Intro to Genetic Testing- Clark

Question 1

What are common structural chromosomal abnormalities (4)? How does a balanced translocation lead to abnormal segregation during the formation of gametes? What method can detect a balanced translocation? Can a balanced translocation be detected by array CGH?

Answer 1

Common structural abnormalities:
-Duplication, deletion, inversion, translocation
Balanced translocation leads to abnormal segregation: Gametes end up:
1. normal 2. balanced carrier 3. partial trisomy + partial monosomy 4. partial monosomy + partial trisomy (blending of different chromosomes because of abnormal separation)
Detect balanced translocation with: FISH
Balanced translocation cannot be detected with aCGH.

Question 2

What are some common deletion syndromes(3)? What is the kb limit for deletions to be detected with high resolution chromosome studies?

Answer 2

1. Wolf-Hirshhorn syndrome:
   •Look like ancient Greek warrior helmet.
2. Prader-Willi syndrome:
   •Deletion of paternal 15q11.
   •Exhibits poor feeding → G.I. tube → scar in stomach.
   •Lack of satiety in childhood.
   •Can lead to stomach rupture at that scar area.
   •Best thing to diagnose is methylation study.
3. Velo-Cardio-Facial Syndrome
   •Deletion of 22q11.2
   •This deletion also leads to DiGeorge and Conotruncal Anomaly Face syndromes (CATCH22)

Kb limit: 5000

Question 3

What are the different genetic defects that give rise to Prader Willi and Angelman syndromes? What is the normal imprinting pattern of paternal and maternal regions of 15q11-q13?

Answer 3

Prader Willi: deletion of paternal 15q11-q13
Angelman: deletion of maternal 15q11-q13
Normal imprinting of paternal (P) and maternal (M) regions of 15q11-q13: In paternal chromosome 15, PWS genes are active and AS gene(s) inactive, whereas in maternal chromosome 15, PWS genes are inactive and AS gene(s) active.

Question 4

What is array CGH? How is the method performed using patient and control DNA? What kind of chromosomal rearrangements does array CGH detect?

Answer 4

Array Comparative Genomic Hybridization:
oPatient’s single stranded DNA (green) and DNA from a reference human (red) compete for hybridization to a target array.
oDetects copy number variation (CNV)
• Deletion in the patient = excess red in the target region on the array
• Duplication in the patient = excess green in the target region on the array

Question 5

What are some advantages of aCGH over conventional cytogenetics and what are some disadvantages?

Answer 5

Advantages over conventional cytogenetics:
• Finer resolution of breakpoints, defines genes involved and higher detection rates
• Cytogenetics can detect 5Mb deletions and duplications but not smaller.

Disadvantages over conventional cytogenetics:
• Higher Cost
• Does not detect balanced rearrangements (inversions, translocations)
• Need to confirm results with FISH
• Many CNVs are of unknown clinical significance
• Some are benign polymorphisms, may need to test parents

Question 6

What types of chromosomal rearrangements cannot be detected by array CGH?

Answer 6

Can’t detect:
      •	Deletions too small to be detected with the probes in the array 
      •	Balanced chromosome rearrangements
      •	Reciprocal translocations
      •	Inversions
      •	Triploidy
      •	Low level Mosaicism
      •	Mitochondrial DNA deletions
      •	Gene mutations

Question 7

How are DNA endonucleases (restriction enzymes) used to detect point mutations? What is an RFLP and how can RFLPs be used in conjunction with Southern blots to detect mutations?

Answer 7

Endonucleases create DNA fragments of different lengths. The expected size of gene fragments may vary when a mutation changes the length of a fragment or affects a cleavage site.

Restriction fragment length polymorphisms (RFLPs) migrate in an electrophoretic gel according to their size, shape and polarity, creating a pattern of normal and abnormal sized fragments.

Southern blots can also determine the number of gene copies in a genome.
oDNA electrophoresis.

Question 8

What is PCR? How was PCR revolutionized by the use of the thermostable Taq DNA polymerase?

Answer 8

Polymerase Chain Reaction (PCR), a method for DNA amplification.
Taq DNA polymerase, from the bacterium Thermus aquaticus, functions at high temperatures without denaturing with each replication cycle.

Question 9

How is Sanger DNA sequencing performed and how is Next Generation Sequencing performed? What is exome sequencing? How is exome sequencing suitable for arriving at a clinical diagnosis

Answer 9

Sanger: a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Next Generation: high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences concurrently
Exome (all exons): Exons contain about 85% of disease causing mutations
-Exons code for proteins
-represent 1% of human genome 180,000 exons in human genome
-Double-stranded genomic DNA is fragmented by sonication.
-Linkers are attached to the DNA fragments
-Hybridized to a capture microarray designed to target only the exons.
-Target exons are enriched, eluted and amplified by ligation-mediated PCR.
-Amplified target DNA is then ready for high-throughput sequencing.
-Since 2010, genes responsible for many syndromes, have been identified by exome sequencing. MLL2, the gene that causes Kabuki syndrome, was one of the first.