Genomics OSFA 2019 Flashcards
Describe culturing of chorionic villi samples/amniotic fluid samples
Which steps do we take to protect from errors?
Villi enzymatically digested (mixing several villi reduces the risk of false positives/negatives due to CPM)
Blood stained amniotic fluids: Centrifugate through gradient to separate amniocytes and RBCs
2-3 cultures are set up in different incubators, so that if a culture is lost due to contamination/incubator fault, back-ups are available and culturing artefacts can be verified/ruled out from a second culture
Infection:
Culture medium contains antibiotics
Change of pH can be a sign of infection (bacteria (pink->yellow), fungi (pink->red), not so much yeasts)
Cloudiness (generally bacterial)
Granular, shimmering appearance (=bacteria), particles, sometimes chains (=yeast), mycelia filaments (fungi) can be seen in x100 phase contrast
When single or multiple abnormal cells are found in CVS or AF cultures, more cells can be analysed. Guidelines describe basic, moderate and extensive workup with a number of cells from 1 or more separate cultures to exclude culturing artefacts.
QF-PCR trisomy result from a CVS shows only biallelic markers - what to do next and why?
Use back-up markers to try and get a tri-allelic result
A bi-allelic result may result from a mitotic event in the placenta and represent confined placental mosaicism which is not representative of the fetus
Confirm the ID with maternal sample (if not available: Confirm result by FISH)
QF-PCR on a CVS or amniotoc fluid shows 1 marker with skewed ratio between 2 peaks or a 3rd allele with lower peak height. What can be done and what may this signify?
Repeat marker at 53 and 58 degrees: Marker may resolve to normal ratio
Check if marker is flanked by normal markers
Run parental sample if available
A SNP under primer binding site may have reduced the primer efficiency producing a peak with lower height
If inconclusive ratio or three alleles persists this could be due to microsatellite instability or submicroscopic duplication
What to do if the QF-PCR result for a prenatal sample shows maternal cell contamination?
In cases without abnormal scan results: Do karyotyping on cultured cells
In cases with abnormal scan results: Do microarray on cultured cells
Culturing may result in amniocytes/chorionic vili digest to outgrow the leucocytes and thereby overcome the MCC
Contact the clinician to inform them of the delay to the result
A QF-PCR result on a CVS shows evidence of trisomy 21 in 3/5 markers (1 biallelic and 2 triallelic). What to do next?
What if it was an amniotic fluid?
CVS: Use the maternal sample for sample ID check to ensure there has been no sample mix-up in the laboratory
Amniotic fluid: Extract from a banked aliquot or a pour-off from the culture and repeat the test to confirm sample ID
Report as a preliminary result and activate long-term cultures for karyotyping to confirm the mode of trisomy (regular/Robertsonian or other translocation)
If translocation involved request parental samples to determine if inherited or de novo to determine recurrence risk
An XY QF-PCR result on a CVS shows evidence of monosomy X. What to do next?
What if it was an amniotic fluid?
Repeat the XY QF-PCR
CVS: Confirm the ID with maternal sample
Amniotic fluid: Extract from a banked aliquot or a pour-off from the culture and repeat the test to confirm sample ID
A XXY result was seen by QF-PCR on an amniotic fluid. The sample was referred due to choroid plexus cysts and duodenal atresia.
What do you do next?
Repeat the QF-PCR on new extraction to confirm the result and sample ID
Look at microarray result to see if this confirms the finding (if this was not an abscan, findings from QF-PCR that does not explain the referral reason could still be followed up by microarray).
Could do karyotyping if suspect mosaicism and would like an estimate of level.
Word the report very carefully to highlight that this is an incidental finding, which does not explain the scan findings
Explain the significance of Klinefelter disease
A CVS sample arrives with referral due to raised NT and raised risk from combined test (1 in 5). Gestation by scan is 13+0 weeks.
What would you do when triaging this sample?
Clarify with clinician if the raised NT is >3.5 mm to determine if this is an abnormal scan finding and needs microarray.
If NT <3.5 mm:
Do QF-PCR on CVS digest (if there is enough material for digest)
Bank a long-term culture
Extract DNA from maternal sample for MCC studies
If NT >3.5 mm:
All of the above and also initiate microarray analysis and XY QF-PCR
Explain QF-PCR
Quantitatively amplify regions of DNA that include tetranucleotide microsatellite markers, which are polymorphic in population. (Tetranucleotide markers are less prone to polymerase slippage than smaller repeats, therefore smaller stutter peaks). Not too many cycles, as want to capture exponential phase. Analysed using fluorescent capillary electrophoresis. Five markers on each chromosome 13, 18 and 21 are analysed. AMEL marker is not polymorphic, but always 103 bp on X and 109 bp on Y). Back-up kits with 4 extra markers are available if first kit only has uninformative markers or are all bi-allelic.
Ratio of 0.8-1.4 (1.5 further apart) is normal. Ratio of <0.65 or >1.8 is trisomic.
If only 1 allele is seen, this could be due to drop-out of other allele of patient is homozygous at this locus (uninformative).
Skewing of AMEL markers may indicate sex chromosome aneuploidy or contamination (e.g. MCC).
If 1 marker only gives a result that conflicts with other markers: Repeat single marker. Can lower annealing temperature (if polymorphism is present under primer binding site). Can do back-up markers if surrounding markers are uninformative. Tri-allelic result in 1 marker only may be true result from somatic instability (MSI) or submicroscopic duplication (SMD).
Run parental samples: Can see if SMD inherited.
Describe the antenatal screening programme (NHS Fetal Anomaly Screening Programme (FASP))
First trimester combined screen (/NT screen): Maternal serum markers (free ß-human chorionic
gonadotrophin (hCG) and pregnancy-associated plasmaprotein-A (PAPP-A)), nuchal translucency and maternal age combined into risk. Offer CVS if high risk (>1:150).
If women book late they can have the Quadruple test: Maternal serum markers (alpha-fetoprotein (AFP),
free ß-human chorionic gonadotrophin (hCG), unconjugated estriol (uE3) and inhibin-A) and maternal age are combined into risk. Offer amniocentesis if high risk (>1:150).
Testing done by QF-PCR, which can detect trisomy 21, 18 and 13, triploidy and monosomy X. They are screened for because they are relatively common and can result in abnormal scan findings, confer a risk of spontaneous abortion or stillbirth and may lead to live born children with mental and physical problems. The test may detect other abnormalities (other sex chromosome abnormalities, mosaic forms or unbalanced translocations).
If QF-PCR is positive: Do karyotyping.
If QF-PCR is negative: Do microarray
What to put in report for a prenatal QF-PCR result showing a trisomy?
What if it was bi-allelic only also in back-up markers?
Provisional result Indicates trisomy Sample identity confirmed Fetal sex No evidence of other trisomies CPM can occur (if CVS, 2% of viable pregnancies) Confirmation by karyotype will follow List features of trisomy
If bi-allelic markers only word more carefully to indicate that could be CPM
When can a CVS be taken?
When can an amniotic fluid be taken?
When can maternal sample be taken for NIPT?
CVS: Usually between 11-13 weeks (must not be performed before 10 weeks)
AF: Usually after 15 weeks (must not be performed before 14 weeks)
NIPT: cffDNA can be detected from 7 weeks, but blood sample not taken until 10 weeks
In which circumstances can NIPT be offered?
In which circumstances can CVS/AF be offered?
NIPT: Following high risk screening for T21, 13, 18
If abnormalities seen on ultrasound scan then CVS or AF may be better as may need microarray
CVS/AF: NIPT high risk Screening high risk (NIPT declined) Previous pregnancy aneuploidy Known familial translocation (e.g. Robertsonian)
What causes Fragile X syndrome?
> 99% of cases: expansion of CGG repeat in 5’-UTR of the FMR1 gene on Xq27.3 leading to abnormal hypermethylation of the promoter region and absence of expression of the RNA-binding protein, FRMP.
The expansion causes a fragile site, which can be seen in approximately 2-40% of blood cells.
Normal: up to 45 repeats
Intermediate: 46 - 58 repeats
Premutation: 59 - approximately 200 repeats, unmethylated
Full mutation: Greater than approximately 200 repeats, methylated
Expansion from a premutation to a full mutation is
invariably on transmission through female meiosis; paternal transmissions can be unstable but
never result in a full mutation.
A small minority of Fragile X cases are due to point mutations or deletions in the coding sequence; these do not exhibit fragile site expression or hypermethylation.
What are the clinical features of fragile X syndrome and other FMR1-related disorders?
Fragile X syndrome: Moderate to severe intellectual and social impairment together with syndromic features
including large ears and head, long face and macroorchidism
Females with full expansion: 50% experience symptoms with varying severity (possibly due to varying X-inactivation in affected tissue)
Affects 1 in 4000-9000 males and 1 in 7000-15000 females
Premutation alleles cause two quite different
disease phenotypes at lower penetrance: primary ovarian insufficiency (POI) in females and
Fragile X-associated tremor/ataxia syndrome (FXTAS)
POI: Amenorrhea in women before the age of 40 for four or more months in association with FSH levels in the menopausal range. Approximately 20% of premutation carriers develop POI compared to 1% in the general
population.
FXTAS is a late-onset neurodegenerative disorder found predominantly in male carriers of FMR1 premutations. Females can also be affected, but less severely. Accumulation of expanded CGG repeat mRNA contributes to intranuclear inclusions. Penetrance is age-related.
Premutation/full mutation mosaicism is not uncommon. This presents a dilemma when a premutation is detected in a patient referred for Fragile X
syndrome: is the premutation itself the cause of symptoms, is it a coincidental finding or could
the patient be mosaic for a premutation and a full mutation.
What is the testing strategy for a patient referred for fragile X syndrome/FMR1-related disease?
Array-CGH is more likely to detect an abnormality of clinical significance, but may lead to increase in reporting time and incidental findings.
Referrals for POI/POF may be tested by conventional karyotype to rule out sex chromosome abnormalities.
UKGTN criteria: Moderate to severe intellectual disability, and does not have profound psychomotor handicap
Prenatal testing: Available if confirmed family history and referred through CG. Offered to all women with an allele of 55 CGG repeats or greater. Test sex first (ffDNA/QF-PCR). Test for MCC, do Amplidex (very sensitive to MCC) and linkage concurrently)
Methylation not always present in DNA from chorionic villi
For female fetuses, there is an additional risk of Turner Syndrome: the incidence of mosaic Turner syndrome is
reported to be up to 5% in female fetuses of mothers carrying the full mutation
Diagnostic testing: Test with flanking PCR. If uninformative female, no result or expansion seen, then do Amplidex or TP-PCR. Expansions can be somatically unstable and smaller peaks representing an expansion can be seen using Amplidex.
How to report:
Premutation allele found in male unaffected relative of Fragile X patient?
Premutation allele found in female relative of Fragile X patient?
Premutation allele found in symptomatic male/female?
Normal result for diagnostic case?
All:
Size
Carrier screening of those relatives at risk
Recommend referral to CG
Premutation allele found in male:
Has risk of developing FXTAS (males >=50 years: 39%, >=80 years: 75%)
Daughters will be obligate carriers with a risk of having children with Fragile X syndrome
Premutation allele found in female:
Premutations are likely to show instability and further expansion in future generations, with a risk of expansion to a full mutation
Prenatal diagnosis can be offered
Has increased risk of developing Fragile X-associated primary ovarian insufficiency (FXPOI)
Premutation allele found in symptomatic:
Does not support diagnosis of FRX
But it cannot be excluded that large mutations may lead to cognitive impairment/autism
Small possibility of mosaicism for a full mutation in other tissues
Normal result in diagnostic case: FXS unlikely, but cannot rule out rare point mutation or deletion or undetected mosaicism
Which types of acute lymphoblastic leukaemia are there?
Precursor B-cell neoplasms (B-ALL), most common, primarily seen in early childhood. FAB categories L1, L2 and L3 (old). WHO categories (2016): Precursor B Lymphoblastic Leukemia/Lymphoma not otherwise specified (NOS) and with recurrent genetic abnormalities:
Hypodiploidy
Hyperdiploidy
t(9;22)(q34;q11.2)[BCR-ABL1] - only one that is more common in adults than children (25% of adult ALL, 2-4% of childhood ALL)
t(v;11q23)[MLL rearranged] - KMT2A (MLL) rearranged is most common in infants - may occur in utero
t(12;21)(p13;q22)[ETV6-RUNX1]
t(1;19)(q23;p13.3)[TCF3-PBX1]
t(5;14)(q31;q32)[IL3-IGH]
intrachromosomal amplification of chromosome 21 (iAMP21)
BCR-ABL1-like ALL
Precursor T-cell neoplasms (T-ALL)
What are the clinical features of acute lymphoblastic leukaemia?
Bone marrow failure, resulting in anemia, neutropenia, thrombocytopenia (low count of red blood cells, neutrophils and platelets (myeloid lineage))
Leukocytosis or leukopenia (raised or low count of lymphocytes)
Bone pain or arthralgia (joint pain)
Splenomegaly and lymphadenopathy (abnormal size of lymph nodes)
Cure rate is 80% in children, 40% in adults.
What are the prognosis of the recurrent genetic findings in acute lymphoblastic leukaemia?
Favourable:
Hyperdiploidy
t(12;21) ETV6-RUNX1
Intermediate:
Normal karyotype
t(1;19) PBX1-TCF3
Poor: Hypodiploidy t(9;22) BCR-ABL1 Most rearrangements involving 11q23 (KMT2A/MLL) t(4;11) AFF1-KMT2A t(17;19) HLF-TCF3 t(8;14) IGH@-MYC iAMP21 >4 abnormalities (complex karyotype)
Describe a possible testing strategy for acute B-lymphoblastic leukaemia presentation case?
Do direct cultures, as ALL cells apoptose easily, do urgently
FISH for common rearrangements with poor prognosis: KMT2A rearrangements (t(4;11) has very poor prognosis) - (break-apart probe) - especially for <1 year old t(9;22) BCR-ABL1 (fusion probe) - especially for adults
FISH for common rearrangements with favourable prognosis:
t(12;21) ETV6-RUNX1 (fusion probe) - can also detect iAMP21 and hyperdiploidy
G band analysis: If normal analyse 20 cells (95% confidence interval from Hook’s table), if abnormal analyse 10 cells
Normal karyotypes may reflect cytogenetically cryptic abnormalities or failed proliferation of blast cells in vitro. SNP array can be used to refine genetic risk category (detect cryptic abnormalities not seen on G-band)
Describe a possible testing strategy for acute lymphoblastic leukaemia post-treatment case?
Monthly monitoring
G-band or FISH if there is no molecular marker
RQ-PCR can be done for:
Favourable:
t(12;21) ETV6-RUNX1
Poor:
t(9;22) BCR-ABL1 (determine breakpoints first with RT-PCR)
What are the clinical features of HNPCC/Lynch syndrome?
Hereditary non-polyposis colon cancer, autosomal dominant
Familial colon or endometrial cancer (rarely also other malignancies)
Mutations in MSH2 and MLH1 account for over 90% of cases
Approximately 80% lifetime risk of colon cancer (average 61 years) – women: 20-60% lifetime risk of endometrial cancer (average 46-62 years)
Mismatch repair deficiency syndrome (MMR-D): Caused by biallelic mutations in MMR genes (additional findings: early onset haematological malignancy, café-au-lait spots)
What is the testing strategy for HNPCC/Lynch syndrome?
Immunohistochemistry (ICH) and/or microsatellite instability (MSI) are cheap and quick ways of pre-screening patients to select only cases with loss of MMR protein function for genetic testing. MSI is more sensitive but less specific than IHC.
ICH: staining for MMR proteins: 2nd hit in tumour DNA resulting in loss of protein can be seen -loss of MLH1 may appear as loss of both MLH1 and PMS2; loss of MSH2 may appear as loss of both MSH2 and MSH6 - missense mutations leading to normal expression of protein, but loss of function may result in false negative IHC
Loss of MLH1 may be due to (inherited) MLH1 mutation (=Lynch) or (sporadic) methylation of the MLH1 promoter (not Lynch) - therefore fo methylation analysis
Loss of MSH2 may be due to EPCAM deletions (predictive testing to at-risk relatives can be offered)
MSI: Detects loss of the function of the MMR proteins - contraction and/or expansion of repeat motifs occur in tumour DNA where a 2nd hit has occurred - seen as hedgehog peaks appearing as 2 hedgehogs - Low instability (1/5 markers) has been linked with high risk of endometrial cancer and MSH6 mutations – IHC analysis can be offered
MSI also occur in 10-15% of sporadic colon cancers.
Do germline testing if 2/5 markers unstable.
Loss of MLH1 or MSH2 activity can be further investigated for presence of p.Val600Glu in BRAF and/or MLH1 hypermethylation studies, which, if positive, indicate the tumour is likely sporadic
If not likely sporadic the patient is then tested for a germline mutation by sequencing of the MMR genes
What causes HNPCC/Lynch syndrome?
Almost all cases: Inheritance of a heterozygous germline mutation in MLH1, MSH2, MSH6 or PMS2,
followed by secondary somatic loss of the remaining copy (therefore autosomal dominant inherited predisposition)
Rare biallelic mutations have been reported in each of the 4 MMR genes that leads to a
more severe phenotype (constitutional MMR eficiency; CMMR-D).
Null mutations that abolish protein translation (frameshift, nonsense and splicing mutations), and missense mutations in the ATP-binding, DNA-binding and dimerization domains of MLH1, MSH2, MSH6 and PMS2 have been described.
What is the pLI score and how can it be used?
the probability that a given gene falls into the Haploinsufficient category
Genes with high pLI scores (pLI ≥ 0.9) are extremely LoF intolerant, whereby genes with low pLI scores (pLI ≤ 0.1) are LoF tolerant.
Name some genetic causes of developmental delay/intellectual disability.
o Common viable trisomies: Down syndrome, Edwards syndrome, Patau syndrome
Mosaic trisomy 8 or mosaic trisomy 9
Sex chromosome abnormalities
Emanuel syndrome (+der(22)t(11;22)(q23;q11) from 3:1 tertiary segregation of balanced t(11;22) from either parent)
Pallister Killian syndrome (very rare, mosaic tetrasomy 12p, (+i(12p)))
Cat-eye syndrome (Extra isodicentric 22 – inv dup(22)(q11.2) – very small (smaller than 21), frequently bisatellited)
+i(15)(q11) proximal region of 15q, two centromeres, bi-satellited, idic(15) = inv dup(15)
Micro-deletion and -duplication syndromes (Wolf-Hirschhorn (4p), cri-du-chat (5p), Williams (7), Miller-Dieker (17p), Smith-Magenis (17p)
Prader-Willi/Angelman syndrome
Fragile X syndrome
Rett syndrome
What is the testing strategy for referrals for developmental delay/intellectual disability?
• ?trisomy or sex chromosome aneuploidy: Test by QF-PCR first (for trisomy and/or XY) – then do array-CGH if QF-PCR negative (for urgent babies set up QF-PCR, array and bank culture)
If ?mosaicism: Do chromosome analysis as can detect mosaicism >10% if 30 cells examined
Extremely urgent: 24 hour FISH protocol
Parental follow-up may be required to further characterise a CNV or determine recurrence risk - can be chromosome analysis, FISH or array PvP
Extra tests required of Prader-Willi/Angelman, Fragile X or Rett syndrome
Describe synchronised cell culturing, harvesting and G-banding
Incubate in medium containing mitogen to stimulate lymphocyte division
Cell cycle: G1 (growth), S (DNA replication), G2 (growth) and mitosis: Prophase, Metaphase, Anaphase, Telophase, Cytokinesis
Thymidine added: Arrests cells in S phase, because inhibits conversion of cytosine. Gathers pool of cells in S phase.
Deoxycytidine added: releases block - cells then enter mitosis at same time
Colcemid: Depolymerises microtubules, preventing cells from progressing from metaphase. Chromosome condensation continues until harvesting
Hypotonic solution creates osmotic gradient and cells swell up (red blood cells burst)
Fixative denatures proteins and stop metabolic processes
Slides are made and aged in oven
Trypsin partially digests proteins in less condensed regions (actively transcribed genes)
Giemsa/Leishman stain is used to dye remaining proteins
What are the minimum requirements for postnatal chromosome analysis?
Clear every pair of homologues at least twice
All ?sex chromosome abnormalities: examine 30 cells (excludes 10% of mosaicism (Hook’s table))
All ?mosaicism: examine 60 cells (excludes 5% of mosaicism (Hook’s table))
For most postnatal referral reasons a minimum banding score of 6 is needed
Banding score 6: At least 3 criteria must be seen: 5q31.2 distinct, 8p21.2 visible, 2 dark bands on 11pter, 22q13.2 distinct
What are the possible gametes from a cross over in an inverted region at meiosis in:
a) Paracentric inversions?
b) Pericentric inversions?
a)
1 normal chromosome - viable
1 inversion chromosome – viable (all genes present)
1 acentric chromosome (lost in mitosis) – non-viable
1 dicentric chromosome (may break due to 2 active centromeres pulling different directions) – non-viable
b)
1 normal chromosome - viable
1 inversion chromosome – viable (all genes present)
1 chromosome with deletion of p-terminal part and duplication of q-terminal part
1 chromosome with deletion of q-terminal part and duplication of p-terminal part
Which has the highest risk of affected viable offspring:
An insertion carrier
or
A carrier of a balanced reciprocal translocation
Unbalanced translocation gametes: Have both loss and gain, therefore lower risk of viable unbalance.
E.g. 46,XX,ins(8;10)(q21;q21q22) Possible gametes: 8 + 10 - normal der(8) + der(10) - balanced 8 + der(10) - extra part of 8 der(8) + 10 - missing part of 8
Describe microarray technical process
Patient and reference samples are labelled with fluorescent dyes in different colours by random prime labelling and mixed together
Combined patient and reference sample is hybridised to slides with attached probes that cover selected areas across the whole genome
Competitive hybridisation ensures approximately equal amounts of patient and control sample to hybridise to each probe – unless a gain or loss of chromosome material is present in the patient, in which case the ratio will be skewed
The slide is scanned - the localisation of the probes on the slide can be mapped to probe location in the genome
Oxford Gene Technology (OGT) CytoSure constitutional arrays have 60,000 probes per slide with high probe density across genes important in developmental delay
What are the QC metrics for microarray analysis?
DLR spread: calculates the probe-to-probe log ratio noise of an array, and is an important QC metric – the value should be <0.3
Red signal to noise ratio and green signal to noise ratio: is calculated by dividing the signal intensity by the background noise (from negative control spots) - an excellent value for signal to noise would be above 100, between 100 and 30 is good but below 30 is poor
Troubleshooting:
Black holes can form in the middle of the slide if a bubble has been present
DNA can be run on a gel to check for fragmentation
What can you use to interpret microarray results?
Categories: Pathogenic, Likely pathogenic, Uncertain significance, Likely benign, Benign
Size (deletions >200kb or duplications > 1Mb are likely pathogenic, if de novo)
Gene content in relation to phenotype
Overlap with recurrent deletions/duplications
Position
Inherited/de novo
Literature
Databases:
Database for Genetic Variants (DGV) - unaffected controls
DECIPHER: Patients with developmental delay, has phenotypical data
DDG2P: Curated list of genes from the DDD study
Gene content (investigate closer using OMIM and GeneReviews)
HI score: Predictive score for how well haploinsufficiency is tolerated - lower values (e.g. 0-10%) indicate a gene is more likely to exhibit haploinsufficiency, higher values (e.g. 90-100%) indicate a gene is more likely to NOT exhibit haploinsufficiency
What are the counselling issues when performing microarray?
CNVs of variable expressivity/incomplete penetrance makes counselling complex, as the expected phenotype for the proband and other family members cannot be predicted (e.g. DiGeorge/22q11 deletion: A mildly affected parent may not have been identified)
CNV of uncertain significance: The patient must be informed that these are a common finding, and that the implication is unknown. Parental samples may aid interpretation.
Incidental findings: E.g. predisposition to cancer may be discovered.
What are the clinical features of Prader-Willi syndrome?
Mild intellectual disability
Hypotonia in infancy
Short stature
Hypogonadism (small penis, undescended testes, delayed puberty)
Hyperphagia and obesity in later childhood
Facial features: Almond-shaped eyes, narrowing of forehead at temples, narrow nose bridge, thin upper lip and downturned mouth
Which disease does the following abnormalities lead to?:
1) Maternal 15q11q13 imprinting centre defect on the maternal allele?
2) Paternal 15q11q13 imprinting centre defect on the paternal allele?
3) Maternal 15q11q13 imprinting centre defect on the paternal allele?
4) Paternal 15q11q13 imprinting centre defect on the maternal allele?
1) Angelman syndrome
2) Prader-Willi syndrome
3) Silent - if passed on by father will lead to PWS
4) Silent - if passed on by mother will lead to AS
What are the clinical features of Angelman syndrome?
Microcephaly Severe intellectual disability Lack of speech Hyperactivity Happy demeanour, inappropriate laughter Gait ataxia (abnormal, uncoordinated movements; unsteady, staggering walk) Seizures
How can UPD occur?
Nondisjunction in meiosis or mitosis causing disomic and nullisomic cells
After fertilisation, trisomic rescue can result in loss of the chromosome from the normal gamete leading to UPD
Fertilisation of a nullisomic gamete can result in monosomy rescue (duplication of the normal chromosome)
Heterodisomy: Results of meiosis I nondisjunction as homologous chromosomes did not separate
Isodisomy: Result of meiosis II or mitotic nondisjunction, as sister chromatids did not separate - or monosomy rescue
How could PWS/AS be inherited?
One parent could have a balanced translocation involving 15q11q13
E.g. carrier of Robertsonian translocation involving chromosome 15 - these are at increased risk of monosomy or trisomy 15, and therefore monosomic or trisomic rescue, which can lead to PWS or AS in the offspring.
What do we need to know in order to use de novo criteria in variant interpretation?
We need to know if there is a good phenotype/genotype match
E.g. classical clinical presentation of Cornelia de Lange (dev. del. and ID with facial gestalt, hirsutism, upper-limb reduction defects, growth retardation and microcephaly): PS2 or PM6 can be used.
Dev. del. only: PS2 or PM6 canNOT be used.
We also need to know if parental relationships have been confirmed (through trio testing or markers) - note that non-paternity could be identified
If confirmed for both parents: PS2 can be used (if phenotype highly specific for gene)
If only 1 or no parental relationships confirmed: PM6 can be used (if genotype/phenotype match)
PS2 can be downgraded to PS2_Moderate if phenotype consistent with gene, but not highly specific (maybe specific to subset of genes, e.g. early infantile epileptic encephalopathy)
It can be downgraded to PS2_Supporting if not highly specific and high genetic heterogeneity (e.g. non-syndromic intellectual disability)
Points system can be used per de novo occurrence - e.g. highly specific phenotype for gene, confirmed parents (2 points), 2 other unrelated patients assumed de novo: 1 point for each = 4 points => use as very strong
Phenotype highly specific for gene: 2 points
Phenotype consistent with gene but not highly specific: 1 point
Phenotype consistent with gene but not highly specific and high genetic heterogeneity: 0.5 points
Phenotype not consistent with gene: 0 points
Divide points by 2 if only assumed de novo
Supporting: 0.5
Moderate: 1
Strong: 2
Very strong: 4
What do we need to know in order to use the phenotype highly specific for a disease criterion in variant interpretation?
In order to use PP4 it is essential that (a) all the known genes associated with the disorder have been analysed and (b) variants in these known genes explain the majority of cases with that clinical diagnosis.
PP4 might be used as a strong piece of evidence if drug enzyme or muscle biopsy analysis that is specifically characteristic of a specific genetic cause of a disorder and would in the absence of genetic confirmation be considered a diagnostic finding
(Evidence from enzymatic assays performed on patient tissue are at gene level and not variant level. Therefore PS3 should not be used, but it could support phenotype specificity, therefore support PP4 at strong level).
How may a patient’s biochemical test results be used for variant interpretation?
Evidence from enzymatic assays performed on patient tissue are at gene level and not variant level. Therefore PS3 should not be used, but it could support phenotype specificity, therefore support PP4 at strong level.
What should be considered when using the null variant criterion in variant interpretation?
There is potential overlap in usage of
PVS1_Moderate and PM4 (protein length changing variant). To prevent double-counting of this evidence type, PM4 should not be applied for any variant in which PVS1, at any strength level, is also applied.
Nonsense and frameshift variants: NMD is not predicted to occur if the premature termination codon occurs in the 3’ most exon or within the 3’-most 50 nucleotides of the penultimate exon. When NMD does not occur, determine if the truncated/altered region is critical to protein function (experimental evidence, e.g. pathogenic variants downstream or assessing tolerance of the exon to LoF variants or length of missing region). Removing >10% of the protein product is more likely to have a loss of function effect (PVS1_Strong).
Canonical ±1,2 splice variants: PP3 (in silico splicing prediction) criterion should not be used to avoid double counting the same predictive evidence. Assess effect of variant by searching for cryptic splice sites, that may reconstitute in-frame splicing. Determine if predicted outcome is in-frame or out-of-frame (is skipped exon number divisible by 3).
LoF should be a disease mechanism for PVS1 to apply. It should only be applied if LoF variants make up at least 10% of the reported pathogenic variants in the gene and a minimum of 3 LoF variants have been classified as pathogenic without using the PVS1 rule. LoF may not have been described if it is lethal.
Low (<10%) haploinsufficiency (HI) score or high (>0.9) probability of LoF intolerance (pLI) suggests significantly lower than expected rate of LoF.
Which tools can be used to support the criterion for missense variants in genes with low rate of benign missense variation?
Missense z-score on gnomAD: If Z>3 then there is a general missense intolerance in the gene - check DECIPHER for constraint in specific region, where variant is located. In DECIPHER open triangles are VUS/benign and golden filled triangles are pathogenic/likely pathogenic. Red/orange/golden bar across top signifies regions with missense intolerance (low values). Grey is not significant. Green less intolerant.
Make sure missense variation is a mechanism of disease, e.g. look in HGMD to see if missense variants are commonly associated with the disease.
Which mechanisms may lead to an autosomal dominant inheritance pattern?
Haploinsufficiency
Gain of function
Dominant negative
