Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15.

A subset of patients with Angelman and Prader-Willi syndrome have apparently normal chromosomes of biparental origin, but abnormal DNA methylation at several loci within chromosome 15q11-13, and probably have a defect in imprinting. Using probes from a newly established 160-kb contig including D15S63 (PW71) and SNRPN, we have identified inherited microdeletions in two AS families and three PWS families. The deletions probably affect a single genetic element that we term the 15q11-13 imprinting centre (IC). In our model, the IC regulates the chromatin structure, DNA methylation and gene expression in cis throughout 15q11-13. Mutations of the imprinting centre can be transmitted silently through the germline of one sex, but appear to block the resetting of the imprint in the germline of the opposite sex.

Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

Genomic imprinting and candidate genes in the Prader-Willi and Angelman syndromes.

The Prader-Willi and Angelman syndromes are now well established as the paradigm of genomic imprinting in human disease. Over the past year, much has been learnt about the mechanisms by which these syndromes arise and molecular diagnostics for the majority of patients are now available. Mouse models for aspects of the syndromes have been established, and the first association between a gene, located in chromosome 15, at 15q11-q13, and a phenotype (albinism) has been proven. Large parts of the critical regions have been cloned and at least six genes identified. Three genes or DNA sequences may be imprinted: two of these demonstrate DNA-methylation imprints and one is functionally imprinted in mouse. While the molecular mechanism of imprinting is not yet understood, it is beginning to yield its secrets to DNA methylation, replication, and chromatin structure studies of the phenomenon.

Imprinting in Prader-Willi and Angelman syndromes.

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

Hormonal and metabolic defects in a prader-willi syndrome mouse model with neonatal failure to thrive.

Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d 2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS.

BAC microarray analysis of 15q11-q13 rearrangements and the impact of segmental duplications.

Chromosome 15q11-q13 is one of the most variable regions of the human genome, with numerous clinical rearrangements involving a dosage imbalance. Multiple clusters of segmental duplications are found in the pericentromeric region of 15q and at the breakpoints of proximal 15q rearrangements. Using sequence maps and previous global analyses of segmental duplications in the human genome, a targeted microarray was developed to detect a wide range of dosage imbalances in clinical samples. Clones were also chosen to assess the effect of paralogous sequences in the array format. In 19 patients analysed, the array data correlated with microsatellite and FISH characterisation. The data showed a linear response with respect to dosage, ranging from one to six copies of the region. Paralogous sequences in arrayed clones appear to respond to the total genomic copy number, and results with such clones may seem aberrant unless the sequence context of the arrayed sequence is well understood. The array CGH method offers exquisite resolution and sensitivity for detecting large scale dosage imbalances. These results indicate that the duplication composition of BAC substrates may affect the sensitivity for detecting dosage variation. They have important implications for effective microarray design, as well as for the detection of segmental aneusomy within the human population.

Distinct phenotypes distinguish the molecular classes of Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a severe neurobehavioural disorder caused by defects in the maternally derived imprinted domain located on 15q11-q13. Most patients acquire AS by one of five mechanisms: (1) a large interstitial deletion of 15q11-q13; (2) paternal uniparental disomy (UPD) of chromosome 15; (3) an imprinting defect (ID); (4) a mutation in the E3 ubiquitin protein ligase gene (UBE3A); or (5) unidentified mechanism(s). All classical patients from these classes exhibit four cardinal features, including severe developmental delay and/or mental retardation, profound speech impairment, a movement and balance disorder, and AS specific behaviour typified by an easily excitable personality with an inappropriately happy affect. In addition, patients can display other characteristics, including microcephaly, hypopigmentation, and seizures. METHODS: We restricted the present study to 104 patients (93 families) with a classical AS phenotype. All of our patients were evaluated for 22 clinical variables including growth parameters, acquisition of motor skills, and history of seizures. In addition, molecular and cytogenetic analyses were used to assign a molecular class (I-V) to each patient for genotype-phenotype correlations. RESULTS: In our patient repository, 22% of our families had normal DNA methylation analyses along 15q11-q13. Of these, 44% of sporadic patients had mutations within UBE3A, the largest percentage found to date. Our data indicate that the five molecular classes can be divided into four phenotypic groups: deletions, UPD and ID patients, UBE3A mutation patients, and subjects with unknown aetiology. Deletion patients are the most severely affected, while UPD and ID patients are the least. Differences in body mass index, head circumference, and seizure activity are the most pronounced among the classes. CONCLUSIONS: Clinically, we were unable to distinguish between UPD and ID patients, suggesting that 15q11-q13 contains the only significant maternally expressed imprinted genes on chromosome 15.

The human TYRO3 gene and pseudogene are located in chromosome 15q14-q25.

Partial cDNAs of the human TYRO3 gene, encoding a putative receptor tyrosine kinase, and its processed pseudogene (TYRO3P) were cloned from human teratocarcinoma cell, bone marrow and melanocyte cDNA libraries. The tyrosine kinase homologous domains of TYRO3 and TYRO3P were sequenced and compared with each other and with the mouse TYRO3 gene. Abundant levels of the 4.2-kb TYRO3 mRNA were detected in human brain, and lower levels in other human tissues. TYRO3 and TYRO3P were both assigned to human chromosome 15q14-q25 by analysis of DNAs from somatic cell hybrids.

Genomic imprinting and uniparental disomy in Angelman and Prader-Willi syndromes: a review.

Although Angelman (AS) and Prader-Willi (PWS) syndromes are human genetic disorders with distinctly different developmental and neurobehavioural phenotypes, they both have abnormalities in inheritance of chromosome 15q11-q13. Whether AS or PWS arises depends on the parental origin of a deletion or uniparental disomy (the inheritance of 2 copies of a genetic locus from only one parent) for 15q11-q13. Normal development requires a genetic contribution for this genetic region from both a male and a female parent. The dependence on parental origin implies that genes in human 15q11-q13 have distinct functions depending upon epigenetic, parent-of-origin differences, known as genomic imprinting. Here, I review the role of uniparental disomy and genomic imprinting in the pathogenesis of AS and PWS, and briefly discuss phenotype-genotype correlations using candidate genes and mouse models, in particular for hypopigmentation.

DNA methylation patterns in human tissues of uniparental origin using a zinc-finger gene (ZNF127) from the Angelman/Prader-Willi region.

In order to further our understanding of the epigenetic modifications of DNA and its role in imprinting, we examined DNA methylation patterns of human tissues of uniparental origin. We used complete hydatidiform moles (CHM), which are totally androgenetic conceptions, to examine the paternal methylation pattern in the absence of a maternal contribution and we used ovarian teratomas to represent the maternal counterpart. We carried out an analysis of DNA methylation of a gene which has been shown to contain sites which are differentially methylated in a parent-specific fashion. The gene, ZNF127, is located on chromosome 15q11-q13 in the region associated with Prader-Willi and Angelman syndromes. The parent-of-origin DNA methylation has been postulated to reflect the presence of an imprint and recent studies have confirmed that ZNF127 is differentially expressed only from the paternal chromosome. We identified a unique pattern of hyper- and hypomethylated sites in androgenetic conceptions which was nearly identical to the paternal pattern found in sperm. This may represent the paternal germ-line methylation imprint. We also studied partial hydatidiform moles, non-molar triploid conceptions, normal chorionic villi, and somatic tissue. These all demonstrated a modified DNA methylation pattern characteristic of normal chorionic villi with only limited findings of the imprint. Our results suggest that human androgenetic conceptions may provide an excellent model to analyze epigenetic DNA modifications, such as methylation, in imprinted genes. The paternal allele-specific methylation imprint will also be useful clinically to confirm the androgenetic nature of suspected molar conceptions in which parental blood samples may not be available.

Comparison of phenotype between patients with Prader-Willi syndrome due to deletion 15q and uniparental disomy 15.

Prader-Willi syndrome (PWS) is a complex multiple anomaly syndrome that has been shown to result from deficient expression of paternal chromosome 15(q11-q13). In most cases, it is caused either by deletion of this region in the paternally inherited chromosome 15 or by maternal uniparental disomy (UPD) of chromosome 15. In order to determine whether there are phenotypic differences between patients whose PWS is caused by these two different mechanisms, 54 affected individuals (37 with deletion, 17 with UPD) were personally examined and studied using molecular techniques. The previously recognized increased maternal age in patients with UPD and increased frequency of hypopigmentation in those with deletion were confirmed. Although the frequency and severity of most other manifestations of PWS did not differ significantly between the two groups, those with UPD were less likely to have a "typical" facial appearance. In addition, this group was less likely to show some of the minor manifestations such as skin picking, skill with jigsaw puzzles, and high pain threshold. Females and those with UPD were also older, on average. Possible mechanisms by which these differences could occur and the implications of these differences for diagnosis are described.

Difference in methylation patterns within the D15S9 region of chromosome 15q11-13 in first cousins with Angelman syndrome and Prader-Willi syndrome.

Abnormalities of chromosome region 15q11-13 are associated with Angelman syndrome (AS) and Prader-Willi syndrome (PWS). Differences between the methylation patterns of the region of chromosome 15q11-13 which hybridizes to the highly conserved DNA, DN34, in normal individuals and in patients with AS and PWS have been described. We report on a family in which first cousins are affected by AS and PWS as a result of a familial paracentric inversion of 15q11-q13. The results of the studies on this family demonstrate the differences in the methylation patterns in the 2 conditions and the phenomenon of genomic imprinting, whereby genetic information is expressed differently dependent on the parent of origin.

Angelman and Prader-Willi syndrome: a magnetic resonance imaging study of differences in cerebral structure.

Recent improvements in magnetic resonance imaging techniques now allow the developing brain to be visualized in sufficient detail to perform "in vivo neuropathology." In this study we compared the cortical morphology in six children with Angelman and four with Prader-Willi syndrome. These two syndromes are of special interest because, although they are both caused by deletions in the same region of chromosome 15, Angelman children are far more severely affected, and do not speak. We measured the length of the banks of the Sylvian fissure in a gapless series of thin sagittal images. Angelman children had a significantly larger proportion (75%) of anomalous fissures than the Prader-Willi children (12%). Anomalous cortical growth could result from mistimed expression and recognition of macromolecules involved in axonal guidance, target recognition, and pruning. We hypothesize that misrouting of long projection axons may be related to the Sylvian fissure anomalies and the language disorder in Angelman syndrome.

Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion.

Many Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients have a cytogenetic deletion of 15q11q13. While AS and PWS share a similar cytogenetic anomaly, they have very different clinical phenotypes. DNAs from 4 AS patients were examined using 5 chromosome 15q11q13-specific cloned DNA segments. With the present level of resolution, the molecular deletions between AS and those previously reported for PWS did not appear to differ. However, in contrast to the paternal inheritance of the deleted chromosome 15 observed in the majority of PWS patients, maternal inheritance of the deleted chromosome 15 was demonstrated in the AS patients by restriction fragment length polymorphisms (RFLPs).

Restriction fragment length polymorphisms within proximal 15q and their use in molecular cytogenetics and the Prader-Willi syndrome.

Restriction fragment length polymorphisms (RFLPs) are described in detail for 6 DNA probes (D15S9-13, D15S18) that localize to the proximal long arm of human chromosome 15 (15q11-15q13: this report and Tantravahi et al., Am. J. Med. Genet. 33:78-87. Multiple RFLPs are detected by the probe that identifies locus D15S13, and these RFLPs are shown by genomic mapping to result from a nearby insertion or deletion of 1.8 kilobases (kb) of DNA. This set of RFLPs detected by proximal 15q probes can be used for studies on the Prader-Willi syndrome (PWS) and on mentally retarded individuals with a supernumerary inv dup(15) chromosome. Five of the polymorphic loci (D15S9-13) map to the region implicated in the cause of the PWS (15q11.2-15q12). Each of 4 families tested with these probes, as well as an additional "PWS-like" patient, was informative by RFLP analysis. The two PWS deletions studied, which occurred de novo, were inherited from the chromosome 15 provided by the father. By contrast, the 2 inv dup(15) chromosomes analyzed were of maternal origin. The use of RFLPs can also simplify the molecular determination of copy number in chromosomal aneuploidy, as exemplified by analysis of individuals with the PWS and a deletion, patients with an inv dup(15), and one patient with a more complex rearrangement involving chromosome 15. Our studies demonstrate the application of DNA probes for both molecular cytogenetic studies on this chromosome region and the development of diagnostic molecular markers to aid early clinical diagnosis of the PWS.

Pericentric inversion of chromosome 16 in a large kindred: spectrum of morbidity and mortality in offspring.

Constitutional pericentric inversions of chromosome 16 are rare in the general population. We report here a large kindred who carry an inv(16)(p13q22) rearrangement. In general, individuals with the inv(16) are in good health but prone to reproductive loss. Two different types of recombinant offspring were identified in this family and analyzed at the molecular level using probes from the alpha-globin and polycystic kidney disease loci. Both were associated with serious major malformations.

Quantitative calibration and use of DNA probes for investigating chromosome abnormalities in the Prader-Willi syndrome.

Ten genomic DNA probes, subcloned from inserts derived from a phage library constructed from the DNA of flow-sorted chromosomes, have now been mapped to locations within 15q11-15q13. By dosage blotting and densitometry, 5 of these probes map to the 15q11.2-15q12 segment missing in one 15 chromosome of a Prader-Willi syndrome (PWS) patient with a prominent cytological deletion. A sixth probe most likely maps to the same region. The other 4 probes map outside of this segment but within 15q11-15q13. Several of the 15q11.2-15q12 probes, and a cDNA probe homologous to one, have been used to test the DNA from 8 patients exhibiting a wide range of the clinical manifestations expected for PWS patients. DNA deletion was observed in all 3 patients with cytological 15q1 deletions as well as in a patient with an unbalanced (Y;15) translocation. DNA from 1 PWS patient with an unbalanced (5;15) translocation and an inverted duplication of the short arm and proximal long arm of 15 showed at least 1 and possibly 2 extra copies of each genomic probe tested. In the other 3 patients with no cytological deletions, no DNA deletions were found. Thus, the molecular probes described can be used in most PWS patients to analyze the region of proximal 15q implicated in this syndrome.

Unexpected familial recurrence in Angelman syndrome.

We report on two instances of familial recurrence of Angelman syndrome which, from pedigree analysis, appear incompatible with currently known mechanisms of inheritance of this disorder. In these two families, deletion-positive Angelman syndrome has recurred in cousins. Several established mechanisms for deletion-positive familial recurrence have been ruled out. In each family, molecular cytogenetic studies show typical chromosome 15 deletions, and DNA methylation analysis verifies the maternal origin of the deleted chromosomes in all four individuals. Since the mothers of the affected individuals in each family are not known to be related, these recurrences appear to be secondary to coincidental, de novo events. This conclusion is consistent with direct and indirect estimates of the population frequency of Angelman syndrome.

Hypopigmentation in the Prader-Willi syndrome correlates with P gene deletion but not with haplotype of the hemizygous P allele.

The Prader-Willi syndrome (PWS) usually results from a paternal deletion of 15q11-q13 or maternal disomy for chromosome 15. Reduced pigmentation of skin, hair, and eyes is common in PWS and was suggested previously to be associated with the 15q11-q13 deletion. The P gene, located in this same region, is associated with OCA2, an autosomal recessive disorder that is the most frequent form of tyrosinase-positive oculocutaneous albinism. We studied 28 individuals with PWS and found that hemizygosity for the P gene was significantly correlated with the occurrence of hypopigmentation among PWS patients. However, we found little or no relationship between the occurrence of hypopigmentation and the polymorphism haplotype of the intact P allele. Thus, our results indicate that hypopigmentation is likely the result of deletion of the P gene in the context of PWS but do not support the linked hypothesis that hypopigmentation results from hemizygosity for variant P alleles with reduced function.