Lecture 7

Question 1

what are some traits of second generation sequencing?

Answer 1

• 10,000 fold drop in the cost of sequencing since 2005
• for typical geneticist needs, only exome sequencing is required
• 160,000 exons is the selective coverage
• newer kits also include 3’ and 5’ UTRs

Question 2

what is the degree of deep sequencing needed when we expect mutations in only in some but not all of sequence reads?

Answer 2

5000X targeted sequencing

Question 3

what is a detailed comparision between WGS and targeted panels?

Answer 3

WGS-most expensive and time costly

-targeted panels-almost evens out by cost with WES

Question 4

what happens in next generation DNA sequencing

Answer 4

• targeted gene panel
• whole exome sequencing
• whole genome sequencing

Question 5

what are the steps in bioinformatics analysis?

Answer 5

1. variant identification
2. exclude synonymous mutations and polymorphisms
3. prioritize variants using the assumed inheritance pattern of the disease
4. in silico predication of variant effects
5. identify previously reported mutations associated with human disease
6. prioritize novel mutations using existing knowledge of gene function

Question 6

what are the parts of functional validation?

Answer 6

• protein expression
• protein function in cells
• animal models of disease

Question 7

what is antenatal bartter syndrome?

Answer 7

• mutations in NKCC2, ROMK, or Barttin
• severe phenotypes
• lab: hypercalciruia and high urinary [Cl-]
• nephrocalcinosis may manifest later in life

Question 8

what is classic bartter syndrome

Answer 8

• mutations of CIC-K
• develop normally during the first 2-5 years of life
• BP is low normal
• lab: hypercalciuria and higher urinary [CL-]
• nephrocalcinosis is usually absent

Question 9

what happened when researchers sequenced only the exome, the most important parts in suspected Bartter syndrome patient, a renal salt wasting disease?

Answer 9

they discovered a congenital chloride diarrhea instead, homozygous mutation in SLC26A3

Question 10

is CCD found by mistake when sequencing the exome for bartter syndrome a treatable disease?

Answer 10

yes, aggressive salt replacement therapy is needed

Question 11

what is the strategy when you have 4 unrelated individuals with Freeman–Sheldon syndrome (FSS) and
8 healthy individuals from 3 different populations for exome sequencing?

Answer 11

find variations common for FSS but absent in controls

Question 12

what is the common variation exome sequencing found for FSS?

Answer 12

MYH3 mutation (heavy myosin 3)

Question 13

what are the symptoms associated with freeman sheldon syndrome FSS?

Answer 13

• artogriposis
• clubfoot due to contracture
• severe face defects
• scoliosis

Question 14

what are the symptoms of sheldon hall syndrome?

Answer 14

• artrogriposis
• clubfoot due to contracture
• severe face defects rare
• scoliosis rare
• calcaneovalgus

Question 15

how did exome sequencing help with Miller syndrome?

Answer 15

from 4 infected individuals from 3 families, ex sequencing found 2 different mutations in DHODH gene, it was then confirmed by regular DHODH sequencing in 3 other families

Question 16

how are Nager and MIller syndrome similar?

Answer 16

symptoms of miller are: missing pinkies, facial features, automosomal recessive
symptoms of Nager are: missing thumbs, facial features, autosomal recessive or dominant. Hoped mutations would be caused on some unknown gene but resequencing of Nager showed no DHODH mutation

Question 17

what are the symptoms of primary ciliary dyskinesia?

Answer 17

lots of bronchitis, headaches, infertility

Question 18

what are the symptoms of Charcot-Marie Tooth Neuropathy

Answer 18

• digital sensory loss and weakness (especially on cold exposure)
• deep tendon reflex abnormalities
• sometimes skeletal deformities

Question 19

what are some of the issues with whole genome sequencing for personalized medicine and for clinical diagnostics?

Answer 19

• error rates, quality scores
• data storage, computational power
• up front database management
• assembling the genome

Question 20

true or false: 23 and me is an example of exome sequencing

Answer 20

false!!!! 23 and me is not exome sequencing, chip based genotyping

Question 21

what is genotyping described as?

Answer 21

refers to sampling specific known positions in the genome of interest, known to vary between the populations or be associated with certain traits/diseases

Question 22

what are the traits of genotyping?

Answer 22

• returns information on genetic variants you know about
• will not question more variants than you originally designed the chip for
• cheaper than sequencing

Question 23

what are the ACMG guidelines for clinicians as it pertains to ordering tests?

Answer 23

• released a minimum list of incidental findings to report
• disorders where prevention and/or treatment are available and where individuals with pathogenic mutations might be asymptomatic for long periods of time
• only variants that met pathogenic criteria
• responsibility for managing incidental findings is placed on the ordering clinician
• labs will bear increased burden of added analysis and confirmation of variants

Question 24

what are some recent advances thanks to whole genome sequencing

Answer 24

• 50 hour differential diagnosis
• intended to be a prototype for use in neonatal intensive care units (examples are severe GJB2 related skin disease and BRAT1 related lethal neonatal rigidity and multifocal seizure syndrome in another infant)

Question 25

what is a key feature in WGS advances

Answer 25

sequencing of parents or affected siblings expedites the identification of disease genes in prospective cases

Question 26

what are CNV’s (copy number variants) associated with?

Answer 26

major cause of complex diseases, represent a novel independent cause of genetic morbidity for neurogenetic disorders

Question 27

what are some of the illnesses associated with CNV’s (copy number variants)?

Answer 27

• intellectual disability
• autism and ASD
• epilepsy

Question 28

what are the ways to detect CNV (copy number variants)?

Answer 28

mapped reads and paired reads

Question 29

what is penetrance?

Answer 29

probability of developing a particular disease given a particular genotype

Question 30

what is age dependent penetrance?

Answer 30

so percentage of people with the genotype developing the disease by age 40, by age 50 and so on

Question 31

what is lifetime risk?

Answer 31

probability that you ever develop the disease before you die. Lifetime risk can never quite be 100%, because you could always die of something else first

Question 32

fill in the blanks: completely penetrant genetic (1)_______ should not be more common in the (2) ________ than the (3) ________ that it causes

Answer 32

(1) variant
(2) population
(3)disease
Completely penetrant genetic variant should not be more common in the population than the disease that it causes

Question 33

what is todays biggest limitation with WGS?

Answer 33

sample size

Question 34

what is the law on sequencing?

Answer 34

it can only be on order of a physician

Question 35

what are the newborn symptoms of antenatal bartter syndrome?

Answer 35

-newborn symptoms: hypokalemia, metabolic alkolosis, vomiting and failure to thrive, history of polyhydramnios and premature delivery, polyuria

Question 36

what are symptoms of classic bartter syndrome?

Answer 36

• vomiting, polyuria, recurrent episodes of dehydration, and fever
• carpopedal spasms and fatigue
• many children show developmental delay