1. Differences from mendelian heredity Imprinting, dynamic mutations RNDr Z.Polívková Lecture No course: Heredity

2. Genomic imprinting
Mendelian principle: autosomal genes have the same expression if transmitted from father or mother
Imprinted genes: maternal and paternal alleles have different expression (activity) according to parental origin
Gene imprinting = epigenetic form of regulation of gene expression

3. Imprinted genes:
Monoallelic expression – expression of only one parental allele (parent-of-origin expression)
Imprinted genes - functionally haploid
Active allele – transcribed
Inactive allele – imprinted – nontranscribed – silent
Imprinting connected with DNA methylation and other changes of chromatin

4. Imprinted genes normally involved in embryonic growth, cell division
Abnormality of imprinting = human pathologies

5. Gynogenesis and androgenesis:
Examples of human pathologies :
Human triploidies:
Additional paternal set of chromosomes = partial mole = hyperplasia of trophoblast
Additional maternal set of chromosomes = hypotrophic placenta
Gynogenesis and androgenesis:
Ovarial teratoma – division of ovum without fertilization
Complete mole – division of only male pronucleus
(without maternal contribution)

6. ∑ : Role of the imprinted genes in early human embryogenesis:
paternally expressed genes → placental proliferation and invasivness
maternally expressed genes → development of embryo

7. Prader- Willi sy (PWS)
MR, short stature ,obesity, hypotonia, characteristic facies, small feet and hands, hypogonadism
Angelman sy (AS)
MR, absence of speech, seizures, jerky gait,
inappropriate laughter, dysmorphic features

8. PWS AS

9. UPD = both chromosomes 15 from one parent
PWS AS
deletion 15q11-13
on paternal chromosome on maternal chromosome
UPD (uniparental disomy)
maternal paternal
mutation maternal active allele
imprinting error
maternal imprint paternal imprint
On both chromosomes on both chromosomes
in PWS region in AS region
UPD = both chromosomes 15 from one parent

10. Deletion 15q11-13
Wysis

11. PWS AS PWS region – active paternal elleles
in proximal region of chromosome 15 – two groups of reciprocally imprinted genes
PWS region – active paternal elleles
AS region - active maternal allele(s)
loss of function of active alleles in PWS region (pat)
loss of function of active allele(s) in AS region (mat)
→ functional nullisomy

12. Imprinted genes on chromosome No 15 normal situation
pat mat }
SNRPN
PWS genes
ZNF127 }
AS gene
UBE3A
active paternal ellele
active maternal allele
„silent“= imprinted paternal allele
„silent“=imprinted maternal allele

13. Deletion in PWS and AS pat mat pat mat
Deletion of paternal active alleles in PWS
Deletion of maternal active alleles in AS
PWS AS

14. UPD - Uniparental disomy in PWS and AS
mat mat pat pat
Uniparental disomy
maternal in PWS
paternal in AS
PWS AS
UPD

15. Mutation in AS
mutated active maternal allele in AS AS

16. Imprinting error pat mat pat mat
maternal imprint of PWS genes on both chromosomes in PWS
paternal imprint of AS gene on both chromosomes in AS
PWS AS

17. Imprinting and Beckwith-Wiedeman syndrome
(EMG – exomphalos - macroglossia – gigantism)
growth abnormalities : macroglossia, gigantism, hemihypertrophy, visceromegaly, abdominal wall defects (omphalocele, umbilical hernia);
hypoglycemia in neonatal period, renal dysplasia, skeletal anomalies
predisposition to embryonal tumors (Wilms´ tumor,… )
Imprinted genes on 11p15:
IGF2 – expressed from paternal allele
H19 – expressed from maternal allele
p57 - expressed from maternal allele ?

18. Changes in BWS:
paternal duplication of 11p (2 x IGF2)
paternal UPD (2 x IGF2)
deletion or translocation of maternal active allele H19→ activation of maternal IGF2 allele („enhancer“ competition model for expression control of IGF2 and H19)
abnormal imprint = biallelic expression of IGF2
Pathogenesis of disease – increased dose of IGF2 (growth factor)
role of H19 and other genes = ?

19. pat mat pat mat 1. paternal duplication
IGF2
IGF2
IGF2
H19
H19
H19
IGF2
H19
normal situation BWS region on 11p15
1. paternal duplication
active and silent paternal alleles
active and silent maternal alleles

20. biallelic expression of IGF2
pat pat
pat mat
IGF2
IGF2
H19
3. del,, transl. of maternal allele H19, expression of paternal IGF2
2. paternal UPD
pat mat
IGF2
H19
biallelic expression of IGF2
4. imprinting error

21. Imprinting and cancer Genetic changes in cancer:
inherited or spontaneous mutations that are not corrected by repair mechanisms – irreversible changes in protooncogenes, tumour suppressor genes
Epigenetic changes: (do not affect the primary sequences of genome) – changes in methylation (imprinting) of these genes
→ activation of protooncogenes, silencing of tumour suppressor genes by aberrant methylation

22. But! in some persons, in some tissues – WT1 is imprinted
Wilms´ tumor (WT)
Locus 11p13 – connected with WAGR syndrome (Wilms´tumor, aniridia, urogenital anomaly, mental retardation)
WT1 gene- transcription factor (tumour suppressor) – biallelic expression in kidney and other organs
But! in some persons, in some tissues – WT1 is imprinted
= polymorphism of imprinting = predisposition to cancer

23. Locus 11p15 – connected with BWS
IGF2 – growth factor – monallelic expression in kidney
biallelic expression in Wilms tumor and other tumors (LOI = loss of imprinting) oncogen
H19 – oncofetal RNA – expressed in embryogenesis- function = ?, loss of H19 expression in WT with biallelic IGF2 expression
H19 - expressed in some tumors (lung, oesophageal, bladder carcinoma)
p57 – inhibitor of cyclin dependent kinase
reduced expression in WT and other tumors (lungs)

24. Imprinting connected with methylation
Imprinted allele = methylated
Imprinted protooncogenes – loss of imprinting (LOI) = activation of imprinted allele = biallelic expression = oncogenes
Imprinted tumor supressor genes = predisposition to tumors – loss of only one allele = loss of gene function

25. Knudson two-hit hypothesis of inactivation of tumor suppressor genes :
1st step: germ mutation or somatic mutation, or imprinting of one allele
2nd step: loss of heterozygosity (LOH) –by mutation in somatic cell

26. Polymorphism of imprinting of some genes in population:
tumour supressor genes WT1 (11p13), IGF2R (=IGF2 receptor on 6q26 - inactive in different tumours, role of IGF2R in extracellular IGF2 degradation)
In population – biallelic expression of these genes
in some people monoallelic expression (imprinted)
= predisposition to cancers
methylation = reversible process – possibility of therapy of tumours caused by aberrant methylation ???

27. Imprinting stage-, tissue-, species- and strain-specific
polymorphism of imprinting (inter-individual differences)
imprinted genes – function in positive or negative regulation of embryonal growth, cell division and differentiation
imprinted genes: receptors, growth factors, regulation proteins, transcription factors, proteins in splicing
= protooncogenes, tumour suppressor genes
role in embryonal development
biallelic expression in some stages of ontogenesis ?
imprinting is reversible

28. Imprinting connected with methylation, histone deacetylation and with remodelation of chromatine structure to inactive state

29. Abnormality of imprinting = growth abnormalities, abnormality of development, behaviour and cancers
Lack of expression (because of mutation, deletion, deficiency) of a gene usually expressed monoallelically from a specific parent
or overexpression (because of duplication, relaxation of imprinting or loss of control) of a normally monoallelically expressed gene

30. Uniparentalní disomy=inheritace of both homologs from the same parent
Mechanisms of UPD origin:
Loss of one chromosome from trisomic zygote
=„correction“ of initial trisomy (trisomy rescue)
gametic complementation = fertilization between nullisomic and disomic gametes (for the same chromosome)
duplication of the single chromosome in monosomic zygote
postfertilisation error – nondisjunction and reduplication of the single chromosome or mitotic recombination

31. Origin of uniparental disomy from trisomic zygote
trizomic zygote
loss of chromosome
loss of chromosome
uniparental disomy
normal

32. Origin of uniparental disomy by gamete complemetation
Fertilization of disomic and nullisomic gametes

33. duplication of chromosome
Origin of uniparental disomy by duplication of chromosome of monosomic zygote
monozomic zygote
duplication of chromosome

34. Origin of partial isodisomy by postfertilisation error
normal zygote-dizomic
nondisjunction and duplication
mitotic recombination

35. Evidence for UPD:
trisomy 15 in CVS, normal karyotype in fetal blood  child with PWS
increased parental age in UPD
transmission of hemphilia from father to son (zygote XXY and loss of maternal X chromosome
transmission od balanced translocation 22/22 in balanced form to healthy child (trisomic zygote and loss of single chromosome 22)
pericentric inversion was present on one homologue in mother and on both homologues in one offspring
maternal UPD in PWS, paternal UPD in AS
maternal UPD of chromosome No 7 in a patient with cystic fibrosis and growth retardation (first detection of UPD)

36. UPD  abnormal development if imprinted genes are present

37. Dynamic mutations

38. mothers od affected males = carriers
Fragile X syndrome
= X-linked mental retardation - 1:1500 of males
cytogenetic manifestation – fragile site Xq27.3 = FRAXA
Clinical signs: mental retardation, macroorchidism (large testicles), long face, large mandible, large everted ears
mothers od affected males = carriers
but: 30% of women = carriers - mentally retarded
20% fraX men mentally normal
deterioration of manifestation through generation = (Sherman paradox)

41. Unstable triplet repeats (CCG)n in FMR1 gene
in normal population copies
premutation (without MR) copies
full mutation (with MR) copies
DNA methylation (promoter region)  FMR1 is not transcribedabsence of proteinMR

42. Premutation=unstable
premutationfull mutation = only through mother carrier (in oogenesis or early in embryonal life)
man with premutation length of element is not increased in the next generation
length of amplification in correlation with cytogenetic expression
gene function ?? – protein expressed in tissues, higer levels in brain and testes

43. gradual origin of mutation = dynamic mutation
Dynamic mutations =
initial change of DNA produce another change
= expansion of triplet repeats

44. Main features od dynamic mutations:
homogenity – no more alleles
somatic variability- different numbers of copies in different tissues
effect of parental origin on manifestation
difference from mendelian principles (low penetrance)
no new mutations – gradual arise through premutation, familiar
expresivity depends on number of copies
anticipation=deterioration of clinical signs through generations

45. Two groups of dynamic mutations:
amplification in noncoding (nontranslated) region of gene (promoters, introns) loss of function
fra X(CCG/GGC), myotonic dystrophy (CTG), Friedreich ataxia (GAA)
amplification in exons (usually CAG repeats)  genes are transcribed abnormal protein
Huntington disease-HD- (abnormal protein huntingtin inactivates associated proteins), spinocerebellar ataxia type 1

46. Expansions depends on the sex of transmitting parent
Fra X, myotonic dystrophy – expansion if disease is inherited from mother
HD - expansion- if inherited from father
Postzygotic origin of amplification on chromosome of specific parental origin - determined in gametogenesis

47. Dynamic mutation Fragile X Myotonic dystrophy Huntington disease
FRAXA DM HD
heredity XD with reduced AD with different age AD with different age
penetrance of onset of onset
parental origin maternal=full mutation maternal –congenital forms paternal – early onset
mechanism abnormal DNA metylation different mRNA level abnormal protein
transcription of FMR toxic for neurons?
is stopped
amplification (CCG)n (CTG)n (CAG)n
normal number
abnormal number premutation
full mutation lower number=reduced penetrance
gene FMR IT15

48. http://dl1.cuni.cz/course/view.php?id=324 presentation
supplementary text to cytogenetics
Thompson &Thompson: Genetics in medicine, 7th ed.
Chapter 5: Principles of clinical cytogenetics: Parent-of-origin effects: Genomic imprinting
Chapter 7: Patterns of single gene i heritance: Imprinting in pedigrees, Unstable repeat expansions
Chapter 12: The molecular, biochemical and cellular basis of genetic disease: Diseases due to the expansion of unstable repeat sequences: Biochemical and cellular mechanisms