Epigenetic contribution to birth defects. David Amor 20 th June 2011

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1 Epigenetic contribution to birth defects David Amor 20 th June 2011

2 Genomic imprinting Genomic imprinting is the biological process whereby a gene or genomic domain is biochemically marked with information about its parental origin. Genomic imprints may be covalent (DNA methylation) or non-covalent (DNA-protein and DNA-RNA interactions, genomic localization in nuclear space), and are maintained throughout the cell cycle.

3 Genomic Imprinting Parental imprints are established during gametogenesis as homologous DNA passes uniquely through sperm or egg, and are maintained into adulthood Genomic imprints have the significant functional consequence of stifling gene expression from one of the parental alleles, resulting in unbalanced gene expression between homologous alleles. As a result of imprinting, there is biased allelic expression that favours expression from one parental locus over the other. Imprinting may not be 100% efficient, and there may be leaky expression from the silenced allele.

4 Imprinted genes Role of imprinting in normal development Cell-cycle regulation- altered growth and development Organogenesis : neurogenesis genes over-represented Cell growth and/or maintenance Regulation of biological and cellular processes Chromatin architecture Errors in imprinting have been implicated in human genetic disease rare birth defects, imprinting syndromes progression of certain cancers some neurological disorders where condition more common in one parental line e.g. AZ, bipolar disorder, male sexual orientation, obesity, schizophrenia

5 How many imprinted genes? Approx 60 identified predicted = approx 1% of the human genome Genome-wide distribution of genes proved (filled triangles) or predicted with high confidence (unfilled triangles) to be imprinted. Red downward triangles, blue upward triangles, and black dots indicate genes predicted to be maternally, paternally, or biallelically expressed, respectively. Philippe P. Luedi et al. Genome Res. 2007; 17:

6 9 Imprinting disorders now recognised Locus 1. Prader-Willi syndrome 15q Angelman syndrome 15q Transient neonatal diabetes 6q24 4. Beckwith-Wiedemann syndrome 11p Russell-Silver syndrome 11p15.5, matupd7 6. matupd14 14q32 7. patupd14 14q32 8. Pseudohypoparathyroidism 1B 20q13 9. Maternal hypomethylation syndrome 6p22

7 Diverse genetic and epigenetic changes underlie recognised imprinting disorders Large deletions or duplications of chromosome regions that contain imprinted genes DNA mutations in genes that are usually imprinted DNA mutations in imprinting control centres Uniparental disomy (UPD) Alteration in epigenetic marks at imprinted loci without alteration in DNA sequence = epimutation For each imprinting disorder, the relative contributions of the above mechanisms is different

8 Uniparental disomy Uniparental disomy: When both copies of a chromosome pair are derived from the same parent. One cause of abnormal imprinting patterns. Typically a result of trisomy rescue in early embryonic life Example: Chromosome 15 x x Methylation status of the SNRPN gene x methylated unmethylated x x Maternally derived chromosome 15 Paternally derived chromosome 15 x x This child would have Prader-Willi syndrome

9 Angelman syndrome Clinical features 1 in 16,000 Intellectual disability Impaired speech Ataxia, seizures, microcephaly Happy demeanour Mechanism 70% deletion 15q 11% UBE3A mutation 7% patupd15 3% epimutations (loss of maternal methylation) 0.3% imprinting centre deletion (Remainder unknown)

10 Prader-Willi syndrome Clinical features 1 in 17,500 Neonatal hypotonia Obesity Cognitive impairment Distinctive behavioural characteristics Hypogonadism Facial appearance

11 Prader-Willi syndrome Mechanism 70% deletion 15q 25% matupd15 <1% epimutations

12 Beckwith-Wiedemann syndrome Clinical features Macroglossia Pre/post natal overgrowth Anterior abdominal wall defects Neonatal hypoglycaemia Ear pits/creases Hemihypertrophy Facial naeveus flammeus Increased risk of abdominal tumours

13 Beckwith-Wiedemann syndrome Mechanisms > 50% have epimutation on maternal allele 50% loss of methylation at DMR2 2-7% have gain of methylation at DMR1 20% patupd11 (mosaic, segmental) 10% mutation in maternal allele of CDKN1C 1-2% cytogenetically visible deletion/duplication Mat Pat H19 Expressed DMR1 H19DMR Meth IGF2 LIT1 DMR2 Meth KvDMR1 CDKN1C Silenced Silenced Expressed Silenced Expressed Expressed Silenced

14 Russell-Silver syndrome Clinical features Growth retardation (IUGR and postnatal) clinodactyly limb length asymmetry triangular facies CALS variable learning difficulties

15 Russell-Silver syndrome Mechanisms 11p locus Mat 45% have epimutation (LOM) of paternal allele of DMR1 Maternally inherited duplications involving DMR1 H19 Expressed DMR1 H19DMR IGF2 LIT1 DMR2 Meth KvDMR1 CDKN1C Silenced Silenced Expressed Pat Meth Silenced Expressed Expressed Silenced Chomosome 7 loci (2 imprinted loci on 7p/7q) 5% have matupd7 maternal duplication of 7p has similar phenoptype epimutations not reported

16 Maternal UPD14 syndrome Clinical features Approx 50 patients reported Pre and postnatal growth retardation hypotonia Facial dysmorphism Early onset puberty Variable learning difficulties Mitter et al. AJMG 2006

17 Maternal UPD14 syndrome Mechanisms Altered gene expression at 14q DMR Most cases have UPD 3 recent reports of paternal chromosome deletions (Kagami et al Nat Genet 2008) 1 report of epimutation (LOM on paternal allele) 10 yo patient with matupd14 syndrome due to epimutation Temple, I K et al. J Med Genet 2007;44:

18 Paternal UPD14 syndrome Clinical features Approx 30 patients reported Polyhydramnios Premature labour Skeletal abnormalies Usually early death Chu et al AJMG 2003 Figure 1. The patient as a neonate. A: Note narrow chest, protuberant abdomen with diastasis recti, and mild rhizomelic shortening of upper limbs. B: Note broad nasal bridge, hirsute forehead. C: Note short AP diameter of chest, short broad thumb. D: Note small thorax with bowing of ribs.

19 Paternal UPD14 syndrome Mechanisms Most pat UPD14 5 chromosome deletions on maternal allele (see Kagami et al Nat Genet 2008) 3 epimutations (GOM on maternal allele) (see Kagami et al Nat Genet 2008) Mattes AJMG (2007) Figure 1. a,b: Chest X-ray at birth demonstrating bell-shaped rib cage with a characteristic arched appearance of the ribs due to caudal bowing anteriorly and cranial bowing posteriorly, elongated clavicles, stippled epiphyses of the humeri, and kyphoscoliosis. c,d: X-ray of the forearms demonstrating contractures of the wrists with ulnar deviation, digit extensor contractures, and swan neck deformity. e,f: Prominent philtrum, prominent midline beak of upper lip, vertical creases under the lower lips, anteverted flared nares, and a broad nasal bridge.

20 Pseudohypoparathyroidism 1B Clinical features hypocalcaemia and hyperphosphataemia due to resistance to parathyroid hormone Mechanism Caused by mutations or epimutations in the regulatory regions of the gene GNAS1 GNAS1 encodes the alpha subunit of the stimulatory G-protein, and in the proximal renal tubule, transcripts of the alpha subunit are derived only from the maternal allele.

21 Pseudohypoparathyroidism 1B PHP-1b occurs in familial and sporadic forms Most familial cases result from a 3-kb microdeletion upstream of GNAS, leading to LOM on the maternal allele Sporadic cases are likely to be caused by epimutation (LOM of the maternal allele) To date there has been only one PHP-1b patient with patupd of 20q Other disorder at GNAS locus Albright Hereditary osteodystrophy Progressive osseous heterotopia McCune-Albright syndrome Inactivating mutations Inactivating mutations Activating mutations

22 Transient neonatal diabetes mellitus (TND) Clinical features presents in the neonatal period with growth retardation and hypoglycaemia Mechanism At 6q24 there is a DMR that is methylated on the maternal allele and unmethylated on the paternal allele TND results from a double dose of the paternal epigenotype 40% paternal chromosome duplication 40% patupd6 20% epimutation: maternal LOM

23 Maternal hypomethylation syndrome From Boonen et al., EJHG 2008 Relatively new syndrome resulting from maternal LOM at multiple loci Ascertained either through BWS (with features of other imprinting disorders) (Rossignol et al., 2006) TND (with features of other imprinting disorders) (MacKay et al., 2006) Homozygous mutations in the gene ZFP57 10 families reported to date, most consanguineous Phenotypes highly variable

24 Maternal hypomethylation syndrome Hypomethylation phenotypes from Mackay, Temple LOM6q24 only LOM multiple Loci Mean birth weight Macroglossia* 1/4 6/9 Abdominal wall defects0/4 2/6 Developmental delay 1/4 2/6 Severe intellectual disability* 2/9 Speech delay* 5/9 Abnormal corpus callosum 2/9 Growth delay 0/4 1/6 Mat age >35 years 0/4 3/6 Infertility/IVF 1/4 1/6 *Also deafness, hypospadius, CHD

25 Other genetic loci that influence imprinting NLRP7 Homozygous (maternal) mutations in the gene NLRP7 cause familial recurrent hydatidiform mole hydatidiform mole is diploid and biparental, but phenotype is that of androgenetic hydatidiform mole NLRP2 Single case report of mother with homozygous mutations in NLRP2 who had two children with BWS due to epimutation at 11p15.5

26 Imprinting disorders and ART Primordial germ cell Factors linked to subfertility? Imprint establishment Use of immature germ cells Ovarian hyperstimulation Germ cells in vitro manipulation Germ cells cryopreservation Mature gametes Mechanical stress Fertilisation IVF/ICSI Zygote Culture conditions Embryo cryopreservation Imprint maintenance

27 AS PWS BWS RSS Mat UPD14 Pat UPD14 PHP-1b TND Mat Hypometh synd Cytogenetic 70% 70% 1-2% <1% 3 patients 5 patients 0 40% 0 UPD 7% 25% 20% 5% >90% >50% 1 patient 40% 0 DNA mutation 5-10% <1% 10%? 0 0 Most familial 0 0 Epimutation Mat LOM 3% % 0 0 0? Most sporadic 20% 100% Mat GOM % patients 0 0 Pat LOM 0 <1% 0 64% 1 patient Pat GOM 0 0% 0 1 patient

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