Asynchronous DNA replication between 15q11.2q12 homologs: cytogenetic evidence for maternal imprinting and delayed replication

DNA replication kinetics of Prader-Willi/Angelman syndrome region of 15q11.2q12 was studied without synchronization in five human amniotic cell and five skin fibroblast strains with a marker 15 chromosome, i.e., 15p+ or der(15), as cytological marker to distinguish between the two homologs. BrdU-33258 Hoechst-Giemsa techniques were used to analyze and compare the late replication patterns in the 15q11.2q12 region between the homologs. Asynchronous replication between the homologs was observed in both amniocytes and fibroblasts. From cells of a marker 15 of known parental origin, the paternal 15q11.2q12 replicated earlier than that of the maternal 15 in 92%–95% of asynchronous metaphases. The remaining 5%–8% of asynchronous metaphases displayed maternal early/paternal late replication. This mosaic pattern of replication in the 15q11.2q12 region may be due to methylation mosaicism of genomic imprinting or a relative lack of self-control of replication. These results provide cytogenetic evidence of maternal imprinting and delayed replication in the 15q11.2q12 region.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication. Received: 7 May 1996; in revised form: 27 January 1997 / Accepted: 31 July 1997     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Parental alleles of an imprinted mouse transgene replicate synchronously

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting. Dev. Genet. 23:275–284, 1998. © 1998 Wiley-Liss, Inc.     

Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader-Willi syndromes.

The D15S9 and D15S63 loci in the Prader-Willi/Angelman syndrome region on chromosome 15 are subject to parent-of-origin-specific DNA methylation. We have found two Prader-Willi syndrome families in which the patients carry a maternal methylation imprint on the paternal chromosome. In one of these families, the patients have a small deletion encompassing the gene for the small nuclear ribonucleoprotein polypeptide N, which maps 130 kb telomeric to D15S63. Furthermore, we have identified a pair of nondeletion Angelman syndrome sibs and two isolated Angelman syndrome patients who carry a paternal methylation imprint on the maternal chromosome. These Angelman and Prader-Willi syndrome patients may have a defect in the imprinting process in 15q11-13. We propose a model in which a cis-acting mutation prevents the resetting of the imprinting signal in the germ line and thus disturbs the expression of imprinted genes in this region.     

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11-q13 deletions. Both also can result from uniparental disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11-q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11-q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, postnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11-q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.     

The impact of imprinting: Prader-Willi syndrome resulting from chromosome translocation, recombination, and nondisjunction.

Prader-Willi syndrome (PWS) is most often the result of a deletion of bands q11.2-q13 of the paternally derived chromosome 15, but it also occurs either because of maternal uniparental disomy (UPD) of this region or, rarely, from a methylation imprinting defect. A significant number of cases are due to structural rearrangements of the pericentromeric region of chromosome 15. We report two cases of PWS with UPD in which there was a meiosis I nondisjunction error involving an altered chromosome 15 produced by both a translocation event between the heteromorphic satellite regions of chromosomes 14 and 15 and recombination. In both cases, high-resolution banding of the long arm was normal, and FISH of probes D15S11, SNRPN, D15S10, and GABRB3 indicated no loss of this material. Chromosome heteromorphism analysis showed that each patient had maternal heterodisomy of the chromosome 15 short arm, whereas PCR of microsatellites demonstrated allele-specific maternal isodisomy and heterodisomy of the long arm. SNRPN gene methylation analysis revealed only a maternal imprint in both patients. We suggest that the chromosome structural rearrangements, combined with recombination in these patients, disrupted normal segregation of an imprinted region, resulting in uniparental disomy and PWS.     

Cytogenetic studies of familial Prader-Willi syndrome

Summary A family in which two first cousins were found to have the Prader-Willi syndrome was investigated cytogenetically. Although G-banding analysis of metaphase chromosomes failed to demonstrate abnormality, close analyses on the fine prometaphase bands by G-banding and the DA-DAPI bands by double stainings revealed a distinct chromosome abnormality in this family. A reciprocal translocation, rep(14:15)(q11.2;q13), was detected in three family members: the mother, the maternal grandmother, and a maternal uncle of the proband. And, the proband and one of the first cousins had an unbalanced translocation that was derived from their carrier parents. The karyotypes of the affected cousins were determined as 46,XY or XX,-15,+der(14),rcp(14;15)(q11.2;q13). Therefore, they were considered to have an identical cytogenetic abnormality: a partial trisomy of the 14pterq11.2 segment and a partial monosomy of the 15pterq13 segment. Detailed clinical features of the proband and his affected cousin are described, main features associated with the Prader-Willi syndrome having been observed in both cousins. These observations support a definite relationship between the Prader-Willi syndrome and chromosome 15.     

Isodicentric X chromosome in a patient with Turner syndrome — implications for localization of the X-inactivation center

Summary Cytogenetic analyses have previously shown that the region Xq11.2–q21 is retained in all structurally abnormal X chromosomes. From these observations the conclusion has been drawn that this critical region on the proximal long arm of the X chromosome contains the locus controlling X-inactivation. Structurally abnormal X chromosomes without the X-inactivation center would allow nullisomy, disomy, or trisomy for genes on the X chromosome, and this condition is presumed nonviable. We studied a 28-year-old woman with primary amenorrhea and features of Turner syndrome who had an unusual isodicentric chromosome of the short arm of X. This patient provided us with the opportunity to more closely define the location of the X-inactivation center. High resolution chromosome analysis showed a 46,X,idic(X)(pterq13.2::q13.2pter) chromosome pattern in 94% of her cells and a 45,X complement in 6%. Replication studies showed this derivative X chromosome to be late-replicating (inactive) in all cells analyzed. DNA analysis confirmed the breakpoint of the isodicentric chromosome to be proximal to PGK1. Based on these results, the locus for the X-inactivation center can be refined to be within Xq11.2–q13.2.     

Prenatal detection of maternal UPD15 in a new case with i(15p) by Timing Replication Test (TRT) and methylation analysis

DNA replication kinetics of the Prader-Willi/Angelman Critical Region (PWACR) was studied with and without synchronisation in human amniotic cell cultures obtained from 20 cases with normal karyotype and 4 cases with a marker of chromosome 15, respectively. A Timing Replication Test (TRT) was performed by synchronisation of amniotic cell cultures and followed by interphase FISH to analyse and compare the early/late replication patterns in SNRPN and UBE3A genes between the homologues of chromosome 15. Asynchronous replication patterns of the analysed genes were observed in both amniotic cell cultures but the percentage of interphase nuclei presenting with asynchronous replication was significantly increased in the cultures with synchronisation (40-51%), as compared to those without synchronisation (20-23%). The evaluations, performed by means of TRT, showed asynchronous replication patterns on control values: between 39% and 46% of cells in all the cases with inv dup(15). In contrast, the percentage of cells with asynchronous replication in the case with i(15p) was significantly decreased (3-6%), as compared to the control value, and it may be indicated by uniparental disomy of chromosome 15 (UPD15). In addition, those results have been confirmed by molecular evaluation, using the methylation diagnostic test for diagnosis of the Prader-Willi Syndrome.     

DNA replication and inactivation patterns in structural abnormality of sex chromosomes

Summary High resolution chromosome analysis and bromodeoxyuridine (BrdUrd) incorporation have been applied to study patterns of chromosomal replication (inactivation) in two cases of unbalanced X-autosome translocations, seven cases of X and Y chromosome rings or fragments, and five cases of dicentric isochromosomes (Xq). Our results indicate the following: (1) In (X-A) translocations, detailed replicational analysis of the translocated autosomal segment is informative. Absence of spreading effect and partial-incomplete spreading effect are the most common observations. (2) Sex chromosome derived fragments and rings can be differentiated based on their replicational features. (3) Dicentric isochromosomes (Xq) can be classified based on intercentromeric distances, replicational asynchrony, and centromere inactivation. (4) A correlation between intercentromeric distance and degree of 45,X mosaicism was observed in dicentric i(Xq) chromosomes.Evidence for spreading effect based on our results and on the review of the literature has been critically analyzed and general rules in evaluating spreading effects (SE) proposed. The cytologic detection of active regions on the late replicating X chromosome and the inactivation capacity of the juxtacentromeric region of Xp is evaluated. It is proposed that centromere suppression and underreplication are related phenomena. Finally, the analysis of informative replicational stages is emphasized and the application of their analysis in basic and clinical cytogenetics demonstrated.     

A t(X;15)(q23;q25) with Xq reactivation in a lymphoblastoid cell line from Fanconi anemia.

A t(X:15)(q23;q25) was detected during cytogenetic investigation of a lymphoblastoid cell line established from a female patient with Fanconi anemia. The translocation was apparently balanced at passage 300 and unbalanced at passage 13. A chromatid exchange between both the normal and the der(15), between the centromere and band 15q25, may explain these results. Replication studies, following BrdU incorporation, indicate that the segment Xq23----qter from the der(15) is early replicating whereas segment Xpter----q23 from the der(X) is late replicating. Since the normal X was early replicating, it is concluded that the segment of the long arm of chromosome X, separated from its inactivation center by the translocation, was reactivated. This interpretation is confirmed by the methylation patterns of the hypoxanthine phosphoribosyltransferase gene (HPRT), mapped on Xq26, which corresponds to that of an active gene, whereas that of phosphoglycerate kinase (PGK1), which remained on the der(X), corresponds to that of an inactive gene. This is the first example of reactivation of a segment of the X chromosome following a structural rearrangement in somatic cells.     

Allele-specific replication of 15q11-q13 loci: a diagnostic test for detection of uniparental disomy.

Allele-specific replication differences have been observed in imprinted chromosomal regions. We have exploited this characteristic of an imprinted region by using FISH at D15S9 and SNRPN (small nuclear ribonucleo protein N) on interphase nuclei to distinguish between Angelman and Prader-Willi syndrome patient samples with uniparental disomy of chromosome 15q11-q13 (n = 11) from those with biparental inheritance (n = 13). The familial recurrence risks are low when the child has de novo uniparental disomy and may be as high as 50% when the child has biparental inheritance. The frequency of interphase cells with asynchronous replication was significantly lower in patients with uniparental disomy than in patients with biparental inheritance. Within the sample population of patients with biparental inheritance, those with altered methylation and presumably imprinting center mutations could not be distinguished from those with no currently detectable mutation. This test is cost effective because it is performed on interphase cells from the same hybridized cytological preparation in which a deletion is excluded, and additional specimens are not required to determine the parental origin of chromosome 15.     

Prader-Willi-Syndrom und Angelman-Syndrom

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic disorders caused by the loss of function of imprinted genes in the chromosomal region 15q11q13. An approximately 2 Mb region inside 15q11q13 is subject to genomic imprinting. As a consequence the maternal and paternal copies in this region are different in DNA methylation and gene expression. The most frequent genetic lesions in both disorders are an interstitial de novo deletion of the chromosomal region 15q11q13, uniparental disomy 15, an imprinting defect or, in the case of AS, a mutation of the UBE3A gene. Microdeletions in a small number of patients with PWS and AS with an imprinting defect have led to the identification of the chromosome 15 imprinting centre (IC) upstream of the SNURF-SNRPN gene, which acts in cis to regulate imprinting in the whole 15q imprinted domain. The IC consists of two critical elements: one in the more centromeric part which is deleted in patients with AS and which is thought to be responsible for the establishment of imprinting in the female germ line, and one in the more telomeric part which is deleted in patients with PWS and which is required to maintain the paternal imprint.     

The similarity of phenotypic effects caused by Xp and Xq deletions in the human female: a hypothesis

Summary We have collected from the literature adult nonmosaic women with the following aberrant X chromosomes: Xp- (52), Xq- (67), idic(Xp-)(10), idic(Xq-)(9), and interstitial deletions (12). Lack of Xp, and especially Xcen-Xp11 (b region), may cause full-blown Turner syndrome. However, individual Turner symptoms, including gonadal dysgenesis, otherwise seem to be randomly distributed with respect to the different Xp and Xq deletions, although breakpoints distal to Xq25 do not give rise to any phenotypic anomalies except in a few cases of secondary amenorrhea or premature menopause. Of the carriers of an Xp- or Xq- chromosome, 65% and 93%, respectively, suffer from ovarian dysgenesis, whereas all idic(Xp-) and idic(Xq-) chromosomes cause primary or secondary amenorrhea. Xq deletions do not induce specific symptoms different from those caused by Xp deletions. Lack of the tip of Xp has led in 46/52 cases to short stature, but 43% of the Xq- carriers are also short. To explain these observations, we propose the following hypothesis. Since deletions of truly inactivated regions do not seem to cause any symptoms, we assume that the b region (Xcen-p11) always stays active in a normal inactive X, but is inactivated in deleted X chromosomes, especially in Xq- chromosomes. In some cases, inactivation may spread to the tip of Xp; this would explain the apparently variable behavior of the Xg and STS genes, and the short stature of some Xq- carriers. Full chromosome pairing seems to be a prerequisite for the viability of oocytes and thus for gonadal development. Deleted X chromosomes necessarily leave a portion of the normal X unpaired and isodicentrics probably interfere with pairing, resulting in atresia of oocytes. The role played by the critical region (Xq13–q24) in ovarian development is still unclear.     

Exclusion mapping of the gene for X-linked neural tube defects in an Icelandic family

Various polymorphic markers with a random distribution along the X chromosome were used in a linkage analysis performed on a family with apparently Xlinked recessive inheritance of neural tube defects (NTD). The lod score values were used to generate an exclusion map of the X chromosome; this showed that the responsible gene was probably not located in the middle part of Xp or in the distal region of Xq. A further refining of these results was achieved by haplotype analysis, which indicated that the gene for X-linked NTD was located either within Xp21.1-pter, distal from the DMD locus, or in the region Xq12–q24 between DXS106 and DXS424. Multipoint linkage analysis revealed that the likelihood for gene location is highest for the region on Xp. The region Xq26–q28, which has syntenic homology with the segment of the murine X chromosome carrying the locus for bent tail (Bn), a mouse model for X-linked NTD, is excluded as the location for the gene underlying X-linked NTD in the present family. Thus, the human homologue of the Bn gene and the present defective gene are not identical, suggesting that more than one gene on the X chromosome plays a role in the development of the neural tube.     

Evaluation of methylation analysis for diagnostic testing in 258 referrals suspected of Prader-Willi or Angelman syndromes

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11–13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing ∼99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations. Received: 10 February 1998 / Accepted: 7 July 1998     

Clinical and cytogenetic studies of the Prader-Willi syndrome: Evidence of phenotype-karyotype correlation

Summary Twenty-seven patients with the presumed diagnosis of Prader-Willi syndrome (PWS) were studied clinically and cytogenetically. The patients were classified into three study groups on the basis of their clinical pictures: group 1 with 12 children meeting the strict diagnostic criteria for PWS; group 2 with nine floppy infants and young children, aged 3 years or less, without obesity and hyperphagia; and group 3 with six older children in whom some characteristic features of the syndrome were absent. High-resolution GTG banding of prometaphase chromosomes revealed del(15)(q11.1;q12) in eleven and t(15;15)(qterp11.2::q12qter) in one of the twelve group 1 patients. In group 2, four patients had del(15)(q11.1;q12), one had t(15;15)(qterp11.1::q13qter), and the remaining four had normal karyotypes. The deleted segment common to the 17 patients with the chromosome aberrations was confined to subband 15q11.2. On the other hand, all six group 3 patients had normal karyotypes. These findings indicated that when strictly defined PWS is absolutely correlated with chromosome 15 aberrations, i.e, there is a positive phenotype-karyotype correlation, and that the aberrations are etiologically related to the syndrome. Parental origin of the deleted chromosome was determined in seven patients, with OFQ-heteromorphisms being used as hereditary markers. The deleted chromosome originated from the paternal chromosome 15 in six patients and from the maternal 15 in one.