Genomic imprinting: Summary of an NICHD conference

American Journal of Medical Genetics 46675-680 (1993)

NICHD Conference

Genomic Imprinting: Summary of an NICHD Conference Golder N. Wilson, Judith G. Hall, and Felix de la Cruz Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas Texas (G.N.W.), Department of Pediatrics and Medical Genetics, University of British Columbia, Vancouver (J.GH.1; the Mental Retardation and Developmental Disabilities Branch, National Institute of Child Health, Bethesda, Maryland (F.d.1.C.).

Compelling evidence for genomic imprinting as a pathogenetic mechanism in humans man- dates re-evaluation of every genetic or multi- factorial disease for parent-of-origin effects. In an expanding list of malformation syndromes, cancers, growth abnormalities, and chromosomal disorders, phenotypes may be determined by source rather than content of transmitted DNA. A multidisciplinary conference held on April 13-14, 1992, reviewed the substantial impact of genomic imprinting in mouse development and discussed in role in human pregnancy, childhood cancers, chromosomal translocations, X-inactivation, and several disorders associated with mental retardation. Topics for future research include the mechanism, timing, reversibility, and homology of the imprinting process. 0 1993 Wiley-Lisa, Inc.

KEY WORDS growth factors, chromosomal translocations, malformation syndromes, childhood cancers, X chromosome

INTRODUCTION Research in mice and humans has demonstrated that

identical genes may be marked differently during maternal versus paternal germ cell development. This phenomenon, termed “genomic imprinting” by analogy to observations in animal behavior, has important implications for childhood cancers, numerous disorders associated with mental retardation, and chromosomal diseases. Evidence for genomic imprinting derives from several disciplines as summarized by Hall [19901.

Received for publication February 24, 1993. Address reprint requests to Golder N. Wilson M.D., Ph.D., Divi-

sion of Pediatric Genetics, University of Texas Southwestern Medi- cal School, 5323 Harry Hines, Dallas TX 75235-9063.

From an NICHD Conference on genomic imprinting, April 1992.

0 1993 Wiley-Liss, Inc.

Mouse Pronuclear ‘Ikansplantation. Shortly after fertilization, the oocyte and sperm pro-

nuclei can be manipulated before they fuse to form the zygote. Zygotes produced from two sperm pronuclei (i.e., no maternal contribution) develop normal placentas but lack embryos; those produced from two oocyte pronuclei (i.e., no paternal contribution) develop embryos without placentas. In each case, the normal genetic complement is present but its usual biparental source is altered. In human tripoloids, the placental hydatidiform mole has two sets of male-derived chromosomes while the ovarian teratomas (germ layers but no placental tissue) have two sets of female-derived chromosomes [Hall, 19901.

Mouse Uniparental Disomies. Breeding of translocation carriers can produce mice

where both chromosomes of a given pair are derived from a single parentuniparental iso- or heterodisomy. Systematic experiments have demonstrated that uniparental disomy for chromosomes 2,6,7,11, and 17 are associated with different phenotypes according to whether the chromosome pair derives from mother or father [Searle and Beechey, 1978; Cattanach, 19861. Growth alteration is common, and the distorted recovery ratios observed when producing uniparental disomies for chromosomes 1, 4, 5, 9, 14, and 17 may indicate early lethality as a consequence of abnormal parental origin [Hall, 19901.

Differences in Mouse ’Ikansgene Expression. Transgenic males may produce fetuses with appropri-

ate tissue expression, while transgenic females may not [Sapienza et al., 19871. Parent-of-origin differences in transgene expression occur in about one-quarter of the mouse transgenes studied, and the expression differences often correlate with altered DNA methylation patterns in maternal versus paternal germ cells [Reik et al., 19871.

Human Syndromes With Mental Retardation. The confusing finding that both Prader-Willi and An-

gelman syndromes are associated with the same chromosome 15 deletion was clarified by studies of parental

676 Wilson et al.

origin. DNA studies have demonstrated that it is the paternal chromosome 15 that is deleted in Prader-Willi syndrome and the maternal chromosome 15 that is deleted in Angelman syndrome [Knoll et al., 19891. As would be expected, maternal disomy 15 (lack of paternal material) has been demonstrated in several cases of Prader-Willi syndrome, but the corresponding paternal disomy 15 has been less frequently observed in Angel- man syndrome [Nicholls et al., 19891. The relevant region of human chromosome 15 is homologous to a region of mouse chromosome 2 that is also known to be imprinted.

The chromosomal segment on l l p that is sometimes deleted in Wiedemann-Beckwith syndrome is also homologous to an imprinted chromosome in the mouse- chromosome 17. Not only do these patients exhibit altered growth, but the discordance among monozygotic twin pairs and the predominance of female transmission in familial cases suggests the operation of imprinting [Reik, 19893. Of note on mouse chromosome 17 is recipro- cal imprinting of the insulin-like growth factor I1 gene and its receptor-only the paternal copy of the former is expressed in embryos and only the maternal copy of the latter [Haig and Graham, 19911. The specificity of these effects emphasizes that small regions of chromosomes may be imprinted, a point relevant to interpreting the effects of uniparental disomies and to uncovering re- sponsible genes within interstitial deletions such as 15qllq13.

Childhood Cancers. The WAGR syndrome of Wilms tumor-aniridia-geni-

tal anomaly-retardation, like that of Wiedemann-Beck- with, has also been associated with del(1lp). Sporadic cases of Wilms’ tumor often exhibit molecular deletions or acquired homozygosity within this area and it is always the maternal l l p region that is deleted or lost [Van Heyningen and Hastie, 19921. Similar imprinting effects are suspected for retinoblastoma and familial glomus tumors [Ponder, 19881.

X-Inactivation and Fragile X Syndrome. Preferential inactivation of the paternally derived X

chromosome in the extraembryonic tissues of rodents and marsupials [Lyon, 19891 but not humans [Migeon et al., 19851 raises the question of whether X-inactivation is a special case of genomic imprinting [Hall, 19901. The processes would be similar in requiring erasure prior to sex-appropriate patterning but different in that imprinting of autosomal regions is non-random. In fragile X syndrome, transmitting but asymptomatic sons of female carriers have been explained by a failure to reac- tivate the fragile X chromosome [Laird, 19871. Others have attributed fragile X expression solely to the number of trinucleotide repeats within the area of DNA instability [Kremer et al., 19911. The outcome of this debate may have broad relevance since trinucleotide repeat amplification is now documented in other inher- ited conditions with parent of origin effects [Harper, 19751.

With this compelling evidence for the operation of genomic imprinting in human disease, the conference was devoted to the following aims:

1. To evaluate the molecular mechanisms involved in imprinting including DNA methylatin, X-inactivation/ reactivation, and alteration in chromatin structure.

2. To survey effects of imprinting on mouse development, including differential expression of transgenes, different phenotypes produced by uniparental disomies, and homologies between mousehuman chromosomal regions and their imprinting disorders.

3. To evaluate effects of parental origin on human chromosomal syndromes according to whether the ex- tralmissing material is paternal or maternal.

PARTICIPANTS Organizers

Felix de la Cruz, National Institute of Child Health and

Judith G. Hall (Conference Chair), University of British

Golder N. Wilson, University of Texas Southwestern

Presentations Opening remarks. Duane Alexander, Director, National

Institute of Child Health and Human Development. Genomic Imprinting Introduction. Judith G. Hall. The Evolutionary Biology of Human Pregnancy. David

Haig, Department of Plant Sciences, University of Oxford, UK.

Genomic Imprinting and Carcinogenesis. Carmen Sapi- enza, Ludwig Institute, La Jolla, CA.

Epidemiology and Molecular Genetics of Childhood My- eloproliferative Syndromes. Kevin Shannon, Univer- sity of California, San F’rancisco, CA.

Imprinting in Mammalian Development. Wolf Reik, In- stitute of Animal Physiology and Genetics Research, Cambridge Research Station, Cambridge, UK.

Intrauterine Development and Uniparental Disomy in Humans. Dagmar K. Kalousek, University of British Columbia, Vancouver, BC.

Genomic Imprinting: Consequences for Human Disease. Human Chromosomal Regions. Albert Schinzel, Uni- versity of Zurich, Switzerland.

Robertsonian Translocations-Clues to Imprinting Ef- fects. Dian Donnai, St. Mary’s Hospital, Manchester, UK.

DNA Methylation and Genomic Imprinting. Howard Cedar, Hebrew University, Jerusalem.

Mammalian Genomic Imprinting: Prader-Willi & An- gelman Syndromes and Mouse Models. Robert D. Nicholls, University of Florida, Gainesville, FL.

DNA Methylation and Genomic Imprinting: The Angel- man (AS) and Prader-Willi (PWS) Syndromes as Models. Daniel J . Driscoll, University of Florida, Gainesville, FL.

The Role of DNA Methylation in X-Inactivation and the Fragile X Syndrome. Barbara R. Migeon, Johns Hop- kins University, Baltimore, MD.

Human X Chromosome Inactivation: The X Inactiva- tion Center and XIST. Carolyn J . Brown, Stanford University, Stanford, CA.

Fragile X Syndrome. Roger E. Stevenson, Greenwood Genetics Center, Greenwood, SC.

Human Development, Bethesda, MD.

Columbia, Vancouver BC, Canada.

Medical Center, Dallas, TX.

Genomic Imprinting 677

may be represented at the gene level by the 5 copies of the chorionic gonadotropin on chromosome 19 that are homologous to the 1 gene for luteinizing hormone. The placental lactogen gene is a duplication of that for growth hormone. Insulin-like growth factor I1 (IGF2) is present at high levels in invasive cytotrophoblast, and it is mod- ulated by three loci that are known to be imprinted in mice-IGF2, IGF2r, and H19. Thus, maternal and paternal imprints may arise in part as alternative strate- gies to gene dosage so that genes of fetal advantage are shut down or enhanced, respectively.

Mechanisms and Developmental Timing Drs. Cedar, Reik, and Sapienza emphasized the poten-

tial variability of the imprinting process and the re- markable embryonic changes in DNA methylation that complicate the idea of an imprint as a simple pattern of methyl groups. Parental modification of transgene expression and the phenotypes produced by mouse uniparental disomies are often strain-dependent. As Dr. Sapienza stated, the question of whether a gene is imprinted or not may be answered: “Relative to what?”

Howard Cedar discussed a methylation-sensitive PCR assay that allowed examination of specific genes in early embryos and gametes. A surprising finding was a mas- sive wave of demethylation shared by several genes at the 16-cell blastocyst stage of mouse embryogenesis. Global remethylation of these genes occurred by the 6.5 day gastrula stage, followed by selected demethylation of certain genes for tissue-specific expression. One allele of the imprinted IGF2 gene escaped this general demethylation and remained partially methylated. Wolf Reik reported minimal differences in methylation or nuclease sensitivity in the CpG islands of embryonic IGF2 genes, but did detect methylation differences some 3 kb upstream.

Using the fluorescent in situ hybridization (FISH) technique to detect genes in embryonic nuclei, Howard Cedar showed that imprinted genes such as IGF2 or its receptor exhibit asynchronous replication-that is, one sees 3 dots of hybridization (one allele replicated, one not) in certain embryonic cells rather than the 4 or 2 dots expected for synchronous replication. As expected from studies of X-inactivation, it was the maternally derived IGF2 allele (repressed in the embryo) that replicated late in hemizygous cells. Thus, the mechanisms of genomic imprinting can be related to allele-specific differences in methylation and replication, but ways in which the imprint is established (imprinter) and maintained (imprintee) remain unknown.

Human Imprinting Disorders The most dramatic but least recognized effect of im-

printing in humans is probably early abortion. Dagmar Kalousek reviewed the poor outcome of pregnancies associated with chromosomal mosaicism at the time of chorionic villus sampling. Full dichotomy between chorionic trisomy and fetal diploidy may occur if there is loss of one chromosome in embryonic progenitor cells. Since one-third of such fetal corrections will result in uniparental disomy, cases with confined placental mosaicism (often observed for chromosomes 7,9,15, and 16)

Molecular Genetics of Fragile X Syndrome. Stephen T. Warren, Emory University, Atlanta, GA.

Imprinting and Imprint Erasure as Viewed Through the Fragile X Syndrome. Charles D. Laird, University of Washington, Seattle, WA.

DISCUSSION Nature and Origins of Genomic Imprinting

Judith Hall suggested imprinting involves a) phenotypic differences that b) depend on parental origin and c) are reversible (i.e., epigenetic). Charles Laird offered definition as a non-genetic process affecting the ability of a gene or chromosome to function in some future cell or generation. According to Dr. Hall, processes exhibiting parent-of-origin differences that may be related to imprinting include meiotic pairing and sex-specific recombination rates, mutation rates as seen with the predominance of single gene mutations in the male germ line, and areas of late replication exemplified by the X chromosome. Although most observations or experiments demonstrating genomic imprinting have concerned abnormal situations, it is important to recog- nize imprinting as a normal process involved in many aspects of mammalian development.

Congenital anomalies are notably lacking in the phenotypes associated with imprinting so far; more promi- nent are alterations in growth, behavior, and survival. It was questioned whether this emphasis on growth and lethality rather than malformation might reflect the higher degree of inbreeding in laboratory mice com- pared to humans. Modifying genes often act in the heterozygous state, meaning that the magnitude of imprinting effects might be less in mice due to increased homozygosity. Until the influence of heterozygosity is clarified, no human developmental anomalies should be eliminated as candidate imprinting disorders.

Central to the imprinting phenomenon is the pla- centa, and in plants it is those organisms with placental homologues that have exhibited parent-of-origin differences. David Haig used this evolutionary perspective to speculate about human maternal/fetal conflicts. Alloca- tion of maternal resources among a mother and her currentlfuture offspring may be highly competitive during the periodic famines and epidemics that character- ize human evolution. Just as a loud cry may win advantage, the fetus that increases its allocation of blood supply or nutrients will favor its own genes relative to those of the mother (one-half of genes shared) or siblings (one- half to one-quarter of genes shared depending on pater- nity). The mother, in turn, may need to dampen fetal demands in order to conserve her nutrients for herself or for more optimal pregnancies.

Examples of this maternofetal “tug-of-war” [Haig, 19921 might include abortion versus continuation, normal versus high maternal blood glucose, low maternal blood pressure versus high placental flow. Phenomena that may correspond to these conflicts include the rela- tion between high chorionic gonadotropin levels (fetal well-being) and maternal nausea, the production of maternal insulin resistance by placental lactogen, and the contrast between hypertension of pregnancy and uter- ine vessel erosion to increase fetal blood flow. Conflict

678 Wilson et al.

present excellent opportunities to examine imprinting. Dr. Kalousek reported maternal uniparental disomy 16 in 2 of 3 growth retarded fetuses with mosaic trisomy 16 in term placentae [Kalousek, 19931. Confined chorionic trisomy 15 mosaicism with correction to paternal uniparental disomy 15 has also been demonstrated in An- gelman syndrome.

Drs. Donnai and Schinzel emphasized that balanced fibertsonian translocations may be a source of uniparental disomy as discussed in companion articles. Un- usual and different phenotypes have been associated with maternal and paternal uniparental disomies for chromosome 14. Important caveats associated with the study of translocations include the need to exclude single gene disruptions or complex rearrangements. Even balanced uniparental disomies, if they derive from 1 rather than both parental homologues (uniparental iso- disomies rather than heterodisomies), may exhibit phenotypes because of autozygous recessive alleles. Two examples have been reported with cystic fibrosis [Spence et al., 1988; Voss et al., 19891.

Using the Oxford Grid as the basis for highlighting human candidate regions, a search for parent-of-origin differences is underway in many human diseases. Al- bert Schinzel and Judith Hall emphasized the uniparental disomy phenotypes of sporadic occurrence, growth retardation, mental retardation, and patterns of minor anomalies with rare major anomalies. Candidate disorders mentioned included the Russell-Silver, Wiedemann- Beckwith, Sotos, Williams, Brachmann-de Lange, and Floating Harbor syndromes. Associated deletions and translocations are valuable in focusing the search for missing parental RFLPNNTR polymorphic alleles, since recombinant chromosomes with uniparental disomy for only one segment can be expected.

Mendelian diseases worth studying for imprinting effects (reviewed by Judith Hall) include dominant disorders where homozygous and heterozygous expression are similar (i.e., Huntington disease), disorders with more than one locus (i.e., tuberous sclerosis or polycystic kidney disease), disorders with early-onset forms (i.e., Huntington disease), and disorders with differences based on parental transmission (i.e., Huntington disease, myotonic dystrophy, diabetes mellitus, psoriasis). By analogy to the differences between mouse strains, variability among families in occurrence or degree of imprinting effects can be expected.

The myeloproliferative syndromes associated with ju- venile chronic myelogenous leukemia and bone marrow monosomy 7 emphasize the complexity of recognizing imprinting effects as discussed by Kevin Shannon. Mul- tiple events contribute to the development of leukemia in this syndrome despite its early onset, and DNA anal- ysis showed no bias for retention of parental alleles in 6 monosomy 7 patients. The increased incidence of juve- nile chronic myelogenous leukemia and bone marrow monosomy 7 in NF-1 is intriguing, considering the pos- sibility of “maternal effect” in both NF-1 and NF-2.

Drs. Nicholls and Driscoll discussed those prototypic disorders for human imprinting, the F’rader- Willi and Angelman syndromes. The University of Florida DNA registry for these disorders has refined the critical re-

gions for both syndromes and highlighted genes of interest including the pigmentation gene DNlO and the GABRB3 aminobutryic acid receptor subunit gene. Mice with corresponding deletions in a homologous region of chromosome 7 (also imprinted in mice) are being constructed for more precise mapping of disomy and deletion phenotypes. Of particular interest within the Prader-Willi and Angelman critical regions is the gene DN34 that exhibits allele-specific methylation reflec- tive of parental origin.

X Chromosome Inactivation and Fragile X Syndrome

Barbara Migeon emphasized that X-inactivation, like genomic imprinting, may be non-random as in marsu- pial somatic cells and rodent extra-embryonic mem- branes. The inactive X chromosome reactivates during human oogenesis and is unstable in human chorionic villi. Correlations with X-inactivation can be made for DNA methylation and developmental timing. CpG islands (regulatory regions, usually upstream) are highly methylated on the inactive X of eutherian mammals but not on the inactivated paternal X of marsupials. The earlier, non-random X-inactivation of marsupials is more reversible than the later, random X-inactivation of mice and humans. The results suggest that imprinting and X-inactivation are mechanisms for cell memory, and that DNA methylation functions secondarily to “lock in” changes in gene activity and maintain them through subsequent cell divisions.

The XIST (X-inactive specific transcription) gene discussed by Carolyn Brown is exceptional in being expressed only from the inactive X chromosome. As such, its expression is female-specific. XIST maps to band Xq13 where the X inactivation center resides. Since DNA sequencing of more than 16 kb of the XIST gene shows an open reading frame of no more than 430 bp, the gene is probably expressed only as RNA. Although the randomness and whole-chromosome distribution of X inactivation differ from autosomal imprinting, potential analogy of the X inactivation center to an imprinted region (perhaps paternally imprinted in marsupials) and of XIST to an imprinting agent make this system intriguing.

A disorder which greatly furthered the imprinting concept by means of the Laird hypothesis was fragile X syndrome. Roger Stevenson reviewed the interesting history and phenotypic diversity of X-linked mental retardation syndromes as frequently updated in the pages of this journal. Among the clinical issues requiring fur- ther study are the ethnic differences in phenotype, the apparent decline in I& with age, and the need for better information on the mental status, behavior, and fecun- dity of those 20% of males with reduced penetrance-the so-called “transmitting males.”

Stephen Warren reviewed the identification of the fragile X retardation-1 (FMR-1) gene and attributed clinical severity to the number of trinucleotide repeats occurring within the gene. Normal individuals have 5-50 trinucleotide repeats (average 29-30) and transmitting males have up to 200 repeats without the re- markable heterogeneity seen in penetrant males. Ex-

Genomic Imprinting 679

C. X-inactivation and fragile X syndrome. 1. What protects CpG islands from methylation in

active genes? 2. What promotes methylation of islands on the inac-

tive X? 3. Role of X-inactivation center and XIST in X-inac-

tivation? 4. Mental functiodmolecular correlation in fragile X

syndrome? 5. Mechanism by which limited augmentation of CGG

triplet occurs to produce transmitting males or unaffected female carriers in fragile X syndrome?

6. Is X-inactivation a specialized form of genomic imprinting?

In addition to these specific suggestions, the attendees left with an enlarged perspective on the relevance of genomic imprinting to human disease. The authors join with them in thanking the National Institute of Child Health for its lasting imprint on our perceptions.

pansion to the 700-1,500 repeats seen in typical fragile X males never occurs in transmitting males; premuta- tion to full mutation transitions occur only in females, where 53% of carriers with full amplification are symp- tomatic. In penetrant female carriers and affected males, the amplified repeats exhibit size variation, producing a smear on Southern blots, and high degrees of methylation. Reverse transcriptase PCR in tissues from affected males demonstrates that FMR-1 repeat amplification and methylation silences the gene. In chorionic villi, on the other hand, the expanded repeats are hypomethy- lated and the FMR-1 gene is expressed. Although FMR-1 is a unique gene whose normal function is not yet known, it is transcribed in the expected tissues of brain and testis. The mouse FMR-1 gene has identical trinucleotide repeats expressed as arginines, and FMR-1 homologues are found in organisms as diverse as non-human primates and yeast.

Charles Laird noted that mutations blocking imprint erasure could lead to persistence of an abnormal imprint. Imprinting implies two states of an abnormal allel-benign and deleterious. When an unaffected daughter transmits a fragile X mutation on her inactive X, then the benign allele becomes deleterious because of incomplete reactivation. Crucial to this perspective of failed imprint erasure (i.e., methylation leading to repeat amplification rather than vice versa) is the fate of the imprint transmitted from affected males. Few offspring of affected males exist to test the prediction that the necessary lack of X inactivation would therefore erase the imprint. Dr. Laird cited 8 female offspring of fragile X fathers (3 with typical Martin-Bell phenotype) of whom 7 showed no fra(X) site by cytogenetics. Unfor- tunately, convincing data regarding trinucleotide repeat size in these families were not available.

Questions for Future Research as Summarized From Conference Participants

A. Nature and mechanisms of genomic imprinting

their associated phenotypes?

printed chromosomal domains?

protein interactions in imprinting?

1. Mechanisms for imprintinglimprint erasure and

2. Extent, stability, and tissue specificity of im-

3. Role of chromatin structure/topology and DNA/

4. Is imprint erasure related to crossing-over? 5. Do imprinted genes share certain developmental

or cellular functions. B. Genomic imprinting in humans

1. Mechanisms and frequencies of uniparental disomy?

2. Liveborn phenotypes associated with uniparental disomy?

3. Role of imprinting in human pregnancy regarding preeclampsia, gestational diabetes, and placental hor- mones released into the maternal blood stream?

4. Mousehuman homology of imprinted regions and imprinting phenotypes? 5. Utility of a genetic register approach? 6. Cytogenetic approaches for delineating imprinted

regions?

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Genomic imprinting: Summary of an NICHD conference