to investigate variations in human genome. It is essential for a pediatrician to know the strengths, limitations and advantages of these testing methods over traditional medical tests to apply optimally in clinical practice of pediatrics.

Contributors: DLN: substantial contributions to design and draft of the work; GKM: substantial contributions to the conception and design of the work, drafting and revising it critically for important intellectual content. Both approve the final version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding: None; Competing interest: None stated.

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