Week 5 - F -Antenatal Genetics 2 - Array-CGH, FISH, Next Gen Sequencing, Sanger (PCR), Central Dogma, Inheritance and DNA testing

Question 1

Which gene mutations are point and which are frameshift? What is the difference between point and frameshift?

Answer 1

Point mutation - this is where the mutation occurs at the single point on the DNA strand - ie only one codon is affected

• * Subsitution and inversion are point mutations

Frameshift mutation - this is where there is an insertion or deletion causing every codon to be affected by the shift

• * Deletion and insertion are framehsift mutations

Question 2

DNA Analysis depends on size of mutation Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes When a whole or part of a chromosome is deleted or duplicated, what test is usually used to identify the mutation?

Answer 2

Array-chromosomal genomic hybridization (Array-CGH) CGH allows for the exploration of all 46 human chromosomes in single test and the discovery of deletions and duplication, even on the microscopic scale - allows for the aetiology of known and unknown conditions

Question 3

When can’t array CGH be used to identify chromosomal mutations?

Answer 3

As array-cgh only identifies unbalanced chromosomal mutations, it cannot be used to identify balanced mutations - ie substitutions Array-CGH allows for duplication/deletions in all 46 chromosomes to be analysed at once Karyotpying can be used for blaanced i think

Question 4

As array is used to discover chromsomal imbalances across all 46 chromosomes, which test can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes?

Answer 4

This would be FISH Fluorescence in-situ hybridisation Used to detect the presence or absence of specific DNA sequences

Question 5

Give an example of 2 diseases and what their mutations are where FISH could be used for the specific diagnoses

Answer 5

Can be used in * Prader Willi Syndrome - deletion of part of paternal chromosome 15 * DiGeorge Syndrome - 22q11.2 chromosome deletion * Angelman syndrome (happy puppet syndrome) - lack of function of part of chromosome 15 inherited from a person’s mother Most of the time, it is due to a deletion or mutation of the UBE3A gene on that chromosome

Question 6

When identifying gene mutations, what is the method that allows you to look at multiple genes to identify which gene may have caused the mutation? - looks at thousands of nucleotides Useful in genetic conditions that can be caused by a variety of mutations and present de novo

Answer 6

This would be next generation sequencing

Question 7

Say next generation sequencing was used to identify an unknown gene mutation in a mother and discovered what the gene mutation was What would then be used to detect if the son had the known gene mutation?

Answer 7

If the son had the known gene - then use Sanger Sequencing (PCR amplification based method)

Question 8

WHat is the most common start codon and what does it code for?

Answer 8

The most common start codon is AUG and it codes for methionine in eukaryotes (nucleus is enclosed within a cell membrane)

The start codon AUG - initiates the translation of the first amino acid in the mRNA polypeptide chain

Question 9

What are the three stop codons? What do they code for?

Answer 9

UGA, UAA, UAG They do not code for an amino acid as they are stop codons

Question 10

What is the central dogma?

Answer 10

The central dogma is the two step process by which DNA is transcripted into mRNA and then translated into protein

Question 11

What else happens in the transcription phase of the central dogma process?

Answer 11

The introns are spliced from the DNA chain - these are the non-coding sections that are spliced out before RNA is translated into a protein

Question 12

The pitfall of next generation sequencing is that it can identify lots of polymorphisms and therefore you need a rough idea of where to look If sequencing the whole genome, how many single nucleotide polymorphisms (SNP) are able to be found?

Answer 12

If sequencing the whole genome - there may be more than 3million (3 000 000) polymorphisms identified and therefore you must have a rough idea of where to look - also expensive to sequence the whole genome

Question 13

At the antenatal care, have to discuss when the patients expected date of delivery is How is this carried out? Using what rule? Take a full history and Also assess risk of diabetes If GDM is a risk, how is it screened for in women?

Answer 13

Assess expected date of delivery by determining when the last menstrual period was - expected delivery date should be 40 weeks after the 1st day of the last menstrual period -

• use Naegel’s rule - add 7 days then add 9months to 1st day of LMP - should be EDD

If GDM is a risk Carry out 75g oral glucose tolerane test at 24-28 weeks If previous GDM - carry this test out at 18+/-28 weeks

Question 14

What is the criteria for diagnosing gestational diabetes mellitus in scotland?

Answer 14

• Fasting venous plasma glucose >/= 5.1mmol/l
• One hour value >/= 10mmol/l or
• Two hour post 75g OGTT - >/= 8.5mmol/l

The guidelines for diagnosing diabetes in a normal patient differ from diagnosing gestational diabetes

Question 15

When is the booking scan carried out? What does it assess? When is the anomaly scan carried out?

Answer 15

Booking scan is carried out around 11-12 weeks - can be anytime from 10 to 13+6 weeks The anomaly (detailed) scan is carried out at 18-20 week

Question 16

What is the likely mode of inheritance in this case?

Answer 16

The likely mode of inheritance is X-linked recessive disease

Question 17

What is the risk that a new child is affected by this disease (ie not just a carrier)? What mutation is responsible for this condition? If the child is a female, what is the likelihood that she is a carrier? If the child is male,w hat is the likelihood that he is affected?

Answer 17

The risk that the new child (no gender identified yet) is affected by the X-linked Duchenne Muscular Dystrophy will be 1in4

A mutation in the dystrophin gene is responsible for this disease

If the child is a female - 1in2 chance that she is a carrier

If the child is a male - 1in2 chance that he is affected

Question 18

With autosomal dominant disease, are any generations skipped in the passing on of the disease? What is the risk of the child being affected if the parent has the disease? Are males or females more likely to be affected?

Answer 18

Autosomal dominant disease Affects all the generations 50% risk of child being affected if one parent has the disease Males and females are equally likely to be affected

Question 19

Describe autosomal recessive conditions? (describe the inheritance) When is there an increased likelihood of autosomal recessive conditions in families?

Answer 19

Autosomal recessive conditions means the child will require both affected chromosomes to have the condition - or the child will be a carrier (still may express some symptoms although variability) 1in4 chance of the parent passing the condition on to the child There is an increased likelihood of autosomal recessive conditions in consanguineous families (incest)

Question 20

What are the two testing methods carried out in pregnancy to test for foetal abnormalities? These tests are invasive

Answer 20

This would be Chorionic villus sampling or Amniocentesis

Question 21

When are both chorionic villus sampling able to be carried out from? What are the miscarriage rates?

Answer 21

Choironic villus sampling - can be carried out after 11.5 weeks gestation Miscarriage rates - 2% Amniocentesis - can be carried out after 15 weeks gestation - before this risks talipes equinovarus Miscarriage rates - 1%

Question 22

What is usually sued to analyse the sample taken in amniocentesis and chorionic villus sampling? Why can results sometimes be taken up to 3weeks to come back?

Answer 22

Usually PCR testing (FISH) is used to analyse the samples for certain conditions such as trisomy 21, 18 and 12 and results will be returned within 2-3 working days For more specific genetic testing, it may tae up to 3 weeks for results to come back

Question 23

What is trisomy 21, 18 and 13 all known as?

Answer 23

Trisomy 21 - Down’s syndrome Trisomy 18 - Edwards’ syndrome Trisomy 13 - Patau’s syndrome

Question 24

Describe some of the features of trisomy 21,18 and 13?

Answer 24

• Trisomy 21 - hypotonia, flat back of head, small mouth, single palmar crease, large first web space, SGA
• Trisomy 18 (Edwards) - low set ears, small jaw and mouth, low birthweight
• Trisomy 13 (patau’s syndrome) - the forebrain often doesn’t divide into 2 halves. This is known as holoprosencephaly. - this failure of the brain to divide in two can cause absence of one or both eyes, cleft lip, problems with nasal passage development

Question 25

If worried about the pregnancy, what test can be carried out at 8 weeks to determine the sex of the foetus - for conditions that are xlinked? If this proves worrying for the genetic condition, can then move on to chorionic villus sampling

Answer 25

Would carry out non invasive foetal sexing by measuring the free foetal DNA in the maternal serum

Question 26

If the mother and father know they have a genetic condition and choose to carry out in-vitro fertilisation? (or in patients who choose this procedure), what can be carried out to look for genetic disease? What day is the embryo implanted in IVF treatment?

Answer 26

Can carry out pre-implantation genetic diagnosis Perform a genetic test on an embryo before re-implanting one with the ‘correct’ genotype. The embryo is usually implantated 5 days after fertilisation of the egg occurs

Question 27

Centres must be licensed to practice Pre-implantation genetic diagnosis (PGD) by the HFEA in accordance with the HFE Act 1990 Increasing numbers of centres in UK Each new condition must be licensed by HFEA. How is the sex of the foetus determined in PGD?

Answer 27

Sex determination of the foetus is carried out using FISH in PGD Different coloured probes to label chromosomes X, Y and 18 (control) Individual chromosomes are not visible

Question 28

What are the advantages and disadvantages of Pre-implantation genetic diagnosis?

Answer 28

Disadvanatges

• * It is expensive - as is any IVF procedure
• * Only 30% take home baby rate per cycle - emotional rollercoaster
• * DNA analysis is less reliable than antenatal testing as only a single cell is sampled
• * Multiple pregnancy risks

Advnatges

• * Pregnancy less likely to be affected from outset
• * Less requiring TOP

Question 29

PGD may be considered when people have genetic conditions which they dont want to pass on to their children Eg X-linked conditions so may tets the gender of the embryo so only female embryo is implanted or conditions such as Huntingtons, CF etc What is the mutation in Huntingotns againand when is disease onset usually?

Answer 29

Mutations in the HTT gene cause Huntington disease. The HTT gene provides instructions for making a protein called huntingtin The HTT mutation involves a DNA segment nown as CAG trinucleotide repeat Normally less than 35 CAG repeats People with Huntington will usually have 40 and can have way more (above 60 in Juvenile Huntingtons) Onset of the condition is usually thirties-fifties