30. Microdeletion and microduplication syndromes: mechanisms, Williams syndrome, 22q11.2 syndromes (DiGeorge/Shprintzen/velocardiofacial) and duplication.

I. Introduction

• Genomic deletions and duplications → copy number variations (CNVs).
• CNVs can involve small regions (single exons) or large chromosomal segments.
• Detecting them is crucial in:
• Congenital malformations.
• Neurodevelopmental disorders (intellectual disability, autism).
• Cancer genetics.
• Two powerful molecular methods:
• MLPA (Multiplex Ligation-dependent Probe Amplification).
• aCGH (array-based Comparative Genomic Hybridization, or molecular karyotyping).

II. MLPA (Multiplex Ligation-dependent Probe Amplification)

Principle

• Detects copy number changes in up to 40–50 DNA sequences simultaneously.
• Works by comparing the relative amplification of DNA probes hybridized to target sequences.

• Relies on ligation of two probe halves → amplification only if both parts hybridize correctly.

Steps

1. Hybridization: Two half-probes bind adjacent to each other on target DNA sequence.
2. Ligation: If both probe halves match perfectly, they are ligated into a full probe.
3. PCR amplification: Ligated probes are amplified with universal primers (same primer pair for all targets).
4. Fragment separation: PCR products separated by capillary electrophoresis.
5. Analysis: Relative peak sizes compared to control DNA →

• Reduced peak = deletion.
• Increased peak = duplication.

Applications

• Genetic disease testing:
• Duchenne/Becker muscular dystrophy (DMD gene exon deletions/duplications).
• Hereditary cancer predisposition genes (BRCA1/2 exon deletions).
• Carrier detection in families.
• Mosaicism detection in some cases.

Advantages

• High sensitivity for single-exon deletions/duplications.
• Relatively cheap and fast.

Limitations

• Only tests pre-selected target regions (not genome-wide).
• Cannot detect balanced rearrangements (translocations, inversions).