Diagnosis of Genetically Inherited disease Flashcards

1
Q

Uses of diagnostic testing

A

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

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2
Q

When testing/screening is appropriate

A

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

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

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3
Q

Chromosome analysis: Karyotyping

A

Can observe chromosomes:

  • finding abnormalities (number/aneuploidy or structure)
  • Only able to observe in actively dividing cells
  • observed using G-banding staining (Giemsa stain)
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4
Q

Types of specimens for chromosome studies

A

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

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5
Q

Chorionic Villus Sampling

A

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

  • intrusive and risky (miscarriage rate = 1.5%
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6
Q

Amniocentesis

A

Done in weeks 18-20

  • sampling the amnion
  • less intrusive & safer
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7
Q

Karyotyping with Giemsa stain

A

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)
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8
Q

Identifying microdeletions using FISH

A

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

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9
Q

Uses of Cytogenetics

A

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

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10
Q

DNA analysis: OLA

A

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
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11
Q

DNA analysis: Sanger (chain termination) Sequencing

A

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)

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12
Q

MEGF10 satellite cell regulator mutation

A

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

Homozygous nonsense mutation, exon 13

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