Rett syndrome

Question 1

Main characteristics of Rett

Answer 1

1. Normal early development 6-18 months
2. Loss of acquired fine and gross motor skills and communication
3. Stereotypic hand movements

Question 2

Genes associated with Rett

Answer 2

MECP2: methyl CpG binding protein (90-95%)

Clinically overlapping phenotype:
CDKL5
Netrin G1

Question 3

MECP2 gene

Answer 3

2 isoforms created by alternative splicing of exon 2 and the use of 2 alternative start codons:

• e1 (contains exon 1 but alternatively splices out exon 2, predominant in brain)
• e2 (exon 1 absent, predominant in fibroblasts/lymphocytes)

Question 4

MECP2 protein

Answer 4

Two functional domains:

1. methyl binding domain (binds specifically to DNA at methylated CpGs)
2. transcription repression domain (recruits corepressor proteins that repress transcription by deacetylation and condensation of chromatin)

E1 form most abundant

Question 5

MECP2 mutations

Answer 5

• Almost all de novo
• Parents can be germline mosaic
• Majority arise on paternal Chr.
• 200 known nucleotide changes
• 8 most common missense and nonsense =70% of mutations
• ~80% mutations in classic Rett in exons 3-4
• Exon4 and intron 2 contain highly repetitive regions, recombination between these can lead to del exons 3-4
• Deletion hotspot in c-terminal 9% of mutations
• Whole exon deletions described
• Mutations identfied in 80-95% females with classic Rett

Question 6

Complication of MECP2 mutations

Answer 6

Mutations in MECP2 do not constitute a diagnosis of Rett
Mutations also seen in individuals with Angelman like phenotype, non-syndromic X-linked MR, autism, neonatal encephalopathy

Question 7

Rett syndrome

Answer 7

Severe neurodevelopmental disorder mostly affecting females

Question 8

Rett in males

Answer 8

Classical Rett is infrequently observed

Neonatal onset encephalopathy with breathing abnormalities and early lethality more common

Deleterious mutations in 1 copy of the X result in severe neonatal encephalopathy and early lethality

May be mosaic or have a 47XXY karyotype (more classical symptoms)

Question 9

Other Rett phenotypes

Answer 9

Abnormal breathing patterns
Seizures
Autonomic nervous system dysfunction

Question 10

Factors influencing severity

Answer 10

Skewed X inactivation

Genotype-phenotype correlation (truncation X more severe)

Question 11

MECP2 duplications

Answer 11

Also results in neurodevelopmental impairment

• early onset infantile hypotonia
• delayed cognitive development
• ID
• epilepsy
• spasticity

Question 12

Mutation spectrum

Answer 12

Missense mutations predominantly found in methyl biding domain (MBD)

Nonsense mutations found in MBD and transcription repression domain (TRD)

Deletions mostly found in hotspot at c-terminal

Question 13

Male MECP2 mutations

Answer 13

Previously thought to be lethal

Three types of mutations observed in males:

• Classic Rett mutations
• Mutations inherited from mother not found in females with Rett
• Males with deletion of the whole gene and neighbouring genes resulting in a severe phenotype
• MECP2 duplication syndrome (most common mutation in males)

Question 14

MECP2 genotype/phenotype correlation

Answer 14

Studies have given conflicting results

Milder mutations:

• missense milder than truncating
• 3’ to TRD region
• Females with missense mutations milder than nonsense/fs
• deletions, if small and towards 3’ end

Skewed X-inactivation may modulate clinical severity

Question 15

Testing strategy

Answer 15

Bidirectional Sanger sequencing detects 85-90% mutations in classic Rett and 30-40% in atypical Rett

MLPA detects deletions/duplications

Question 16

Possible testing problems

Answer 16

reports of false negative results due to primer site polymorphisms

SNPcheck used regularly to look for new SNPs (assess freq info)

PCR primers tagged for use of universal sequencing primers

Analyse sequencing data using Mutation Surveyor

Question 17

Diagnostic referrals (male and female)

Answer 17

• accepted from GPs, paediatrics, clinical genetics
• parental samples requested if variant identified in child
• offer testing to other female relatives of proband
• test male relatives with possible Rett phenotype

Question 18

Referral types

Answer 18

Diagnostic
Parental testing following testing in proband
Prenatal

Question 19

Parental referrals

Answer 19

• only accepted from clinical genetics
• following identification of a variant pathgenic of VUS in proband
• determine recurrence risk
• risk of germline mosaicism

Question 20

Prenatal diagnosis

Answer 20

• if mother carries mutation risk is 50%

- test for MCC

Question 21

X inactivation detection

Answer 21

Females can show skewed X inactivation

If mother is found to have a mutation, analysis to determine degree of skewing may help in counselling

PCR of polymorphic CAG repeat in exon1 of the Androgen receptor

DNA subject to restriction digest HpaII and CfoI

Can detect ration of 75:25 or unilateral inactivation

may aid in determining the phenotype

Question 22

Alternative testing

Answer 22

CDKL5
Angelman
NTNG1
FOXG1
SLC9A6

Question 23

CDKL5

Answer 23

atypical Rett

group characterised by infantile spasms and jerks, early onset seizures

Question 24

Angelman syndrome

Answer 24

Present in early childhood and presents with global dev delay, speech and communication problems, stereotypical hand movement, autistic

Question 25

NTNG1

Answer 25

NetrinG1- dev of CNS

atypical Rett

Question 26

FOXG1

Answer 26

Congenital form presenting within first month

Question 27

SLC9A6

Answer 27

X-linked ID
microcephaly
epilepsy
ataxia
phenotype mimics Angelman

Question 28

Pathophysiology of Rett

Answer 28

Regression arises from altered neuronal function rather than neurodegeneration

Maintenance of an appropriate level of MECP2 essential for neuronal function (enhanced as well as reduced function results in impairment)

Brains have smaller and more densely packed neurons

Question 29

Impaired epigenetic transcriptional regulation

Answer 29

• MECP2 binds 2 alternatively methylated forms of DNA (methylated cytosine adjacent non G base and hydroxymethylcytosine)
• These methylation patterns accumulate postnatally during neuronal maturation
• This coincides with surge in MECP2 expression
• Binding of MECP2 may regulate transcription
• timing coincides with increased synaptogenesis
• mechanism may contribute to delayed onset of symptoms

Question 30

Altered chromatin architecture

Answer 30

MECP2 is able to compact chromatin and influence the higher-order structure of DNA

Impacts the ability of other nuclear proteins to interact with chromatin

e.g. chromatin remodelling protein ATRX, shown to lose normal localisation at heterochromatic foci in null mice (this correlates with disease onset)

Question 31

Neuronal circuit dysfunction

Answer 31

Third aspect in timing of Rett onset

Progressive molecular and nuclear changes in neurons impair neuronal function and culminate in altered circuit connectivity (may explain delay)