Diagnosis of Genetically Inherited disease

Question 1

Uses of diagnostic testing

Answer 1

1) confirming/refuting clinical diagnosis
2) Assessing carrier status
3) Prenatal diagnosis (confirming ultrasound results)
4) Predictive testing (adult onset inherited disorders)

Question 2

When testing/screening is appropriate

Answer 2

If intervention is available and if early diagnosis can reduce disease morbidity or mortality

-e.g. BRCA1 (5000-10000 carriers) or CFTR

Question 3

Chromosome analysis: Karyotyping

Answer 3

Can observe chromosomes:

• finding abnormalities (number/aneuploidy or structure)
• Only able to observe in actively dividing cells
• observed using G-banding staining (Giemsa stain)

Question 4

Types of specimens for chromosome studies

Answer 4

1) Containing spontaneously proliferating cells:
- bone marrow, lymph nodes, solid tumours, chrorionic villi

2) Specimens routinely cultured in the lab:
- blood lymphocytes, amniotic fluid samples, tissue biopsies, long term CVS

Question 5

Chorionic Villus Sampling

Answer 5

Done in weeks 12-14 of pregnancy
- sampling of the extra embryonic tissue (chorion) derived from conceptus

• intrusive and risky (miscarriage rate = 1.5%

Question 6

Amniocentesis

Answer 6

Done in weeks 18-20

• sampling the amnion
• less intrusive & safer

Question 7

Karyotyping with Giemsa stain

Answer 7

Bright-field G-banding done to observe chromosomes

• G-banding involves trypsin digestion of chromosomes followed by DNA staining with Giemsa (G bands stain dark)

Patterns of banding shown in idiograms

• Differentiates G bands, R bands, Centromere (C bands) and non-centromeric heterochromatin
• chromosomes can then be classified by their topography (size, centromere position, chromomere, heterochromatin patterns)

Question 8

Identifying microdeletions using FISH

Answer 8

Fluorescent In Situ Hybridization performed on dividing or non-dividing cells

3 types of FISH probe:

1) Repetitive sequences (inc. centromeres)
2) DNA segments that will bind to & cover entire length of chromosome
3) DNA segments from specific genes on a chromosome that have been previously identified

Fluorescent tag localises specific microdeletion positions

Question 9

Uses of Cytogenetics

Answer 9

Pregnant women:
- diagnosing Trisomy 21 when indicative features of Down’s are observed

Children with developmental/phenotypic problems
- understanding cause of disease

Couples with reproductive problems

Question 10

DNA analysis: OLA

Answer 10

Oligonucleotide Ligation Assay
- non-PCR method relying on DNA hybridization (very specific technique relying on ligation of 2 strands with a ligase (no polymerases))

If microdeletion is present, ligation fails (mis-match of oligonucleotide strand with chromosome)

• gel electrophoresis shows if ligation occured (bigger band = ligation)
• fluorescent tagging also used

Question 11

DNA analysis: Sanger (chain termination) Sequencing

Answer 11

Synthesising new DNA strands complementary to single-stranded template but employing dideoxynucleotides:

• ddNTPs prevent further DNA extension
• tagged ddNTPs loaded into sequencing trace to identify DNA sequence (order of ddNTPs)

Identifies exons and mutations (e.g. MEGF10)

Question 12

MEGF10 satellite cell regulator mutation

Answer 12

Causes a myopathy known as EMARDD (Early onset Myopathy, Areflexia, Respiratory Distress & Dysphagia)

Homozygous nonsense mutation, exon 13