Microarray and DNA Sequencing for Diagnosis

Chromosomal Microarray (CMA)

• Chromosomal microarray relies on the principle of complementary DNA hybridization to screen the entire genome for copy number variations (CNVs), including both losses (deletions) and gains (duplications).
• Array comparative genomic hybridization (aCGH) is a type of CMA that differentially labels the patient’s DNA with a fluorescent dye (e.g., green) and a normal reference DNA with another dye (e.g., red).
• These labeled samples are mixed and hybridized to oligonucleotides spotted onto a microarray grid.
• A green-to-red fluorescence ratio of 1:1 indicates normal representation, whereas an excess of green indicates amplification (duplication) and an excess of red indicates a loss (deletion).
• Single nucleotide polymorphism (SNP) arrays are another form of CMA that detect polymorphic variations between nucleotides; they are highly useful for identifying uniparental disomy and areas of consanguinity (loss of heterozygosity).
• CMA offers a diagnostic resolution up to 50-fold higher than conventional karyotyping, enabling the detection of abnormalities down to the single-exon level.
• Unlike traditional karyotyping, CMA does not require dividing cells or cell cultures, making it a faster option for diagnosis.
• Major limitations of CMA include its inability to detect balanced chromosomal translocations, inversions, and low-level chromosomal mosaicism.

Clinical Applications of Microarray

• CMA is recommended as the first-tier clinical diagnostic test for individuals with unexplained intellectual disability (ID), global developmental delay (GDD), multiple congenital anomalies, and autism spectrum disorders (ASD).
• It effectively detects submicroscopic microdeletion and microduplication syndromes (such as Williams syndrome and DiGeorge syndrome) that involve segments of DNA smaller than 5 million base pairs, which are missed by conventional G-banded karyotyping.
• While highly sensitive, CMA can detect benign familial variants; therefore, parental testing is often required to determine if a discovered variant is inherited or a clinically significant de novo variant.

DNA Sequencing

• Sanger Sequencing: Considered the gold standard method for mutation screening, it relies on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro replication. It can only sequence a part of a gene at a time and is ideal when a distinct clinical phenotype points to a single specific gene (e.g., PAH for phenylketonuria).
• Next-Generation Sequencing (NGS): A high-throughput technology that can run thousands or millions of sequences in parallel at lower costs and higher speeds.
• Targeted Gene Panels: NGS panels simultaneously test tens to hundreds of genes associated with overlapping phenotypes, such as deafness, epilepsy, or muscular dystrophy.
• Whole Exome Sequencing (WES): Sequences the protein-coding regions of the genome (the exome), which represent only 1-2% of the human genome's 3 billion base pairs.
• Whole Genome Sequencing (WGS): Sequences both coding and noncoding (intronic or regulatory) regions, providing roughly 3,000 times more data than a microarray and offering improved detection of structural variations, CNVs, and repeat expansions.

Clinical Applications of DNA Sequencing

• NGS is indicated for conditions exhibiting extreme locus heterogeneity (where multiple genes cause the same condition) or indistinct phenotypes (such as undiagnosed inborn errors of metabolism or overlapping congenital anomalies).
• It is used to identify single nucleotide variants (SNVs), missense mutations, nonsense mutations, and small insertions/deletions (indels) that disrupt gene expression.
• WES or WGS is frequently performed using a "trio" approach, simultaneously testing the patient and both biological parents to determine the segregation of variants and accurately identify de novo pathogenic mutations.
• Bioinformatics analysis filters thousands of identified variants against population databases and classifies them into five categories: pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, and benign.
• Current NGS techniques have limitations; they may not be useful for detecting methylation disorders, triplet repeat expansions, and certain large structural rearrangements, although WGS capabilities are continually improving.

Comparison of Diagnostic Modalities