First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

Comprehensive methylation analysis in typical and atypical PWS and AS patients with normal biparental chromosomes 15.

Imprinting defects in 15q11-q13 are a rare but significant cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Patients with an imprinting defect have apparently normal chromosomes 15 of biparental origin, but are recognised by @parental DNA methylation at D15S63 (PW71) or SNURF-SNRPN exon 1. We have investigated the methylation status of five additional loci in 12 such patients with or without a deletion in the imprinting centre. In each patient, the imprinting defect affected all loci tested. During routine diagnostic testing we identified four patients who had a normal methylation pattern at SNURF-SNRPN exon 1, but an abnormal pattern at D15S63. In two of these patients, who were suspected of having PWS, this change was restricted to D15S63. In two patients suspected of having AS, several but not all loci were affected. Using a newly developed methylation-specific PCR test for D15S63 we found that these methylation changes are rare in patients suspected of having AS. Although we can not prove that the methylation changes in the four patients are causally related to their disease, our findings demonstrate that spatially restricted changes in methylation can occur. In some cases, these changes may reflect incomplete imprint spreading.

Disruption of the bipartite imprinting center in a family with Angelman syndrome.

Imprinting in 15q11-q13 is controlled by a bipartite imprinting center (IC), which maps to the SNURF-SNRPN locus. Deletions of the exon 1 region impair the establishment or maintenance of the paternal imprint and can cause Prader-Willi syndrome (PWS). Deletions of a region 35 kb upstream of exon 1 impair maternal imprinting and can cause Angelman syndrome (AS). So far, in all affected sibs with an imprinting defect, an inherited IC deletion was identified. We report on two sibs with AS who do not have an IC deletion but instead have a 1-1.5 Mb inversion separating the two IC elements. The inversion is transmitted silently through the male germline but impairs maternal imprinting after transmission through the female germline. Our findings suggest that the close proximity and/or the correct orientation of the two IC elements are/is necessary for the establishment of a maternal imprint.

Maternal methylation imprints on human chromosome 15 are established during or after fertilization.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the lack of transcripts expressed from the paternal copy of the imprinted chromosomal region 15q11-q13 (refs. 1,2). In some patients, this is associated with a deletion of the SNURF-SNRPN exon 1 region inherited from the paternal grandmother and the presence of a maternal imprint on the paternal chromosome. Assuming that imprints are reset in the germ line, we and others have suggested that this region constitutes part of the 15q imprinting center (IC) and is important for the maternal to paternal imprint switch in the male germ line. Here we report that sperm DNA from two males with an IC deletion had a normal paternal methylation pattern along 15q11-q13. Similar findings were made in a mouse model. Our results indicate that the incorrect maternal methylation imprint in IC deletion patients is established de novo after fertilization. Moreover, we found that CpG-rich regions in SNURF-SNRPN and NDN, which in somatic tissues are methylated on the maternal allele, are hypomethylated in unfertilized human oocytes. Our results indicate that the normal maternal methylation imprints in 15q11-q13 also are established during or after fertilization.

A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15.

Balanced translocations affecting the paternal copy of 15q11--q13 are a rare cause of Prader-Willi syndrome (PWS) or PWS-like features. Here we report on the cytogenetic and molecular characterization of a de novo balanced reciprocal translocation t(X;15)(q28;q12) in a female patient with atypical PWS. The translocation breakpoints in this patient and two previously reported patients map 70-80 kb distal to the SNURF-SNRPN gene and define a breakpoint cluster region. The breakpoints disrupt one of several hitherto unknown 3' exons of this gene. Using RT--PCR we demonstrate that sequences distal to the breakpoint, including the recently identified C/D box small nucleolar RNA (snoRNA) gene cluster HBII-85 as well as IPW and PAR1, are not expressed in the patient. Our data suggest that lack of expression of these sequences contributes to the PWS phenotype.

Imprinting centre deletions in two PWS families: implications for diagnostic testing and genetic counseling.

Prader-Willi syndrome (PWS) is a complex genetic syndrome involving imprinted genes on chromosome 15. It is usually sporadic, and very few affected siblings have been described. Here, we report the clinical and molecular findings in two families with a microdeletion affecting the chromosome 15 imprinting centre (IC). Carrier males have a 50% risk of having children with an imprinting defect leading to PWS, and in one of the two families, a father has two affected daughters. In the other family, diagnostic testing was confounded by the presence of a neutral microdeletion close to the IC. The silent transmission of PWS IC deletions through the female germline and the occurrence of neutral microdeletions close to the IC can impose considerable problems on diagnostic testing and genetic counselling in affected families.

A 28-kb deletion spanning D15S63 (PW71) in five families: a rare neutral variant?

Methylation analysis with probe PW71 (D15S63) is an established procedure to test patients suspected of having Prader-Willi syndrome or Angelman syndrome. Using this test, we have identified a 28-kb deletion spanning D15S63 in five independent families. Sequence analysis revealed identical breakpoints in all the families. The haplotype data are compatible with a common ancestral origin of the deletion in at least two families. The deletion was not found in 1, 000 unrelated controls. Although the deletion maps within the imprinting-center region, neither maternal nor paternal inheritance of the deletion appears to affect imprinting in proximal 15q. We conclude that the deletion is a rare neutral variant that can lead to false-positive results in the PW71-methylation test.

A previously unrecognised phenotype characterised by obesity, muscular hypotonia, and ability to speak in patients with Angelman syndrome caused by an imprinting defect.

The clinical features of Angelman syndrome (AS) comprise severe mental retardation, postnatal microcephaly, macrostomia and prognathia, absence of speech, ataxia, and a happy disposition. We report on seven patients who lack most of these features, but presented with obesity, muscular hypotonia and mild mental retardation. Based on the latter findings, the patients were initially suspected of having Prader-Willi syndrome. DNA methylation analysis of SNRPN and D15S63, however, revealed an AS pattern, ie the maternal band was faint or absent. Cytogenetic studies and microsatellite analysis demonstrated apparently normal chromosomes 15 of biparental inheritance. We conclude that these patients have an imprinting defect and a previously unrecognised form of AS. The mild phenotype may be explained by an incomplete imprinting defect or by cellular mosaicism.

A 5-kb imprinting center deletion in a family with Angelman syndrome reduces the shortest region of deletion overlap to 880 bp.

Imprinting on human chromosome 15q11-q13 is controlled by a bipartite imprinting center (IC) that maps to the SNRPN locus. Deletions of the IC result in an imprinting defect and Prader-Willi syndrome or Angelman syndrome (AS). We have now identified a 5-kb IC deletion in an English AS patient (AS-LO); this represents the smallest microdeletion found in AS and narrows down the shortest region of deletion overlap to 880 bp.

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.

Different mechanisms and recurrence risks of imprinting defects in Angelman syndrome.

Angelman syndrome (AS) is a neurogenetic disorder that appears to be caused by the loss of function of an imprinted gene expressed from maternal chromosome 15 only. Approximately 6% of patients have a paternal imprint on the maternal chromosome. In the few cases, this is due to an inherited microdeletion, in the 15q11-q13 imprinting center (IC), that blocks the paternal-->maternal imprint switch in the maternal germ line. We have determined the segregation of 15q11-q13 haplotypes in nine families with AS and with an imprinting defect. One family, with two affected siblings, has a microdeletion affecting the IC transcript. In the other eight patients, no mutation was found at this locus. In two families, the patient and a healthy sibling share the same maternal alleles. In one of these families and in two others, grandparental DNA samples were available, and the chromosomes with the imprinting defect were found to be of grandmaternal origin. These findings suggest that germ-line mosaicism or de novo mutations account for a significant fraction of imprinting defects, among patients who have an as-yet-undetected mutation in a cis-acting element. Alternatively, these data may indicate that some imprinting defects are caused by a failure to maintain or to reestablish the maternal imprint in the maternal germ line or by a failure to replicate the imprint postzygotically. Depending on the underlying cause of the imprinting defect, different recurrence risks need to be considered.

A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus.

The analysis of allelic methylation differences in 15q11-q13 has been established as a valid test for the Angelman and Prader-Willi syndromes. Current tests use methylation-sensitive restriction enzymes and Southern blot analysis. Here we describe a single-tube PCR test. It is based on sodium bisulfite treatment of DNA, which converts unmethylated, but not methylated cytosine residues to uracil, and PCR primers specific for the maternal and the paternal allele. The method was validated in a blinded retrospective study on 87 DNA samples from normal controls and patients. Prospective studies by independent laboratories will be needed before this assay can replace Southern blot analysis in routine diagnostic procedures.

Familial translocations involving 15q11-q13 can give rise to interstitial deletions causing Prader-Willi or Angelman syndrome.

A de novo interstitial deletion of 15q11-q13 is the major cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Here we describe two unrelated PWS patients with a typical deletion, whose fathers have a balanced translocation involving the PWS/AS region. Microsatellite data suggest that the deletion is the result of an unequal crossover between the derivative chromosome 15 and the normal chromosome 15. We conclude that familial translocations involving 15q11-q13 can give rise to interstitial deletions causing PWS or AS and that prenatal diagnosis in such families should include fluorescence in situ hybridisation or microsatellite studies or both.