Testing for Single Gene Disorders

Introduction

Single gene (monogenic) disorders are caused by heritable pathogenic variants perturbing protein structure, function, gene dosage, or regulation.
Associated mutations encompass single base substitutions (missense, nonsense, silent mutations), deletions, insertions, splice-site variants, and triplet repeat expansions.
Follow predictable Mendelian inheritance patterns: autosomal dominant, autosomal recessive, X-linked, and mitochondrial.

Step 1: Clinical phenotyping combined with basic investigations (biochemical/enzyme assays, imaging, electroencephalogram).
Step 2: Rule out chromosomal anomalies utilizing karyotype and chromosome microarray (CMA).
Step 3: Targeted molecular testing if clinical phenotype strongly points to a specific gene or disease group.
Step 4: Broad genomic testing (targeted gene panels, whole exome sequencing, or whole genome sequencing) if non-specific or targeted testing is negative.
Step 5: Functional validation for variants of uncertain significance (VUS) and periodic reanalysis every 6–12 months.
Trio testing involving proband and both parents is preferred to identify de novo variants and compound heterozygotes effectively.

Sanger Sequencing: Gold standard for mutation screening. Relies on selective incorporation of chain-terminating dideoxynucleotides during in vitro DNA replication. Ideal for evaluating small genes when clinical phenotype strictly points to a distinct monogenic etiology.
Next-Generation Sequencing (NGS): High-throughput technology analyzing millions of DNA sequences in parallel at high speed and lower cost.
- Targeted Gene Panels: Simultaneously tests a predefined subset of genes associated with overlapping clinical phenotypes (e.g., epilepsy or neuromuscular panels).
- Whole Exome Sequencing (WES): Selectively targets protein-coding regions (exome), representing 1-2% of the genome but harboring the vast majority of identified disease-causing pathogenic variants.
- Whole Genome Sequencing (WGS): Evaluates the entire genomic sequence, including coding and noncoding (intronic or regulatory) regions. Offers improved detection of structural variations, copy number variations (CNVs), and repeat expansions.

Amplifies target DNA sequences to rapidly detect known mutations.
Size Analysis: Utilized for testing cystic fibrosis.
Restriction Fragment Length Polymorphism (RFLP): Applied in testing for spinal muscular atrophy.
Amplification-Refractory Mutation System (ARMS) PCR: Used for detecting founder mutations like thalassemia.
Real-Time PCR: Rapid technique yielding results within one hour utilizing special kits.

Resolves CNVs (deletions or duplications) of several kilobases within one or more genes.
Detects small intragenic deletions and duplications missed by traditional chromosome analysis and Sanger sequencing.
Limitation: Cannot detect sequence-level insertions or deletions, balanced translocations, or low-level mosaicism.

Tracks genetic traits through extended families utilizing closely linked polymorphic markers as a surrogate for the disease trait.
Deployed when specific genetic variants cannot be identified or are impractical to sequence.
Limitations: Vulnerable to genetic recombination between marker and gene, locus heterogeneity, and incorrect proband clinical diagnosis. Requires multiple affected family members.

Testing Modality	Clinical Indications	Examples
Single Gene Sequencing	Minimal locus heterogeneity; distinctive clinical findings pointing to a specific gene	CFTR for cystic fibrosis; PAH for phenylketonuria
Targeted Gene Panel	Locus heterogeneity; overlapping phenotypes; disorders sharing a common biological pathway	Muscular dystrophy panel; cardiomyopathy panel; epilepsy panel
Whole Exome Sequencing (WES)	Extreme heterogeneity; de novo mutations; indistinct phenotypes; nondiagnostic initial tiered tests	Autism, intellectual disability, Kabuki syndrome
Whole Genome Sequencing (WGS)	Suspected noncoding variation; suspected structural variation; critical illness requiring rapid data; nondiagnostic WES	DiGeorge syndrome; severe infantile intensive care presentations

Interpretation relies on assessing analytic validity (test accuracy), clinical validity (prediction of disease presence), and clinical utility (guidance for medical management).
Variants are classified into five functional categories according to American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines:
- Pathogenic
- Likely pathogenic
- Variant of unknown significance (VUS)
- Likely benign
- Benign
Negative DNA sequence test does not entirely rule out single gene disorders, as pathogenic variants may reside in unsequenced noncoding regions or result from undetected large structural variants missed by the employed assay.