Molecular and clinical studies in 8 patients with Temple syndrome.

Temple syndrome (TS14, #616222) is a rare imprinting disorder characterised by phenotypic features including pre- and postnatal growth retardation, muscular hypotonia and feeding difficulties in infancy, early puberty and short stature with small hands and feet and often truncal obesity. It is caused by maternal uniparental disomies, paternal deletions and primary imprinting defects that affect the chromosomal region 14q32 and lead to a disturbed expression of imprinted genes in this region. Here, we present detailed clinical data of 8 patients with Temple syndrome, 4 with an imprinting defect, 2 with an imprinting defect in a mosaic state as well as 1 complete and 1 segmental maternal uniparental disomy of chromosome 14.

Expanding the clinical spectrum of the 'HDAC8-phenotype' - implications for molecular diagnostics, counseling and risk prediction.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin-related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of 11 patients carrying 10 distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS-overlapping features. The phenotype of our patients is very broad, whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X-inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counseling and risk prediction.

Molecular and Clinical Aspects of Angelman Syndrome.

The Angelman syndrome is caused by disruption of the UBE3A gene and is clinically delineated by the combination of severe mental disability, seizures, absent speech, hypermotoric and ataxic movements, and certain remarkable behaviors. Those with the syndrome have a predisposition toward apparent happiness and paroxysms of laughter, and this finding helps distinguish Angelman syndrome from other conditions involving severe developmental handicap. Accurate diagnosis rests on a combination of clinical criteria and molecular and/or cytogenetic testing. Analysis of parent-specific DNA methylation imprints in the critical 15q11.2-q13 genomic region identifies 75-80% of all individuals with the syndrome, including those with cytogenetic deletions, imprinting center defects and paternal uniparental disomy. In the remaining group, UBE3A sequence analysis identifies an additional percentage of patients, but 5-10% will remain who appear to have the major clinical phenotypic features but do not have any identifiable genetic abnormalities. Genetic counseling for recurrence risk is complicated because multiple genetic mechanisms can disrupt the UBE3A gene, and there is also a unique inheritance pattern associated with UBE3A imprinting. Angelman syndrome is a prototypical developmental syndrome due to its remarkable behavioral phenotype and because UBE3A is so crucial to normal synaptic function and neural plasticity.

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.

Silver-Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues.

In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome-specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non-affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.

Malignant melanoma and Wiedemann-Beckwith syndrome in childhood.

Patients with Wiedemann-Beckwith syndrome (WBS, MIM 130650), a congenital overgrowth syndrome, have a known increased tumor risk especially for embryonic tumors. WBS belongs to the "imprinting" syndromes caused by overexpression of IGF2 and/or loss of CDKN1C on chromosome 11p15.5. A 13-year-old boy with WBS developed a spitzoid malignant melanoma (Clark level V, Breslow index 4.8 mm) on the right cheek. Genetic analyses of the patient's blood showed hypermethylation at the H19 locus on chromosome 11p. The (epi)genetic changes of the WBS locus might have played a role in the pathogenesis of melanoma development.

Slow progression of a small Wilms' tumor.

We report the uncommon clinical course of a female with right-sided hemi-hyperplasia. At the age of 2 years and 2 months, a small spherical lesion of the right kidney was detected by ultrasound and magnetic resonance tomography. When the patient was 4 years and 7 months, the very slowly growing tumor was removed completely and diagnosed as intermediate risk stage I nephroblastoma. The case demonstrates that even small renal lesions require diagnostic work-up and adequate treatment.

Use of multiplex ligation-dependent probe amplification increases the detection rate for 11p15 epigenetic alterations in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) describes a malformation syndrome with severe intrauterine and postnatal growth retardation. Currently, two major (epi)mutations have been described: while approximately 10% of patients carry a maternal uniparental disomy of chromosome 7 (UPD7), 35-60% show a hypomethylation at the H19 differentially methylated regions (DMRs) in 11p15. Until recently, a Southern-blot based test was routinely used to identify epimutation carriers. Nevertheless, this test was time consuming and hampered by the huge amount of genomic DNA needed. With the methylation-specific multiplex ligation-dependent probe amplification assay (MLPA) for SRS, a PCR-based test is now available, allowing the analysis also of small amounts of DNA. Probes in this assay hybridize to the H19 DMRs but do not cover the genomic target of the Southern-blot probe. We now screened 72 patients with SRS by MLPA. Hypomethylation of the H19 DMRs was confirmed in all patients analyzed by Southern blot. In addition, we identified six individuals with hypomethylation of the H19 DMR who had previously normal blot results. This discrepancy can be explained by the observed generally lower degree of demethylation in this group, possibly not detectable by the less sensitive Southern-blot method but also with a varying degree of methylation at different DMRs in the same individual. Apart from hypomethylation in the H19 DMR, we observed a slight demethylation for one of the IGF2 probes. The total detection rate of 11p15 hypomethylation is now increased to >38%. Considering maternal UPD7 and chromosomal aberrations, (epi)genetic alterations now account for more than 50% of SRS patients. In summary, MLPA represents an easy, low cost and reliable system in the molecular diagnostics of SRS.

Imprinting defects on human chromosome 15.

The Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurogenetic diseases that are caused by the loss of function of imprinted genes on the proximal long arm of human chromosome 15. In a few percent of patients with PWS and AS, the disease is due to aberrant imprinting and gene silencing. In patients with PWS and an imprinting defect, the paternal chromosome carries a maternal imprint. In patients with AS and an imprinting defect, the maternal chromosome carries a paternal imprint. Imprinting defects offer a unique opportunity to identify some of the factors and mechanisms involved in imprint erasure, resetting and maintenance. In approximately 10% of cases the imprinting defects are caused by a microdeletion affecting the 5' end of the SNURF-SNRPN locus. These deletions define the 15q imprinting center (IC), which regulates imprinting in the whole domain. These findings have been confirmed and extended in knock-out and transgenic mice. In the majority of patients with an imprinting defect, the incorrect imprint has arisen without a DNA sequence change, possibly as the result of stochastic errors of the imprinting process or the effect of exogenous factors.

Molecular characterization of a unique de novo 15q deletion associated with Prader-Willi syndrome and central visual impairment.

We report a 2-year-old boy with Prader-Willi Syndrome (PWS) caused by a deletion of the PWS critical region as a result of an unbalanced translocation t(3;15). Additional features, including central visual impairment, relative macrocephaly, retrognathia, preauricular tags, and bilateral club-feet, were noticed. The extension of the deletion was determined by fluorescence in situ hybridization (FISH) analysis using 11 region-specific YAC clones. Nine YACs were found to be deleted, allowing us to determine that the deletion is larger than in patients with typical PWS deletions. The karyotype of this patient can thus be designated: 45,XY,-15,der(3)t(3;15)(qter;q14).ish der(3)t(3;15)(qter;q14) (wcp3+,wcp15+,D15S10-,PML+,D15Z1-,D3S4560+,801_f_9x1, 815_e_6x2) de novo. Molecular analyses using seven polymorphic markers helped to narrow down the breakpoint between marker ACTC.PC3 and the distal end of the YAC 815_e_6. These results provide evidence that haploinsufficiency for genes in 15q13-q14, not affected in common PWS deletions, is associated with the additional features found in the patient, including a central visual impairment.

The IC-SNURF-SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A.

The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the coordinated control of an imprinting center (IC) at the 5' end of the SNURF-SNRPN gene. One gene cluster spans the centromeric part of this domain and contains several genes that are transcribed from the paternal chromosome only (MKRN3, MAGEL2, NDN, SNURF-SNRPN, HBII-13, HBII-85 and HBII-52). Apart from the HBII small nucleolar RNA (snoRNA) genes, each of these genes is associated with a 5' differentially methylated region (DMR). The second gene cluster maps to the telomeric part of the imprinted domain and contains two genes (UBE3A and ATP10C), which in some tissues are preferentially expressed from the maternal chromosome. So far, no DMR has been identified at these loci. Instead, maternal-only expression of UBE3A may be regulated indirectly through a paternally expressed antisense transcript. We report here that a processed antisense transcript of UBE3A starts at the IC. The SNURF-SNRPN sense/UBE3A antisense transcription unit spans more than 460 kb and contains at least 148 exons, including the previously identified IPW exons. It serves as the host for the previously identified HBII-13, HBII-85 and HBII-52 snoRNAs as well as for four additional snoRNAs (HBII-436, HBII-437, HBII-438A and HBII-438B), newly identified in this study. Almost all of those snoRNAs are encoded within introns of this large transcript. Northern blot analysis indicates that most if not all of these snoRNAs are indeed expressed by processing from these introns. As we have not obtained any evidence for other genes in this region, which, from the mouse data appears to be critical for the neonatal Prader-Willi syndrome phenotype, a lack of these snoRNAs may be causally involved in this disease.

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.

Marked differences in unilateral isolated retinoblastomas from young and older children studied by comparative genomic hybridization.

Although it is established that the loss of function of both alleles of the RB1 gene is a prerequisite for the development of retinoblastoma, little is known about the genetic events that are required for tumor progression. We used comparative genomic hybridization (CGH) to search for DNA copy number changes in isolated unilateral retinoblastomas. From a series of 66 patients with retinoblastomas with somatic mutations in both RB1 alleles, tumor samples from 13 children with the youngest (2.0-9.8 months) and 13 with the oldest (36.2-84.1 months) age at operation were studied. Loss at 13q14, the location of RB1, was demonstrated in two tumors only. Recurring chromosome imbalances included gains at 6p (11/26), 1q (10/26), 2p (4/26), and 17q (4/26), gains of the entire chromosome 19 (3/26), and losses at 16q (9/26). A commonly gained region at 1q32 was identified. Increased dosage of GAC1, a candidate oncogene located in 1q32, was found in two of four tumors by Southern blot analysis. Comparison of the CGH findings revealed that retinoblastomas from children with an older age at operation showed significantly more frequent (13/13 cases vs 4/13 cases; P = 0.0005) and more complex genetic abnormalities (median, 5 changes/abnormal tumor vs median, 1.5 changes/abnormal tumor; P = 0.003) than retinoblastomas from children with a young age at operation. Gains at 1q, 2p, 17q, of the entire chromosome 19 and losses of 16q were restricted to the older age group. Our results suggest that the progression of retinoblastomas from older patients follows mutational pathways different from those of younger patients.

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.

The 28-kb deletion spanning D15S63 is a polymorphic variant in the Ashkenazi Jewish population.

D15S63 is one of the loci, on chromosome 15q11-q13, that exhibit parent-of-origin dependent methylation and that is commonly used in the diagnosis of Prader-Willi or Angelman syndromes (PWS/AS). A 28-kb deletion spanning the D15S63 locus was identified in five unrelated patients; in each of them the deletion was inherited from a normal parent. Three of the five families segregating the deletion were reported to be of Jewish Ashkenazi ancestry, and in the other two families the ancestral origin was unknown. To determine whether the 28-kb deletion is a benign variant, we screened for the deletion in 137 unselected Ashkenazi individuals and in 268 patients who were referred for molecular diagnosis of PWS/AS, of whom 89 were Ashkenazi and 47 were of mixed origin (Ashkenazi and non-Ashkenazi Jews). In the control group, three individuals were carriers of the deletion; among the patients, three were carriers, all of whom were Ashkenazi Jews. There was no significant difference between the control group and the Ashkenazi patients, indicating that the deletion is not a cause of PWS- and AS-like syndromes. The frequency of the 28-kb deletion in the Ashkenazi population was 1/75. Since methylation analysis at the D15S63 locus may lead to misdiagnosis, we suggest the use of SNRPN, either in a PCR-based assay or as a probe in Southern hybridization, as the method of choice in the diagnosis of PWS/AS.