What is imprinting?
Differential expression of genes by parent of origin
May be cluster of genes controlled by IC (cis acting)
Most commonly methylation of cytosine at CpG nucleotides
What is the imprinted region associated with these disorders?
15q11-q13
Paternal 15q11-q13
-critical region unmethylated
- several genes preferentially expressed: MAGEL2 MKRN3 NDN PWRN1 SNURF-SNRPN snoRNA genes
- Deficiency or SNORD116 results in key characteristics of PWS
Maternal 15q11-q13
- CpG islands associated with paternally expressed genes are methylated
- methylation prevents TF binding and assembly of transcriptional machinery
- UBE3A expressed (brain)
Genetic mechanism of PWS
Loss of paternally expressed genes within 15q11-q13
De novo deletion 75-80% Maternal UPD 20-25% Imprinting defect (excluding del) 1% IC deletion 10-15%
Genetic mechanisms in AS
Loss of maternally expressed genes
- de novo deletion 70-75%
- paternal UPD 3-7%
- Imprinting defect (excluding del) 2-3%
- IC del 10-15%
- UBE3A mutation 10% (normal methylation pattern)
- Unknown 10%
PWS clinical phenotype
Hypotonia Failure to thrive neonatal Mild LD Hyperphagia and obesity later in dev Male hypogonadism Short stature Small hands and feet Behavioural problems
AS clinical phenotype
- Severe mental retardation
- Lack of speech
- Hyperactivity
- Inappropriate laughter
- Gait ataxia
- Seizures
- Microcephly
Mechanism of deletion (common break points)
Non-allergic homologous recombination between low copy repeat regions
UPD
Both Chr originate from same parent
Heterodisomy (MI) or isodisomy (MII)
Most commonly maternal due to non-dysjunction (maternal age effect)
Can result from early mitotic error, may result in somatic mosaicism
Isodisomy may result from gamete complementation
Low recurrence risk unless carry Robertsonian translocation
Other imprinting disorders
- Beckwith-Weidemann syndrome (11p15)
- Silver-Russell syndrome (11p15)
- UPD14 (14q32)
Describe bisulphite PCR
- Methylation pattern at the SNRPN locus within the PWS/AS critical region
- Treat DNA with sodium bisulphite
- Converts unmethylated C residues to uracil (deamination)
- PCR primers specific for methylated/unmethylated
- Common primer in unmethylated region acts as conversion control
- detects 99% cases of PWS and 80% AS
- cannot establish disease mechanism
maternal = methylated paternal = unmethylated
Heterodisomy
Pair of non-identical chromosomes inherited from one parent (meiosis I error)
Isodisomy
Single chromosome from one parent duplicated (meiosis II error)
Trisomy rescue
Conception trisomic, 1 homologue is lost by anaphase lag in early cell division
Robertsonian translocation
Carriers of Robertsonian translocations involving chromosome 15 at increased risk of having an affected child
Can lead to UPD via monosomy or trisomy rescue
Imprinting defects
- Failure to set the correct parental expression pattern
- Often de novo
- 10-15% IC microdeletion
- Maternal IC deletion inherited from mother = AS
- Paternal IC deletion inhertied from father = PWS
- Deletion up to 50% recurrence risk
Describe the PWS/AS IC
It contains two critical regions:
AS-SRO (shortest region of deletion overlap)
PWS-SRO
In what situation is an IC mutation mosaic in AS?
A third of AS patients with a primary epimutation show somatic mosaicism (occurred after fertilisation).
PWS pathogenesis
No individual protein coding gene linked to phenotype
Knockout mice for each gene may show some features of PWS
Loss of expression of SNORD116 snoRNA cluster thought to cause phenotype
A microdeletion in this cluster has been identified in a child with PWS
May regulate alternative splicing
AS pathogenesis
Caused by loss of UBE3A expression in the brain
UBE3A encodes a E3 ubiqutin ligase protein that targets certain proteins for degradation
Aberrant protein degradation interferes with correct neuronal development
Disadvantages of bisulphite PCR
- false positive due to snp under primer
- false positives due to incomplete DNA modification removed by control common primer that binds converted DNA
- no information about disease mechanism
- cannot detect mosaicism (false neg)
MS-MLPA
Allows for detection of methylation pattern and copy number changes
Can detect larger deletions and IC deletion
Requires 5 control samples
Methylation sensitive restriction enzyme added to half of the ligation products (Hha1)
Unmethylated DNA is digested and not amplified
Methylation ratio established by comparing probes in digested/undigested reactions
MS-MLPA disadvantages
-sensitive to PCR contaminants
-sensitive to DNA quality
-Cannot detect UBE3A mutations
Can generate false positives due to snp under probe site (confirm single probe del)
-Expensive
-Cannot differentiate between UPD and imprinting defect (no IC del)
