Specialist OSFA (genetics) Flashcards
What are the risks for a carrier of a balanced Robertsonian translocation?
- Infertility (male)
- Offspring with trisomy: (if chromosome 13 or 21 involved) - risk 10% if female carrier, 1% if male carrier - if isochromosome: risk is 100%
- Offspring with UPD: Trisomy or monosomy rescue could lead to UPD: 0.5-1% risk - if 14 or 15 involved this could lead to UPD 14mat (Temple syndrome) or UPD 15mat (Prader-Willi syndrome) - and UPD 14pat or UPD 15pat (Angelman syndrome)
What does standard analysis in constitutional cytogenetics work consist of?
- Minimum 2 cells analysed and cleared - if mosaic: minimum 1 cell from each cell line
- Minimum quality:
CVS/AF: 3 (2 if confirming aneuploidy, 4-5 if looking for small structural rearrangements)
Blood: 6
Microdeletion syndromes: 7
What are the cytogenetic reporting targets?
Prenatal: Rapid QF-PCR/FISH 3 working days; karyotype: 14 calendar days
Postnatal: Rapid aneuploidy: 3 working days; Karyotype: Urgent: 10 days; Routine: 28 days; Tissue: 28 days
Haematology: Rapid PCR/FISH: 3 working days; karyotype: Urgent: 14 days, Routine: 21 calendar days
What should be done in postnatal constitutional analysis if mosaicism is suspected?
In which cases should mosaicism be investigated?
Extended scoring or analysis protocols for G banding:
• A minimum of 30 cells
• Duplicate cultures
• More than one tissue type
FISH may be more suitable if probes are available
Referring clinicians should be made aware that it is not possible to reliably exclude mosaicism from any analysis and specifically those not
targeted for extended counts
Extended analysis should be considered in cases presenting with the following
reasons for referral:
• Ambiguous genitalia /indeterminate sex
• Clinical details suggestive of known autosomal mosaic syndromes
e.g. trisomy 8; + i(12)(p10); +dic(15)(q12)
• Clinical features suggestive of a specific aneuploidy syndrome, but which have a normal karyotype on standard analysis
• Diagnosed or suspected cases of sex chromosome aneuploidy, known to be associated with mosaicism e.g. Turner syndrome
• Follow-up of a prenatal diagnosis of a possible mosaicism of
clinical significance in an affected child after birth
• Karyotypically normal parents of more than one child with the
same, or a related chromosome abnormality
What should always be included in reports?
- Recipient
- Lab identification
- Patient and sample identification
- Result summary - Use correct nomenclature (ISCN/HGVS)
- Restate clinical question
- Description of findings in words
- Name of associated disease/syndrome/prognosis
- Interpretation: In context of relevant information (e.g. clinical features, family history, ethnic background, other investigations)
- Assessment of recurrence risk (use Bayes if possible)
- Further testing required
- Basis of test and limitations
- Authorised by
What are the minimum FISH analysis recommendations for constitutional genetics?
Metaphase: Minimum 5 cells analysed (2 of which are checked)
Interphase: Aneuploidy: 30 examined, microduplication/deletion: 15 examined
Which sources of information are available for cytogenetic analysis?
- Departmental SOPs
- Association for Clinical Genomic Science (ACGS) best practice guidelines
- Gardner and Sutherland
For which referrals can microarray be used as a front line test?
Learning difficulties Developmental delay Dysmorphic features Congenital abnormalities (If baby/child with features of trisomy then do QF-PCR first)
What are the requirements for excluding mosaicism in CVS and AFs?
Depending on the abnormality, a basic, moderate or extensive work-up is required. All of them require more than one culture to be investigated.
Abnormalities in single cells normally only require basic work-up (except autosomal trisomies).
For chromosomes that are known to be involved in UPD (7, 11, 14, 15), further studies
should be considered.
For level III mosaicism in a CVS, follow up amniocentesis or fetal blood
sampling along with detailed ultrasound assessment of fetal morphology should be
advised.
The report must include a statement, that the level of mosaicism is not necessarily reflect the proportion in the fetus.
Use Hsu guidelines.
For which chromosomes should we be concerned about UPD?
If there is apparent CPM for chromosome 7, 11, 14, 15 or marker chromosomes originating from these.
Mosaic chromosome 16:If UPD is excluded, there is still a significant risk of an adverse outcome.
Which referral reasons are there for CF testing?
Diagnostic (newborn screening, meconium ileus, chest infections, CBAVD/infertility/obstructive azoospermia, bronchiectitis, pancreatitis, classical CF)
Confirmation of CF
Relatives of CF patients (risk of having children with CF)
Partners (especially if accurate pregnancy risk required)
Gamete donors
Fetal echogenic bowel (test parents first for carrier status)
First cousins
Not recommended to test children under 16 for carrier status
When should reflex testing for the intron 8 polyT tract in the CFTR gene be carried out?
- All males with obstructive azoospermia (could be 5T homozygote)
- Patients with bronchiectasis or pancreatitis where 1 mutation has been found
- R117H mutation detected (can modify severity)
What can be detected on 1st trimester scan and what is the significance?
- Raised nuchal translucency (indicator of genetic testing if >3.5 mm) - can indicate trisomy 13, 18 or 21 or 45,X.
- Cystic hygroma - can indicate 45,X or Noonan syndrome
- Echogenic bowel - can indicate trisomy 21 or CF - important to know local CF positive echogenic bowel cases for calculation of residual risk
- Choroid plexus cysts - can indicate +18 or +21
- Atrioventricular septal defect (AVSD) (+21)
- Ventricular septal defect (VSD) (+13,+18,+21)
- Diaphragmatic hernia (+18,+21, i12p)
What can be detected on 2nd trimester scan and what is the significance?
- Cardiac abnormalities (21, 18, 13 and 45,X)
- Features of Patau (+13): Holoprosencephaly (forebrain fails to develop into two hemispheres), Bilateral cleft lip, Polydactyly (extra digits), Talipes/club foot
- Features of Down syndrome (+21): Duodenal atresia (absence or closure of first section of small intestine) – is seen as two tubes appearing on ultrasound scan (“double bubble”)
- Features of Edwards (+18): Unilateral cleft lip, Micrognathia, Fisted hand/clenched fist, Talipes/club foot
What sizes of bands are expected on a EcoRI/EagI Southern blot of a:
1) Normal female patient
2) Normal male patient
3) FRX premutation carrier female
4) FRX premutation male
5) FRX full mutation carrier female
6) FRX full mutation male
1) 2.8 (EcoRI+EagI) and 5.2 kb (EcoRI only)
2) 2.8 kb
3) 2.8 and 5.2 kb + >2.8 and >5.2 kb
4) >2.8 kb
5) 2.8 and 5.2 kb +»_space;5.2 kb or smear
6)»_space;5.2 kb or smear
What are the expansion ranges in Fragile X syndrome?
<46: Normal
46-58: Intermediate allele - normal phenotype, very rare expansion to premutation in 1 generation, to full mutation in 2 generations
59-200 and unmethylated: Premutation - can expand to full mutation in 1 generation, may be affected with FXTAS, females: FXPOI, children with FXS
>200 or methylated (no expression): Full mutation, males affected, females: 50-60% affected
Reporting FRX results:
1) Normal/intermediate allele male
2) Normal/intermediate allele female
3) Premutation male
4) Premutation female
5) Full mutation male
6) Full mutation female
1) FRX unlikely (rare point mutation/deletion), possible mosaicism - refer to clinical genetics if >50
2) FRX unlikely (rare point mutation/deletion), possible mosaicism - FXPOI/FXTAS excluded - refer to clinical genetics if >50
3) FRX unlikely (rare point mutation/deletion), possible mosaicism, possible tissue mosaicism
4) Carrier of FXS - clinical symptoms of FXTAS or FXPOI may be attributable to expansion, but could be due to something else
5) FXS due to full mutation confirmed - Males with a full mutation rarely have children, all daughters are expected to carry premutation - male offspring are normal
6) Patient is a carrier of FXS - Symptomatic females generally less affected than males - Risk for female transmitting full mutation is 50%
Genetic counselling to mother of patients and her family should be recommended.
What is the location of the PWS/AS critical region?
15q11.2q13.1
What are the possible mechanisms of PWS and their frequency?
Deletion: ~70-75%
Uniparental disomy: ~25-29%
Imprinting centre defect: <1%
UBE3A gene mutation: n/a
What are the possible mechanisms of AS and their frequency?
Deletion: ~68%
Uniparental disomy: ~7%
Imprinting centre defect: ~3%
UBE3A gene mutation: ~11%
Testing strategy for PWS/AS referrals?
1) Microarray
2) If microarray normal - MS-PCR/MS-MLPA to see if maternal/paternal methylation present
3) If microarray deletion in 15q11-q13 region - MS-PCR/MS-MLPA to see if maternal or paternal allele missing - request parental samples to see if balanced rearrangement in a parent to assess recurrence risk (but most often sporadic)
4) If abnormal MS-PCR - repeat and request parental samples for UPD studies - if UPD then unlikely recurrence - IC defect: Normally sporadic, but deletions can be inherited (silent if mat allele with pat del)
Testing strategy for DMD referrals?
1) Diagnostic:
MLPA (2 kits) to detect deletions and duplications in dystrophin gene (70% of patients)
Sequencing available on request to detect point mutations
If a single exon deletion or duplication is found confirm by multiplex PCR or sequencing
Frameshift model is correct in 90% of cases (frameshift/nonsense mutations cause DMD, in-frame mutations cause BMD - use frame checker)
2) Carrier testing:
Look for familial mutation by appropriate method (MLPA/sequencing) or if no mutations has been identified do linkage
3) Prenatal testing:
Available to female carriers and females with affected sons (risk of mosaicism)
First do fetal sexing (invasive/non-invasive) - if male do invasive test (MLPA/sequencing) or NIPD
How can a female be affected by classical DMD?
1) Non-random X-inactivation due to X-autosome translocation unmasking a pathogenic dystrophin variant or unexplained non-random X-inactivation (in skeletal muscle tissue)
2) Uniparental disomy for X with pathogenic dystrophin variant
3) Turner syndrome with pathogenic dystrophin variant
4) Homozygosity or compound heterozygosity for pathogenic dystrophin variant
What are the clinical features of DMD and BMD?
DMD:
Progressive muscular weakness (mainly proximal) - wheelchair dependency before 13 years
Waddling gait, difficulty climbing
Developmental delay and learning difficulties (30-50%)
Dilated cardiomyopathy
Mean age of death at 25 years (main causes cardiomyopathy and respiratory insufficiency)
Gower’s sign
Serum creatine kinase (CK or SCK) levels raised
BMD: Maybe late learning to walk Muscle weakness around 11 years Muscle cramps Lose ability to walk at 40-50 years No learning difficulties Survive to middle age and beyond (mean age of death mid-40s, main cause: cardiomyopathy) Serum creatine kinase levels >5x normal
Carrier females:
5-10% of carriers have cramps or mild muscle weakness
Usually not noticed until in 30s
Carriers of BMD less affected than DMD carriers
20% of carriers have evidence of dilated cardiomyopathy – should have 5 yearly echo/ECG
Serum creatine kinase (CK) levels 2-10x normal in 50% of cases
