Rare disease genetic testing

Question 1

What is Sanger sequencing? (4)

Answer 1

• First type of sequencing
• Requires a PCR primer set for every exon
• Time consuming and resource heavy because you sequence one gene at a time
• Used now to sequence single exons for familial testing

Question 2

What are the 2 types of microarrays?

Answer 2

• Oligonucleotide microarrays
• Single nucleotide polymorphism arrays

Question 3

What are oligonucleotide microarrays? (3)

Answer 3

• Detects gain or loss of patient DNA vs control (competitive hybridisation)
• 0 = normal
• > 0.5 = gain of patient DNA
• <-0.5 = loss of patient DNA

Question 4

What are single nucleotide polymorphism arrays? (3)

Answer 4

• Analysing normal polymorphisms
• Probe for possible polymorphisms, probes bind to the patient DNA
• Amount of binding tells you homozygous AA/BB or heterozygous A/B

Question 5

What is genomic sequencing? (2)

Answer 5

• (Next generation sequencing)
• DNA is sequenced many times rather than just once in Sanger, more accurate

Question 6

How much of the genome is protein-coding?

Answer 6

1%

Question 7

What are the types of genomic test? (4)

Answer 7

• Single gene sequencing (Sanger)
• Targeted gene panels (a set of genes linked to a particular condition)
• Exomes (NGS for protein-coding only)
• Genomes (NGS of the whole genome)

Question 8

What are the benefits of NGS? (4)

Answer 8

• Allows analysis of thousands of genes in one assay
• Whole genome sequencing enabled
• Can run multiple patients in one assay
• Many more diseases diagnosed via one test

Question 9

What are the types of DNA variants? (8)

Answer 9

• STOP and START variants
• Missense
• Nonsense
• Synonymous (doesn’t change the amino acids)
• Splice site
• Duplications/deletions (frameshift)
• May be a single nucleotide change
• May be multiple nucleotide changes

Question 10

What is the start sequence?

Answer 10

ATG coding for methionine

Question 11

What is the most common variant?

Answer 11

Missense - changes the amino acid that is being coded for

Question 12

Which nonsense variants may escape NMD? (2)

Answer 12

• DNA variant is present in the last exon
• DNA variant is located in the last 50 nucleotides of the penultimate exon

Question 13

What is a copy number variant (CNV)?

Answer 13

Large deletions/duplications that encompass more than one gene

Question 14

What is the acceptor splice site? (2)

Answer 14

• The 5’ end of the intron
• Always an AG dinucleotide

Question 15

What is the donor splice site? (2)

Answer 15

• The 5’ end of the intron
• Always a GU dinucleotide

Question 16

How does splicing occur? (3)

Answer 16

• Small nuclear RNA (snRNA) molecules bind to specific proteins forming a small nuclear ribonucleoprotein complex (snRNP)
• snRNPs combine to form the spliceosome
• Spliceosome identifies the acceptor and donor splice sites and removes the intron

Question 17

What are splice variants? (4)

Answer 17

• Variants that occur in introns
• If the variant occurs in the donor splice site the intron won’t be removed which changes the protein sequence and can cause alterations to the reading frame
• Variant in the acceptor splice site means a whole exon is removed which may be vital to the protein function
• Splice changes are often disease causing

Question 18

What are the symbols for each type of DNA sequence? (5)

Answer 18

• c. = coding DNA (includes introns)
• g. = genomic sequence
• m. = mitochondrial DNA
• r. = RNA
• p. = protein (amino acid) sequence

Question 19

What can variants be in terms of alleles? (3)

Answer 19

• Heterozygous (one allele has the variant)
• Homozygous (both alleles have the variant)
• Hemizygous (if X-linked and a male carrier)