Genomics NHS

Question 1

Main techniques and example of application?

Answer 1

• PCR (QF, RT, triplet), Haemophilia A
• karyotyping, structural rearrangements, AML
• pyrosequencing, EDS, OI
• arrays (microarrays, CGH, SNP, methylation), fragile X
• sequencing (panels, WES, WGS), Wilson disease
• FISH, PWS, ALL
• MLPA, BWS
• NIPD, CF

Question 2

Types of PCR used, and application?

Answer 2

• QF-PCR = for common aneuploidies (13/18/21/X), on fetal tissue samples (amnio/CV) or blood samples from newborns
• RT-PCR = specific AML inversions
• triplet PCR = for repeat expansions, eg. HD
• STR analysis = STR regions of DNA extracted and amp to find no. repeats of these variable seqs

Question 3

Applications of karyotyping?

Answer 3

• poss structural rearrangement or chromosomal mosaicism
• ambiguous genitalia –> sex chromosome karyotype
• aneuploidies –> eg. Down’s Syndrome (trisomy 21), Turner Syndrome (XO), Patau Syndrome (trisomy 13)
• AML –> for structural variant detection and copy number variant detection

Question 4

How does pyrosequencing work?

Answer 4

• DNA broken up into 100bp ss fragments
• PCR creates millions of copies of each fragment, split across thousands of wells, with just one type of fragment per well
• DNA fragments incubated with DNA pol, ATP sulfurylase, and apyrase enzymes, and adenosine 5’ phosphosulfate and luciferin substrates.
• 1 type of nts added to wells and incorp by DNA pol at 3’ end, releasing pyrophosphate, ATP sulfurylase converts this to ATP, which takes part in the luciferase-mediated conversion of luciferin to oxyluciferin, thus emitting light proportional to amount of ATP, which is picked up by a detector
• unused nucleotides and ATP degrade to apyrase, allowing the reaction to start again with another nucleotide, process is repeated, adding each nucleotide until the synthesis is complete
• detector picks up the intensity of light emitted, used to infer the number and type of nucleotides added

Question 5

Applications of pyrosequencing?

Answer 5

• for connective tissue disorders, eg. EDS = heterogenous group of disorders, investigate for vascular/classical dependant on presenting symptoms as underlying genes varies
• osteogenesis imperfecta (autosomal dominant and recessive) –> 90% patients have defect in type I collagen and usually inherited autosomally dominantly, but severe OI often result of de novo mutation (can also get parental mosaicism causing familial recurrence)
• oncology: eg. non-Hodgkin’s Lymphoma, Ig gene and rearrangement and hypermutation detection

Question 6

Types of arrays and applications?

Answer 6

• microarrays , eg. for intellectual disabilities like fragile X –> method: denature DNA, fragment DNA, label each fragment w/ fluorescent dye, sample and control DNA labelled w/ diff colours, both sets of DNA inserted into chip and allowed to hybridise to synthetic DNA on chip, if there is a mutation, sample DNA won’t bind correctly to normal sequence on chip, but will bind sequence representing mutated DNA
• CGH (comparative genomic hybridisation) arrays = microarray, for analysing CNVs relative to ploidy levels in sample comp to reference, w/o need to culture cells –> used for prenatal stuff
• SNP arrays, eg. ALL, for analysing CNVs, similar to CGH but also look at loss of heterozygosity (looking to switch to these completely)
• methylation arrays for some pediatric tumours and some neurological tumours (CpG sites) –> DNA methylation at promoters reduces gene expression
• methylation Testing (constitutional) → PWS/AS critical region for Prader Willi and Angelman

Question 7

Advantage of arrays over sequencing?

Answer 7

• low cost and high throughput

Question 8

Types of sequencing and applications?

Answer 8

• small, medium or large panels depending on disease/area looking at mutations
• Wilson disease = bi-directional sequencing of whole protein coding region identifies 99.9% of disease alleles
• Haemophilia A = sequencing of protein coding region detects over 90% disease alleles in those negative for inversions with moderate/severe disease
• single gene sequencing, eg. factor X deficiency
• WES, eg. glycogen storage disease (metabolic disorder) = more targeted, so cheaper and can return results quicker, most disease causing variants thought to be in exomes
• WGS, eg. neonatal diabetes = can look at SNVs, indels, SV and CNVs, includes promoters and enhancers unlike WES

Question 9

How does FISH work?

Answer 9

• helps identify where particular gene falls w/in individuals chromosomes
• method: prepare short seqs of ssDNA matching gene looking for (=probes), label probes w/ fluorescent dye, will bind complementary DNA when added, so location of fluorescence shows gene
• types of probe: locus specific, centromeric repeat, whole chrom probes
• largely replaced by microarrays for many apps

Question 10

Applications of FISH?

Answer 10

• constitutional, eg. Prader-Willi microdeletion/microduplication
• oncology, eg. for AFF1/MLL dual fusion (acute lymphoblastic leukemia), BCL2 breakapart (B cell lymphoma 2)

Question 11

What is MLPA and applications?

Answer 11

• variation of PCR that allows amp of multiple targets w/ single primer pair
• can detect CNVs in whole chroms to single exons
• haemophilia A = in some testing for exon duplications/deletions is appropriate using MLPA
• BWS = 11p15 growth imprinted region

Question 12

What is NIPD and applications?

Answer 12

• by CV/amnio
• works by analysing cfDNA in mothers blood (most is from mothers cells, but some from placenta will contain fetal DNA (cffDNA = cell-free fetal DNA)
• haplotype testing for CF and BMD/DMD
• haplotype = set of mutations/polymorphisms that tend to be inherited together
• Rhesus testing

Question 13

Example of autosomal dominant and testing?

Answer 13

• Huntington’s (HTT) = STR for those w/ clinical indications, or linkage analysis for families w/ confirmed diagnosis
• Osteogenesis Imperfecta (usually AD) = WES or medium panel of genes/loci

Question 14

Example of autosomal recessive and testing?

Answer 14

• CF (CFTR) = NIPD by haplotype testing or mutation testing when parents are carriers, or for child w/ suspected CF can do targeted mutation testing, single gene sequencing or MLPA, carrier testing also by targeted mutation testing

Question 15

Example of X-linked dominant and testing?

Answer 15

• Fragile X (FMR1) = STR testing, microarrays for intellectual disabilities overlap

Question 16

Example of X-linked recessive and testing?

Answer 16

• DMD/BMS = single gene sequencing or MLPA, linkage testing when appropriate, also NIPD haplotype testing when have at risk pregnancy