18.01.22 Origin of UPD

Question 1

What is UPD and how frequent is it?

Answer 1

Two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent.

Frequency ~1 in 3,500 (Robinson et al., 2000). Orphanet definition of a rare disease is 1 in ≥2000.

Question 2

When do abnormalities arise from UPD?

Answer 2

UPD for some chromosomes does not exert any adverse effect on an individual

Abnormalities arise when:

1) Genes within the UPD region are subject to genomic imprinting
2) Homozygosity for recessive mutations: two copies of a recessive mutation from a parental carrier
3) UPD resulting from a somatic mutation can cause LOH or LOI (loss of imprinting)
4) UPD in conjunction with mosaicism for a chromosomally abnormal cell line e.g. placental or sometimes foetal mosaicism due to trisomy rescue

Question 3

What is genomic imprinting, what effect on these genes can be caused by UPD?

Answer 3

Differential expression dependent on parent of origin

Leads to monoallelic expression of either the maternal and paternal allele of a diploid locus (‘parent of origin effect’)

UPD for an imprinted region results in two active, expressed parental alleles or two silent, repressed parental alleles, depending on the contributing parent

UPD results in abnormal dosage of the imprinted gene products

Question 4

Give examples of how UPD resulting from a somatic recombination can cause loss of heterozygosity (LOH) or loss of imprinting

Answer 4

Somatically acquired UPD (aUPD) in cancer e.g. retinoblastoma, Wilms tumour.

Pre-existing driver mutation is converted to homozygosity, providing an additional clonal advantage

Somatic recombination leading to mosaic segmental UPD is probably common to all individuals and is more likely to occur with increasing cell divisions. Results in late onset conditions [Robinson 2000].

Question 5

Give examples of three conditions associated with UPD

Answer 5

1. [upd(6)pat] - transient neonatal diabetes

2, [upd(7)mat] - Russell-Silver syndrome

3. [upd(11p15.5)pat] - Beckwith-Wiedemann syndrome
4. [upd(14)mat] - short stature and precocious puberty with mild delay
5. [upd(14)pat] - distinct skeletal dysplasia with asphyxiating thorax
6. [upd(15)mat] - Prader-Willi syndrome
7. [upd(15)pat] - Angelman syndrome

Question 6

What are the two different forms of UPD?

Answer 6

1. Uniparental isodisomy (UPID): The presence of two identical copies of one parental homologue. (Likely Meiosis II nondisjunction or mitotic error)
2. Uniparental heterodisomy (UPHD): The presence of both homologues from the transmitting parent. (Likely Meiosis I nondisjunction).

Question 7

Why might UPD along the length of an involved chromosome pair can be UPID for certain loci and UPHD for others?

Answer 7

Recombination in meiosis I e.g. in the distal long arm, followed by meiosis I non- disjunction could lead to a disomic gamete isodisomic for the distal long arm and heterodisomic for the proximal long arm. Or vice versa if the nondisjunction was in meiosis II.

Don’t confuse this with segmental UPD, which is UPD for only part of the chromosome and occurs by recombination during mitosis rather than meiosis.

Question 8

What are the three categories of UPD?

Answer 8

1. UPD for the entire chromosome complement
2. UPD for a complete chromosome.
3. UPD for a Segmental UPD (11% of all known UPD cases)

Question 9

Give examples of disease associated with UPD of the entire chromosome complement.

Answer 9

1. Complete hydatidiform mole: UPD for the entire diploid complement, exclusively paternal in origin (UPDpat). Predominantly 46,XX karyotype due to endoreduplication of a single 23,X sperm [Gardner and Sutherland].
2. Benign cystic ovarian teratoma: Maternal UPD (UPDmat) for the entire diploid complement. Arises in a germ cell due to failure of a premeiotic or meiotic cell division [Gardner and Sutherland].
3. Triploidy (partial hydatidiform mole): Extra set of chromosomes can have either a maternal origin (digynic triploidy) or paternal origin (diandric triploidy) [Devriendt 2005], usually paternal [Gardner and Sutherland].

Can get mosaic UPD/triploidy [Liehr 2014].

Mosaicism for UPD of the entire diploid complement (maternal or paternal) with a biparentally-inherited karyotype is a rare finding in living newborns.

Question 10

Mosaicism for UPD of the entire diploid complement (maternal or paternal) with a biparentally-inherited karyotype is a rare finding in living newborns, resulting from one in three mechanisms, describe these.

Answer 10

Major mechanisms of formation of genome wide uniparental disomy (UPD) mosaicism:

(A) fertilisation of an egg by one sperm, and irregular reduplication and division of the male pronucleus followed by cell division and diploidisation of the male haploid genome resulting in a cell line with a normal biparentally inherited karyotype and a genome wide uniparental cell line;

(B) fertilisation of an egg by two sperms followed by cell division and diploidisation of the haploid male genome; or

(C) fertilisation of an empty egg and subsequent diploidisation of the male haplotype as well as of a normal egg with a haploid chromosomal complement by a normal sperm followed by amalgamation of both.

Question 11

How can UPD for a complete chromosome arise?

Answer 11

1) Trisomy rescue (most common)
2) Gamete complementation (very rare)
3) Monosomy rescue
4) Mitotic error

Question 12

Describe trisomy rescue.

Answer 12

Meiotic nondisjunction in one parent results in a disomic gamete.

Fertilisation of this disomic gamete with a normal haploid gamete results in a trisomic conceptus.

‘Rescue’ through loss of one homologue (perhaps through anaphase lag) at a very early postzygotic stage (possibly even in the zygote), results in UPD in 1/3 cases.

Question 13

What is the result of trisomy rescue at meiosis I and meiosis II.

Answer 13

Meiosis I nondisjunction uniparental heterodisomy (UPHD)

Meiosis II nondisjunction uniparental isodisomy (UPID)

Because the loss occurs postzygotically, mosacism in such conceptuses is often observed, with the trisomic cell line often confined to the placenta. Not uncommon to observe 100% trisomy in placental tissues with complete absence of trisomy in the fetal tissue [Robinson 2000]. UPD phenotypes may therefore be complicated by the effects of compromised placental function and/or fetal trisomy mosaicism.

Question 14

What is the proposed reason why the conversion of trisomic tissue to disomy is rare during embryogenesis?

Answer 14

it may be that conditions present in the first few mitoses are conductive to correction, and this is soon lost post inner cell mass formation. i.e. occurs at a very early post-zygotic stage.

Question 15

When do most non-disjunction events occur? What type of UPD does this result in?

Answer 15

Most aneuploidy results from maternal meiosis I nondisjuncton, therefore most UPDs arising from a trisomic rescue will be maternal heterodisomy.

Question 16

What is the chance that carriers of a Robertsonian translocation that has arisen through trisomy rescue will have UPD?

Answer 16

50%

Question 17

Describe gamete complementation.

Answer 17

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.

A chance meeting of these gametes will result in a diploid zygote with UPD for that chromosome.

Although very unlikely that a meiotic error would happen coincidently in both parents, at least three cases have been recognised in the literature to result from this mechanism. In these cases, at least one parent carried a translocation for the chromosome involved [Shaffer 2003].

Hard to differentiate whether UPD has occurred by gamete complementation, or by trisomic rescue very early in development and no trace of the original trisomic cell.

Question 18

Describe monosomic rescue.

Answer 18

Meiotic nondisjunction in one of the parents results in a nullisomic gamete.

Fertilisation of this nullisomic gamete with a normal haploid (monosomic) gamete results in a monosomic conceptus.

‘Rescue’ of the remaining homologue results in UPD (specifically isodisomy).

Monosomic rescue is rare compared to trisomy rescue because there is a smaller window of opportunity for a lethal monosomic conceptus to rescue (as compared to most trisomic conceptuses).

Question 19

What are the mechanisms of monosomic rescue?

Answer 19

Mechanisms of monosomic rescue [Shaffer]:

mitotic nondisjunction

duplication

mitotic misdivision leading to isochromosome formation

Question 20

Why are most UPDs arising from monosomic rescue paternal isodisomy?

Answer 20

Because most nullisomic gametes are the result of maternal meiosis I nondisjunction, most UPDs arising from a monosomic rescue will be paternal isodisomy.

Question 21

Describe how mitotic error can lead to UPD.

Answer 21

Conceptus and subsequent cell line are initially normal.

Mitotic error leading to either trisomy or monosomy is then ‘rescued’ using the same mechanisms as outlined previously.

Leads to UPID in both monosomy and trisomy rescue.

Somatic, mitotic recombination results in segmental UPD

Question 22

What is segmental UPD?

Answer 22

UPD for part of one chromosome pair with biparental inheritance for the rest of the pair – due to recombination e.g. Mosaicism for partial paternal isodisomy of 11p15.5 seen in 10-20% of cases with Beckwith Wiedemann syndrome [Kotzot 2008].

Question 23

How is segmental UPID thought to be formed?

Answer 23

Segmental isodisomy is assumed to be formed postzygotically by a mitotic exchange between non-sister chromatids. According to this mechanism the UPD should be present in each daughter cell, maternal UPID in one and paternal UPID in the other one

The only exception would be the loss of the opposite UPD either because of occurrence in a very early mitosis and subsequent splitting in embryonic and extraembryonic tissues or, as assumed in Beckwith-Wiedemann syndrome, because of lethality of cells with maternal isodisomy.

In an alternative mechanism, trisomy rescue may take place as described previously, but the mitotic exchange between maternal and paternal chromatids takes place prior to the rescue, and the chromosome that took part in recombination and was from the disomic gamete is lost.

If the recombination occurs in a cell after the formation of the inner cell mass (which gives rise to the embryo) there will be mosaic segmental UPD. As with other UPD conditions, segmental UPD may result in conversion of a recessive mutation to homozygosity.

Question 24

What factors predispose to UPD?

Answer 24

Structural rearrangements - Robertsonian or reciprocal trasnlocation that are at risk of 3:1 segregation. ]

Chromosomes involved

Question 25

Why might structural rearrangements predispose to UPD?

Answer 25

UPD could also occur from correction of monosomy originating from 3:1 segregation through replication of the homologue (as in monosomy rescue) or in the case of an acrocentric chromosome, isochromosome formation.

Isochromosomes, by definition, are abnormal chromosomes derived from a single chromosome through a sister chromatid exchange or duplication event; whereas, Robertsonian translocations are true translocations between two (different) homologous chromosomes [Shaffer 2003].

Thus, isochromosomes are comprised of two arms that are genetically identical (homozygous), while Robertsonian translocations are comprised of nonidentical arms and would show heterozygosity for most markers tested.

UPD could occur after postzygotic correction of an interchange trisomy.

Question 26

What is the most common mechanism of UPD in Robertsonian translocation carriers?

Answer 26

In the case of a parent with a Robertsonian translocation, the most common mechanism leading to UPD is trisomy rescue after non-disjunction.

Question 27

What is compensatory UPD?

Answer 27

Ring chromosomes and other structural abnormalities may be corrected by ‘compensatory UPD’ – structurally abnormal homologue is replaced with a copy of the normal homologue resulting in UPD [Robinson 2000].

Question 28

What is contraposed UPD?

Answer 28

Very rare form of UPD is complementary isochromosomes, e.g. isochromosome i(2p) from one parent and isochromosome i(2q) from other parent. This results in ‘contraposed UPD’.

Question 29

When is UPD testing advised?

Answer 29

The patient’s features are consistent with a UPD-related disorder (i.e. imprinting syndrome).

There is a familial chromosomal rearrangement (numerical or structural) involving imprinting-related chromosomes.

There is a rare recessive disorder or unexplainable parent-child transmission e.g. an apparently homozygous mutation in an affected individual, where one parent is shown not to be a carrier, and the de novo mutation rate is low.

Question 30

Why should methylation-based tests be used with caution in CVS samples?

Answer 30

Methylation pattern may not be set at CV sampling stage

Placental methylation may not reflect fetal methylation.

Question 31

How should test be designed to account for possibly UPD mosaicism?

Answer 31

Appropriate tissue selection

Techniques based on 1 band (uniparental) vs 2 bands (biparental) would not detect mosaicism for UPD/BPD.

Microsatellite analysis may require calculation of maternal to paternal allele ratios (rather than simply presence of absence of maternal or paternal peaks).

MS-MLPA ratios and ratios of mat/pat nucleotide differences (SNP-array/sequencing) may not pass the typical ‘threshold’ used for non-mosaic disorders. A consistent skewing from normal could indicate mosaic UPD.

Question 32

What is the effect of treating DNA with sodium bisulphite?

Answer 32

Treating DNA with sodium bisulphite converts unmethylated cytosine nucleotides to uracil. Methylated cytosines (e.g. those in CpG islands of the methylated parental chromosome) remain unchanged.

These differences can be used to distinguish methylated and unmethylated DNA

Question 33

Which technique use a stage of sodium bisulphite treatment?

Answer 33

MS-PCR
Bisulphite restriction analysis and PCR
MS melt curve analysis
Pyrosequencing

Question 34

What other methods exist for UPD detection that do not require DNA to be pre-treated with sodium bisulphite?

Answer 34

Microsatellite analysis
Southern blotting
SNP array
WES/WGS
Cytogenetics

Question 35

How can microsatellite analysis be used for UPD detection?

Answer 35

Uses PCR to amplify DNA repeat sequences that are specific for a particular chromosome/region.

The sequences that are used are usually short tandem repeats (STRs). STRs are stable, short repetitive DNA sequences that are comprised of repeated elements. They are highly polymorphic and vary in length between individuals depending on the number of repeats.

Fluorescently tagged primers for microsatellites within the chromosome of interest are amplified and quantified using QF-PCR (with products sized on capillary electrophoresis).

Parental bloods are also analysed to determine the inheritance of the microsatellites in the patient.

Question 36

What are the advantages and disadvantages of using microsatellite analysis for UPD detection?

Answer 36

Advantages: Can potentially detect deletions, UPD and segmental UPD. Can distinguish hetero and isodisomy.

Disadvantages: Requires parental bloods, markers may not be informative.

Question 37

What are the advantages and disadvantages of using MS-MLPA for UPD detection?

Answer 37

Advantages: MLPA is simple and robust. Does not require parental bloods. Can distinguish UPD/IC defect from a deletion by comparing the results from the MLPA relating to copy number to that relating to methylation status. Only small amount of DNA required in comparison to Southern Blotting. Can also detect duplications.

Disadvantages: This type of MLPA cannot distinguish UPD from an imprinting centre defect.

Question 38

How can Southern Blotting be used to detect UPD?

Answer 38

This method used to be common practice for PWS/AS detection; now methylation sensitive PCR or MLPA are commonly used.

This is a gene specific method, which 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.

Using two restriction enzymes will therefore result in two different size fragments, a larger fragment for methylated DNA (cut by the standard enzyme only) and a smaller fragment for unmethylated DNA (cut by the standard and MS enzyme).

Normal individuals will show 2 bands. Those with UPD will show only 1 band – which band is present will depend whether the mat or pat chromosome has been inherited.

Question 39

What are the advantages and disadvantages of using Southern blotting for UPD detection?

Answer 39

Advantages: Does not require parental bloods.

Disadvantages: Low throughput, poor sensitivity, requires large quantities of DNA.

This method cannot distinguish UPD/deletion or imprinting centre (IC) defect – additional testing maybe required to rule out a deletion.

Question 40

How can SNP arrays be used to detect UPD?

Answer 40

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, if parents are heterozygotes). If parents are homozygotes, SNP array testing cannot distinguish between isodisomy and heterodisomy. This method therefore relies on testing large numbers of SNPs to maximise how many are informative.

Question 41

What are the advantages and disadvantages of using SNP arrays for UPD detection?

Answer 41

Advantages: Can detect deletions, UPD, segmental UPD, hetero/isodisomy (if the SNPs are informative). Genome-wide rather than targeted to a single imprinting-related region.

Disadvantages: Requires parental bloods to detect UPD, expensive compared to targeted methods.

Question 42

How can WES/WGS be used to UPD detection?

Answer 42

Increasingly trio whole/clinical exome sequencing and whole genome sequencing are being applied to diagnose rare diseases (e.g. DDD and 100KGP).

Long regions of homozygosity can be identified through WES/WGS and resolved to UPD events [King 2014].

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).

Using bioinformatic searching of trio sequencing data, enrichment of genotypes that are only compatible with uniparental inheritance can be detected. This enables 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.

Unlike SNP array which requires prior knowledge of the SNP locations, WES/WGS is a true exome/genome-wide approach. Parental samples are essential for interpretation of results.

Another method involves sequencing a small portion of the genome (evenly distributed SNPs and small tandem repeats) termed Selected Target Regions (SeTRs)

Question 43

When might UPD be indicated from cytogenetic analysis?

Answer 43

Indications of UPD can be provided by molecular methods, but a comprehensive case characterization should include cytogenetic analyses as well.

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