20.01.22 Origins of UPD

Question 1

What is UPD?

Answer 1

• Two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent
• Requires the simultaneous occurrence of two very rare events:
  1) nondisjunction of the same chromosome in gametes that join
  2) trisomy followed by chromosome loss (very rare)

Question 2

Clinical consequences for UPD

Answer 2

1) Imprinted genes on chromosome
- Parent of origin exp required
- Normally have monoallelic exp of these genes
- UPD causes both alleles either to be silenced or both to be expressed
- causing phenotype
2) Creates HOM variants (‘isozygosity’)
3) UPD resulting from somatic recombination can cause LOH or LOI (loss of imprinting)
- Somatically acquired UPD in cancer
- Pre-existing diver mutation converted to HOM which gives cell clonal advantage
4) UPD in conjunction with mosaicism for an abnormal cell line
- placental/fetal trisomy mosaicism due to trisomy rescue

Question 3

Syndromes associated with UPD

Answer 3

• UPD(6)pat - transient neonatal diabetes
• UPD(7)mat - Russell-Silver syndrome
• UPD(11p15.5)pat - Beckwith-Wiedemann syndrome
• UPD(14)mat - short stature and precocious puberty with mild delay
• UPD(14)pat - distinct skeletal dysplasia with asphyxiating thorax
• UPD(15)mat - Prader-Willi syndrome
• UPD(15)pat - Angelman syndrome

Question 4

Name the two different types of UPD

Answer 4

1) Uniparental isodisomy (UPID)
- two identical copies of one parental homologue
- Likley Meiosis II nondisjunction or mitotic error
2) Uniparental heterodisomy (UPHD)
- Both homologues from one parent (but one of each)
- Likely meiosis I nondisjunction

Question 5

What is segmental UPD?

Answer 5

• UPD for only part of the chromosome
• Occurs by recombination during mitosis (NOT meiosis)
• Not to be confused with when you see UPD where the chromosome is partly UPID and partly UPHD (this is due to recombination during meiosis)

Question 6

Name the three forms of UPD?

Answer 6

1) UPD for the entire chromosome complement
2) UPD for a complete chromosome
3) Segmental UPD

Question 7

UPD for the entire chromosome complement - how does this arise?

Answer 7

1) Complete hydatidiform mole
- PAT UPD for entire diploid complement
- Caused by endoreduplication of a single 23,X sperm
2) Benign cystic ovarian teratoma
- MAT UPD for entire diploid complement
- Arises in germ cell due to failed meiotic cell division
3) Triploidy (partial hydatidiform mole)
- Can have MAT (digynic triploidy) or PAT (diandric triploidy) origin
- Most likely PAT
4) Can get mosaic UPD/triploidy
- Very rare
- Caused by 3 mechansisms:

A) Normal fertilization, then replication without division occurs of male pronucleus leading to normal biparental cell line and haploid PAT cell line, then get haploid rescue and a mosaic UPD PAT cell line

B) Egg fertilized by 2 sperm, cells divide into biparental diploid and haploid PAT cells, then get haploid rescue and a mosaic UPD PAT cell line

C) Fertilization of an empty egg, get haploid rescue and UPD PAT (at the same time also have a normal fertilization of a second egg with a normal diploid complement)

Question 8

UPD for a complete chromosome - Name the 4 formation mechanisms by which this arises

Answer 8

1) Trisomy rescue
2) Gamete complementation
3) Monosomic rescue
4) Mitotic error

Question 9

1) Trisomy rescue

Answer 9

• Most common mechanism
• Meiotic nondisjunction in one parent results in a disomic gamete
• This is then fertilized with a normal haploid gamete = trisomic conception
• Rescue occurs as one homologue is lost at a very early postzygotic stage causing UPD
• Meiosis I nondisjunction = UPHD
• Meiosis II nondisjunction = UPID
• Post-zygotic correction so most are mosaic (trisomy can remain only in the placenta (CPM) or affecting the fetus)
• Most common is MAT meiosis I nondisjunction (mat UPHD)

Question 10

2) Gamete complementation

Answer 10

• Very very rare
• Meiotic nondisjunction in both parents results in a disomic gamete from one parent and a nullisomic gamete for the same chromosome from the other parent
• If these gametes form embryo then get a diploid zygote with UPD for that chromosome

Question 11

3) Monosomic rescue

Answer 11

• Rarer than trisomy rescue
• Meiotic nondisjunction in one parent = nullisomic gamete
• If fertilized with normal haploid gamete = monosomic conception
• Get rescue event of remaining homologue = UPID
• As most nullsomic gametes occur due to MAT meiosis I nondisjunction, most UPD cases will be PAT UPID

Question 12

4) Mitotic error

Answer 12

• Conception is normal
• Then get mitotic error (introduces monosomy or trisomy)
• This is then rescued causing UPID

Question 13

Segmental UPD

Answer 13

• UPD of only one section of a chromosome
• Arises due to recombination
• Segmental isodisomy is assumed to be formed postzygotically by a mitotic exchange between non-sister chromatids
• Therefore each daughter cell will show UPID (one MAT and the other PAT) creating mosaicism
• Can get loss of one of these UPID cell lines (e.g. mat UPID for 11p15.5 is lethal in BWS)
• alternatively can also arise by mitotic exchange from pat and mat chromatids, followed by trisomy rescue (chr that is lost took part in the recombination)
• Segmental UPD can be mosaic (if recombination occurs after formation of inner cell mass)

Question 14

What increases the chance of UPD?

Answer 14

• Carriers of structural rearrangements can get meiotic malsegragation causing UPD
  1) Robertsonian translocations (non-identical homologues) - most commonly causes UPD by trisomy rescue after non-disjunction
  2) translocations with a risk of 3:1 nondisjunction
• can then get monosomy rescue
• OR can get isochromosome formation if it involves an acrocentric chromosome (derived from a single chromosome through a sister chromatid exchange or duplication event) - they have two arms that are identical to each other
• With no family history, recurrence risk of sporadic UPD is very low

Question 15

When do we need test for UPD?

Answer 15

• The patient’s features are consistent with a UPD-related disorder
• familial chromosomal rearrangement (numerical or structural) involving imprinting-related chromosomes
• There is a rare recessive disorder or unexplainable parent-child transmission

Question 16

Methods to detect UPD?

Answer 16

• most are methylation based assay
• Therefore be careful testing CVS (methylation pattern not set at CVS stage and placental meth may not reflect fetal meth pattern)
  1) Bisulphite modification methods
• MS-PCR
• Bisulphite restriction analysis and PCR
• MS melting curve analysis
• Pyrosequencing
  2) Microsatellite analysis
  3) MS-MLPA
  4) Southern blotting
  5) SNP array
  6) Whole exome/genome sequencing
  7) Cytogenetic analysis

Question 17

What is bisulphite modification?

Answer 17

• DNA treated with sodium bisulphite (SB) converts unmethylation cytosines to Uracils
• If cytosine is methylation (i.e. in a CpG island) then it remains unchanged
• This helps to distinguish methylation from unmethylated DNA

Question 18

1) Bisulphite modification methods
- MS-PCR

Answer 18

• DNA is treated with SB - so parental chromosomes will now differ in DNA seq
• PCR with two sets of primers (one for meth DNA seq and one for unmeth DNA seq)
• Gel electrophoresis - meth and unmeth DNA products differ in size
• Normal result = 2 bands
• UPD = 1 band

Pros - does not require parental bloods

Cons - Doesn’t detect mechanism causing loss of one allele ( can’t tell the difference between UPD, deletion, IC deletion) and can’t detect segmental UPD

Question 19

1) Bisulphite modification methods
- Bisulphite restriction analysis and PCR

Answer 19

• Treat DNA with SB
• Produces differential restriction enzyme sites for the methylated and unmethylated alleles
• Either creation of new restriction sites, or retention of methylation-dependent restriction sites
• PCR with standard primers (both meth and unmeth seq are amplified)
• Treat with restriction enzymes (cut unmodified meth DNA only)
• Gel electrophoresis (meth and unmeth products are different sizes)
• Normal result = 2 bands
• UPD = 1 band

Pros - does not require parental bloods

Cons - Doesn’t detect mechanism causing loss of one allele ( can’t tell the difference between UPD, deletion, IC deletion) and can’t detect segmental UPD

Question 20

1) Bisulphite modification methods
- MS melting curve analysis

Answer 20

• DNA treated with SB
• PCR with fluorescently-tagged primers specific for either meth and unmeth seq
• The PCR products cooled to 40oC, then heated at 0.05oC per second to 95oC
• Fluorescence in monitored
• Unmeth PCR product = lower CG dinucleotide content = lower melting temp

Normal = two peaks (one for meth and one for unmeth product)

UPD = only 1 peak

Pros - does not require parental bloods

Cons - Doesn’t detect mechanism causing loss of one allele ( can’t tell the difference between UPD, deletion, IC deletion)

Question 21

1) Bisulphite modification methods
- Pyrosequencing

Answer 21

• DNA is treated with SB
• Methylation differences can be detected and quantified by analysing the bisulphite-induced C/T differences at CpG sites
• Commonly only look st several CpG sites within specific imprinting regions (targeted analysis)

Question 22

2) Microsatellite analysis

Answer 22

• PCR to amplify DNA repeat sequences that are specific for a particular chromosome/region.
• Short tandem repeats (STRs) - stable, highly polymorphic, short repetitive DNA sequences that are comprised of repeated elements
• Use QF-PCR methodology
• Parental bloods are also analysed to determine the inheritance
• Normal = 2 het peaks of the same height, or 1 hom peak
• Example abnormal results:

Mother – repeats 150 and 160

Father – homozygous 166 repeats

1) 150 and 160 = mat UPD heterodisomy
2) 150 homozygous = mat UPD isodisomy
3) 150 and 166 = normal disomy, biparental inheritance
4) 166 homozygous = paternal UPD but not informative as to if heterodisomy or isodisomy

Pros - Can detect deletions, UPD and segmental UPD.

Cons - Can distinguish hetero and isodisomy, requires parental bloods, and can get lots of uninformative markers

Question 23

3) MS-MLPA

Answer 23

• detects dosage and meth status
• DNA is denatured, MS-MLPA probes added and hybridised for 16hrs, and bound probes ligated.
• The probe-DNA mix is then split into two tubes -
  1) processed as standard MLPA for dosage (undigested)
  2) incubated with a methylation-sensitive enzyme: Probes ligated to unmethylated DNA will be digested by the enzyme and therefore not amplified. Methylated DNA will not be digested and the ligated probe will generate a signal
• Pros - MLPA is simple and robust. Does not require parental bloods. Can distinguish UPD/IC defect from a deletion. Only small amount of DNA required. Can also detect duplications

Cons: Can’t distinguish UPD from an IC defect

Question 24

4) Southern blotting using methylation specific restriction enzymes

Answer 24

• This is a gene specific method
• Uses methylation sensitive restriction enzymes to allow detection of UPD
• MS restriction enzymes will not digest DNA with a methyl group attached to the C nucleotide of the CpG islands
• Uses two restriction enzymes which generates:
  1) a larger fragment for meth DNA (cut by the standard enzyme only)
  2) a smaller fragment for unmeth DNA (cut by the standard and MS enzyme)
• Normal = 2 bands.
• UPD = only 1 band

Pros - Does not require parental bloods

Cons - Low throughput, poor sensitivity, requires large quantities of DNA. Can’t distinguish UPD/deletion/IC defect

Question 25

5) SNP array

Answer 25

• SNP arrays are capable of detecting methylation differences across several CpG sites.
• UPD can be detected by using homozygosity profiling with a SNP array (although homozygosity will flag regions of isodisomy, but not heterodisomy)
• This method therefore relies on testing large numbers of SNPs to maximise how many are informative.
• One method using SNP arrays: Probe extension assay
  1) array contains probes attached to a bead which are complementary for the adjacent DNA sequences of a variety of SNPs
  2) Nucleotides that are complementary to the SNP nucleotide are added by a polymerase, and these nucleotides are labelled either green or red
  3) The fluorescence from each bead is measured and the levels of red, green or both measured
  4) These levels of fluorescence indicate an AA, BB or AB genotype, and can then be compared to parental samples

Pros - Can detect deletions, UPD, segmental UPD, isodisomy (if the SNPs are informative). Genome-wide analysis.

Cons: Expensive compared to targeted methods, and can’t detect heterodisomy

Question 26

6) Whole exome/genome sequencing

Answer 26

• Long regions of homozygosity can be identified through WES/WGS and resolved to UPD events
• SNP data also facilitates detection of mosaic UPD by detecting minor allele fractions with systematic departures from diploid genotypes (that are not associated with copy number change).

BI can detect enrichment of genotypes in trio data that are only compatible with UPD, allowing discrimination between biparentally inherited homozygosity and isodisomy, and also detection of heterodisomy

• WES/WGS is especially powerful in detecting UPD which results in homozygosity for AR disease.
• Parental samples are essential for interpretation of results.

Question 27

7) Cytogenetic analysis

Answer 27

• Not commonly used
• Can indicate UPD but not diagnose it (molecular follow up is required)
• i.e. Abnormal karyotypic findings like chromosomal rearrangements may be indicative of UPD; this may include (Robertsonian) translocations, complementary isochromosomes, heteromorphisms, deletions and duplications, sSMC presence, mosaic triploidy, and trisomy