Genetic Variants and Testing

Some genetic conditions can be diagnosed clinically. However, when possible, confirmation of such entities with molecular diagnostics is ideal. This means identifying the DNA variant(s) causing the disease.

A “variant” refers to a DNA sequence that differs from the reference human genome. Adjectives are added before a variant to express the likelihood of the DNA change to be disease-causing:

benign variant: very unlikely to cause disease
pathogenic variant (or “mutation”): very likely to be disease-causing
variants of uncertain significance (VUS): not enough is known to determine whether these are disease-causing

No single genetic test can identify all types of potentially pathogenic genetic variation. Therefore, it’s important to order the right test based on the genetic diagnosis you are considering. For example, if you are strongly considering Turner syndrome (45, X), only tests that can identify whole-chromosome anomalies (aneuploidies) are appropriate.

Types of Genetic Variants

Type of Variant	Description of Category
Aneuploidy	An abnormal number of chromosomes in each cell (e.g., 45 or 47 chromosomes instead of 46)
Translocation	A piece of a chromosome has moved from its original location and has become attached to another chromosome; a translocation may disrupt a gene
Deletion, duplication, copy number variant (CNV)	Part of a chromosome (tens to millions of nucleotides) is missing or extra
Point mutation, single nucleotide variant (SNV)	Change in a single nucleotide
Insertion-deletion (indel)	A few nucleotides are inserted and/or deleted, which may alter the protein-coding sequencing after the change Can include in-frame deletions or frameshift variants
Triplet (trinucleotide) or other polynucleotide repeat expansion	Three nucleotides (or other repeat length) are repeated too many times
Epimutation	Change to molecules covalently attached to DNA, without changing the DNA sequence itself (e.g., methylation); such changes are epigenetic

The following table lists the genetic tests used in clinical genetics and the types of mutations they detect. Note that karyotypes, microarrays, whole-exome sequencing, and whole-genome sequencing are genome-wide analyses that can be used as screening tests when you suspect the presence of a genetic condition but are uncertain about what it might be.

Genetic Variants and Tests

Type of Test	Description	Type of Variants the Test Can Detect
Karyotype	Picture of chromosomes taken under a light microscope	Triploidy (69 chromosomes instead of 46) Aneuploidy Translocation Deletion/duplication (large only)
Chromosomal microarray analysis (CMA)*	A high-resolution karyotype that uses lasers and a computer instead of a microscope Two varieties: oligonucleotide and single nucleotide polymorphism (SNP)	Aneuploidy Deletion/duplication Absence of heterozygosity (only with SNP array)
Fluorescence in situ hybridization (FISH)	A fluorescent probe targeted to a specific section of a chromosome counts how many copies of that section are in a cell	Aneuploidy Deletion/duplication Translocation
Polymerase chain reaction (PCR)	A specific stretch of DNA is amplified to determine its presence or size	Deletion/duplication Triplet repeat expansion
Southern blot	A specific stretch of DNA is isolated to determine if that genomic region is present or its size	Deletion/duplication Triplet repeat expansion (rarely in clinical use at this time)
Multiplex ligation- dependent probe amplification (MLPA)	A special PCR test that measures the number of copies of a section of DNA that are present	Deletion/duplication
Gene sequencing	Checks the sequence of a gene for variants	Point mutation Indel
Methylation assay	Ascertains methylation status of a gene	Epimutation DNA mutations in genes encoding epigenetic regulators or in imprinting control centers
Methylation array**	Ascertains methylation status of multiple regions of the genome	Epimutations DNA mutations in genes encoding epigenetic regulators or in imprinting control centers
Panel test	Simultaneously examines multiple disease genes for related phenotypes	Point mutation Indel Deletion/duplication
Whole-exome sequencing***	The coding sequence of every gene is checked for variants	Point mutation Indel Deletion/duplication
Whole-genome sequencing**	The sequence of the entire genome (coding and noncoding) is checked for errors	Point mutation Indel Deletion/duplication
RNA sequencing**	Ascertains the sequence of mRNA	Suggest defects in gene splicing or gene expressions

Other tests that do not specifically look at DNA are also used to confirm diagnoses. Examples include enzyme assays for biochemical disorders and protein studies for disorders of collagen.

Secondary Findings

When whole exome sequencing or whole genome sequencing is undertaken, patients and families have the opportunity to learn about secondary findings. Secondary findings are mutations identified in one of 81 genes, selected by the American College of Medical Genetics and Genomics, that may be unrelated to the patient's presentation and reason for testing, but are thought to be medically actionable. Examples include BRCA1, when identification of a mutation may lead to high-risk cancer screening, or FBN1, when identification of a mutation may lead to cardiology and ophthalmology evaluations. For secondary findings, only pathogenic variants are reported; variants of unknown significance are not.

Research

Landmark clinical trials and other important studies

Research

Exome Sequencing Compared with Standard Genetic Tests for Critically Ill Infants with Suspected Genetic Conditions

Stevens Smith H et al. Genet Med 2020.

In this retrospective cohort study, the utility of exome sequencing is compared to that of standard genetic testing in critically ill infants and is found to have higher yield as a single test, although it does not lead to overall improved diagnostic yield or patient survival.

Reanalysis of Clinical Exome Sequencing Data

Liu P et al. N Engl J Med 2019.

A laboratory going back to its previous whole exome sequencing reports and updating them with current knowledge of disease genes and variants can make new diagnoses and clarify others.

Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions

Aref-Eshghi E et al. Am J Hum Genet 2019.

A methylation array enables the diagnosis of epigenetic disorders.

Analysis of Cell-Free Fetal DNA in Maternal Blood in Screening for Aneuploidies: Updated Meta-Analysis

Gil MM et al. Ultrasound Obstet Gynecol 2015.

Meta-analysis demonstrating that cell-free fetal DNA analysis of maternal blood is a high-sensitivity and high-specificity method for identifying trisomies 13, 18, and 21

Comprehensive Evaluation of the Child with Intellectual Disability or Global Developmental Delays

Moeschler JB et al. Pediatrics 2014.

Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders

Yang Y et al. N Engl J Med 2013.

The first publication of a large cohort of patients analyzed by clinical whole-exome sequencing, demonstrating a diagnosis rate of approximately 25% and a few patients with two separate mendelian disorders