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Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome

Molecular breakpoint cloning and gene expression studies of a novel translocation... Background: Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2). Methods: We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Results: Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. Conclusion: As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype. Page 1 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 copy snoRNA genes (HBII-436, HBII-13, and HBII-437), Background Prader-Willi syndrome (PWS) is a complex neurodevelop- and one snoRNA gene (HBII-438) present in two copies mental disorder and a classic example for genomic that are 240 kb apart [19]. Since the snoRNAs are derived imprinting in humans. The incidence is about 1 in 10– from processed spliced-out introns, their expression is 20,000, and the clinical manifestations include decreased controlled by the SNRPN promoter and is highest in fetal activity, neonatal hypotonia, neonatal feeding diffi- brain. The known function of other C/D box snoRNAs is culties, hyperphagia with obesity, hypogonadism, short to guide 2'- O – ribose methylation of ribosomal RNA or stature, small hands and feet, characteristic facial features, small nuclear RNA. This post-transcriptional modification and mild to moderate mental retardation. Diagnostic cri- is conserved throughout evolution and is thought to con- teria have been proposed [1] and revised recently [2]. fer increased stability to the small RNA molecules [23]. The modification targets of the imprinted C/D box snoR- About 70% of individuals clinically diagnosed with PWS NAs in the PWS/AS region are still unknown. have a ~4 Mb interstitial deletion at 15q11-13 of paternal origin, with clustered breakpoints (BP) at either of two Spontaneous chromosome translocations can be proximal sites (BP1 or BP2) and one on the distal site extremely valuable for assessing the contributions of indi- (BP3) (Fig. 1a). The majority of the remainder have mater- vidual loci to the phenotype of microdeletion syndromes. nal uniparental disomy 15. In about 1 % of the cases, the Five individuals with features of PWS have been reported disease is due to aberrant imprinting and gene silencing. who have balanced reciprocal translocations with break- Of these, 14% have small deletions in the imprinting points in the PWS/AS deletion region. All of them involve center (IC) region of the paternal allele that abolish the the SNRPN locus. The breakpoints are located in intron 2 expression of all imprinted paternally-expressed genes in (proximal, n = 2), disrupting the SNURF/SNRPN coding cis. In the remainder no demonstrable DNA sequence region, or in exon 20a/intron 20 (distal, n = 3) within the changes have been observed [3-7]. In Angelman syn- 3'-untranslated region of the long SNRPN transcript. One drome (AS, MIM#105830), which is usually caused by the individual with a proximal and two of three patients with same mechanisms affecting the maternal chromosome a distal breakpoint meet the diagnostic criteria for PWS 15, mutations in the maternal copy of a single gene, (score of 8 or more points) [20,24-28]. UBE3A (MIM#601623), encoding a ubiquitin ligase, are detected in about 5% of cases, whereas in PWS, no dis- Here we report the clinical, cytogenetic and molecular ease-causing mutations in a single imprinted gene have characterization of a 22 year old male with features of yet been reported. PWS who has a different de novo balanced reciprocal trans- location t(4;15)(q27;q11.2). We mapped the breakpoint Three paternally expressed genes have been identified to SNRPN intron 17 (position on chr 15: 22803227, between BP2 and SNRPN. These include MKRN3/ UCSC Genome browser May 2004) and determined the ZNF127 (MIM# 603856; Makorin 3 or Zinc finger protein expression of snoRNAs on both sides of the breakpoint in 127) [8,9], MAGEL2/NDNL1 (MIM# 605283; MAGE-like cultured fibroblasts and lymphoblasts. 2 or Necdin-like 1) [10,11], and NDN (MIM# 602117; Necdin) [12,13] (Figure 1a). The small nuclear ribonucle- Methods oprotein polypeptide N (MIM# 182279; SNRPN) gene Cytogenetic and FISH analysis Metaphase spreads obtained from short-term blood lym- was the first gene with a known function to be mapped to the PWS/AS deletion region, and is expressed from the phocyte cultures and Epstein-Barr virus (EBV)-trans- paternal chromosome only [14-17]. Multiple alternatively formed lymphoblastoid cells (LCL) were processed for spliced transcripts originate at the SNPRN promoter [18- high-resolution GTG- banding by standard methods. For 20]. The major SNRPN transcript is bi-cistronic encoding FISH studies, Bacterial Artificial Chromosomes (BACs) two mRNA species. Exons 1–3 encode a protein product were sourced from the RPCI-11 library and selected using of unknown function called SNURF (SNRPN upstream the UCSC Genome Browser, Assemblies: July 2003 and reading frame). Exons 4–10 encode SmN, a homolog of May 2004). Fluorescence labelling, hybridization proce- the SmB/B' protein that binds small nuclear RNAs dures and imaging were performed as previously involved in pre-mRNA splicing. The largest transcripts described [29]. extend over a ~460 kb genomic region and include a large 3'UTR comprising up to 148 exons [19]. DNA methylation study Genomic DNA was purified by phenol-chloroform extrac- Multiple introns downstream of the SNURF-SNRPN cod- tion from LCLs from the study subject, a normal control, ing region contain C/D box small nucleolar RNA and a PWS individual (Patient E in [6], Coriell Human (snoRNA) genes. There are two multi-copy snoRNA clus- Mutant Cell Repository # GM12134). To investigate ters (HBII-52 and PWCR1/HBII-85) [21,22], three single methylation at exon 1 of SNRPN, 50 µg DNA were used Page 2 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Map Figure 1 ping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes Mapping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes . a . Schematic map of human chromosome region 15q11-q13. Black and gray circles represent imprinted genes, expressed from the paternal or maternal allele, respectively. White circles designate bi-allelically expressed genes. BP1, 2, and 3 indicate the locations of the deletion breakpoint hotspots [43]. b . FISH results placed BAC RP11-160D9 highlighted in green (nucleotide position 22577151-22735621) proximal to the translocation breakpoint and RP11-876N20 highlighted in blue (position 22857334- 23036552) distal to the breakpoint. Intron 17, comprising nucleotides 22795282 to 22811656, thus is located ~ 63.4 kb down- stream of RP11-160D9 and ~ 42 kb upstream of RP11-876N20. c . On representation of the SNRPN region (not drawn to scale) boxes represent exons and ESTs, lines represent snoRNA copies. Orange boxes and lines indicate exons, ESTs or snoR- NAs tested for expression either by RT-PCR or quantitative RT-PCR. Black flash indicates the breakpoint in intron 17 of the SNRPN locus. for the bisulfite reaction and PCR with primers according DNaseI (Roche) and RT-PCR was performed using Super- to standard protocols [30,31]. PCR products were sepa- script II (Invitrogen). Primers were designed for exon-to- rated on a 3% agarose gel, stained with ethidium bro- exon amplification in an overlapping fashion – where mide, and visualized under UV illumination. possible – for SNRPN, MKRN3, MAGEL2, NDN, snoR- NAs, and two ESTs within Intron 20 of SNRPN (Table 1). Expression studies by RT-PCR and quantitative RT-PCR Total RNA was extracted from LCL and primary fibroblast For a subset of exons in the SNPRN gene and the snoRNAs cultures (FB) using RNA Stat 60. The RNA was treated with HBII-13, HBII-437, and PWCR1/HBII-85, quantitative Page 3 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 1: Primers and conditions for PCR RT-PCR Gene/Exon fwd (5'-3') rev (5'-3'); complement strand size T ann. ZNF127 GGG TTG CGG TTT TGC TAT TA TTT CTC GTG TGC TTC AAT GC 168 bp 59C MAGEL2 CTG AAG CCT GGG ACT TTC TG GGA CCT TGG CCA CAA ACT TA 225 bp 59C Necdin GAA GAA GCA CTC CAC CTT CG CCA TGA TTT GCA TCT TGG TG 164 bp 59C SNRPN Ex 1–3 ATG GAG CGG GCA AGG GAT CGC GGT ACA ACT GAC ACT CTT GG 124 bp 53C SNRPN Ex 14/16 CTG CAA ACA TAG GAG ATG ATA GTT CC CTT ATG AAA GCA CTG AGA TGA AGC C 459 bp 53C SNRPN Ex 16/17 GAA AGT GAC CTA AAG AGT GTC ATT G CTT GCA GTT GGA CAG CCG ACT C 515 bp 53C SNRPN Ex 17/18 AGA TAT CTT TAA AAT TGA GTC TTC TGT CCA TGA AGA TGC AGC ACT TTT GAA GAA 218 bp 53C SNRPN Ex 19/20a CAT TGT GCT TAT TTA CTA TTT TTG TAG ACG CTG CAG GTG GTG ACC ATG TG 150 bp 53C AK094315 TCT TCT CTA CCC TCA TTC CCA GC TCG CTA CAC CCC TTT GCT TAT G 222 bp 53C AB061718 AGG AGG GGT TCA AAG ATG C CTG GTA AAC AAA CTG GTA AAG GTG 204 bp 50C HBII-85/PWCR1 CGA TGA TGA GTC CCC CAT AAA AAC CAG TTC CGA TGA GAA CGA CG 79 bp 53C HBII-13 GGA TTT GTG ATG AGC TGT GTT TAC GGA CTT CAG AGT AAT CAC GTT G 67 bp 54C HBII-438A/B GGA TCG ATG ATG AGA ATA ATT ATT G GGA CCT CAG ATT GAC ATC TG 67 bp 53C GABRB3 TCA GGC GGC ATT GGC GAT ACC ATA AAA ACT TGA CAG GCA GAG 352 bp 52C GABRA5 AAT ATT GCC TTA ATG TTT CTA GCC TAT TCT ATT TCT TCG TGT 425 bp 48C GABRG3 GCG TAT TCA CAT AGA CAT CTT G GAT TGG TCA CTA CTG GTC TGG 188 bp 52C GAPDH TGG GCT ACA CTG AGC ACC AG GGG TGT CGC TGT TGA AGT CA 50 bp 53C Quantitative RT-PCR Gene/Exon fwd (5'-3') rev (5'-3');complement strand size T ann. SNURF Ex2 ACG AAC TAC AGA ACA GCA CGT ACC CTG CGT TTG ACT TGG ACT TCC 50 bp 60C SNURF Ex3 TTC TCA GCA GCA GCA AGT ACC T TGC CTC AGT TCA GCC TGG A 50 bp 60C HBII-437 ATC ATT ATT TCT TGA ATT GG CCC TCA CGC TCC CTT TGC 50 bp 60C SNRPN Ex 14/15 CTG CAA ACA TAG GAG ATG ATA GTT CC CAA AGA CGA TAA AAT GTT CCT TCT TG 50 bp 60C SNRPN Ex 19/20a GGA ACC ACC ATT TGT CTA TGA TCC CTG CAG GTG GTG ACC ATG TG 50 bp 60C HBII-438 ATA ATT GTC TGA GGA TGC T GAT TGA CAT CTG GAA TGA GTC 50 bp 60C HBII-85/PWCR1 TCG ATG ATG AGT CCC CCA TAA CAT TTT GTT CAG CTT TTC CAA GG 50 bp 60C PCR to generate Southern probes in intron 17 Gene/Exon fwd (5'-3') rev (5'-3');complement strand size T ann. SB 1 ACC ATC AGT GAA TGA CCT GTT GC CCC AGC CTC TTT CCT ATG TCT TG 565 bp 53C SB 3 TGG TAA ACT GAT GAG AGC ACA GCC GCC TGG GAG ACA GAA TGA GAA AC 416 bp 53C RT-PCR assays were performed with SYBR Green I™ dye in Southern blot analysis an ABI 7700 cycler (Applied Biosystems) by using Southern blot analysis was performed according to stand- standard protocols [32,33]. Primers were designed to ard methods with ExpressHyb™ solution (BD Bio- amplify products of 50 bp in length. GAPDH expression sciences). Genomic DNA from a normal individual and was used as a reference. Each sample was run at least in the t(4;15) carrier was cleaved in a double digestion with triplicate. The results were interpreted as described previ- restriction enzymes NheI and BsaWI to release a 6.4 kb ously [28]. fragment, and with NheI and ApaI to release a 10 kb frag- ment in the normal chromosome. The DNA probes were LCL RNA samples from a PWS individual with a microde- synthesized by PCR from genomic DNA and cloned into letion of the imprinting center (GM12134), a normal a pCRII T/A-vector (Invitrogen). The probes were individual, an individual with an intrachromosomal trip- designed to hybridize within intron 16 (SB-1) and lication of the PWS region on the paternally-derived chro- upstream of the ApaI restriction site (SB-3) (Table 1). mosome 15 (Patient 1 in [34], Coriell Human Mutant Cell Repository # GM12135), and fibroblast RNA from Breakpoint cloning with a PCR-based method another t(4;15) PWS individual with the breakpoint in Genomic DNA from a normal individual and the t(4;15) intron 2 of SNRPN [27,28] served as controls. carrier was cleaved in a double digestion with restriction Page 4 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 t(4;15) ca Figure 2rrier at 15 years of age t(4;15) carrier at 15 years of age . Note absence of typical PWS facial features and presence of mild truncal obesity. enzymes EcoRV and ApaI, followed by adapter ligation undescended testes were observed. During infancy, he had according to the manufacturer's instructions (BD Genome poor suck and prolonged feeding times, but his weight Walker Universal Kit) [35]. A nested PCR reaction with gain was satisfactory and he did not require tube feeding. adapter primers and sequence-specific primers was per- He was suspected to have absence seizures of about 20 formed and the amplification products were cloned into seconds duration, along with proneness to giggling, some- the pC2.1 T/A-vector (Invitrogen) after gel purification. times with eye-rolling. These episodes resolved by four The clones were sequenced from both directions with uni- years of age, and an EEG was normal. versal primers from the vector (M13) and sequence spe- cific primers. He had a left esotropia that was surgically corrected. Dur- ing childhood, sticky saliva, dry mouth, skin picking and Results a marked tolerance to pain were noted and have persisted. Clinical case report The patient (Fig. 2) was born at 41 weeks of gestation with Excessive daytime somnolence continued beyond infancy a birth weight of 8 lb. Pregnancy was uneventful, but fetal and treatment with amphetamine was started at 9 years of movements were somewhat reduced. In the newborn, age. A sleep study, at 13 years of age, was normal. In 2002, poor muscle tone, weak cry, excessive sleepiness, and a further sleep study and a multiple sleep latency tests Page 5 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 confirmed the diagnosis of narcolepsy. His daytime sleep- Developmentally he had a mild delay in comparison to iness has continued to respond to dexamphetamine. his older siblings. He attended normal school but had some difficulties due to rigid behaviours and poor peer Regarding his body weight, there was no rapid weight gain interactions. Psychological testing (WISC 111, Wide between 1 and 6 years. Around 8 years of age, his interest Range Achievement test and BASC self report) revealed an in food increased, and now he would keep eating if he had overall normal intellect. However, he had some involun- unrestricted access to favorite sweet foods. He lives with tary fluctuation in attention and significant visual percep- his parents who help to control his food intake. At 14.5 tual difficulties, e.g. deficits in visual organization, in th years, he had small hands and feet, at the 20 percentile making sense of his visual world and transcribing visual th and 5 percentile, respectively, and showed mild truncal material. These perceptual problems have had a signifi- th obesity. His head circumference of 56.7 cm was at the 98 cant effect on his learning and social life. At the age of 22, percentile. Brain MRI scan was normal. At age 16 years, his he is attending a mainstream high school requiring extra height was 155.7 cm and weight 65 kg. At the age of 22 time and assistance in completing a diploma in informa- years, his height is approximately 164 cm and his weight tion technology. He is good at dismantling computers and has increased to 90 kg (BMI = 33.5). installing hardware, and prefers working on his computer to socializing. Hyperphagia and skin picking are still a At 13 years of age, he was found to have delayed puberty challenge for him. and reduced linear growth velocity with his height falling rd Cytogenetic analysis below the 3 centile. Treatment with testosterone resulted in improved height gain and genital development. At 15 High-resolution chromosome analysis showed an appar- years of age, he had a left orchidopexy and removal of a ently balanced reciprocal translocation between the long dysplastic intra-abdominal right testis. He remains on 6 arm of chromosome 4 and the proximal long arm of chro- monthly testosterone implants because of reduced mosome 15. The breakpoints were assigned to chromo- hypothalamic function. He has never been on growth hor- some bands 4q27 and 15q11: 46, XY, t(4;15)(q27;q11) mone treatment. (Fig. 3a). Parental chromosomes were normal, indicating that the patient's translocation was de novo. ho Figure 3 a. H mio gh lo resolution G-ba gs nded ideograms and prometaphase chromosomes of the translocation derivatives and their normal a. High resolution G-banded ideograms and prometaphase chromosomes of the translocation derivatives and their normal homologs . An apparently balanced translocation t(4;15)(q27;q11) was identified with arrows indicating band location of breakpoints. b. DNA methylation analysis of CpG island of SNRPN promoter and exon 1. 1. The 174 bp PCR product is derived from the methylated maternal chromosome. 2. The 100 bp product is derived from the paternal chro- mosome. PWS: PWS control, Normal: normal control, and t(4;15) carrier; H O: no template control. The t(4;15) carrier shows the normal bi-parental methylation pattern. Page 6 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 2: SNRPN and snoRNA expression analysis with quantitative RT-PCR Amplification product PWS Normal control t-PWS (4;15) PWS triplication t-PWS intron 2 t-PWS (4;15) LCL LCL LCL LCL FB FB SNURF Ex 2 0.0001 0.53 0.54 2 0.002 0.56 SNURF Ex 3 0.0002 1.1 1.43 6.4 0.0004 0.56 HBII-437 0.00003 0.86 1.13 4.9 0.00008 0.1 SNRPN 14/15 0.0013 4.7 4.91 17.23 - - SNRPN 19/20a 0.005 1.34 0.003 4.7 0.007 0.007 HBII-438 0.03 1.5 0.02 6.2 0.07 0.07 PWCR1/HBII-85 0.03 3.7 0.02 16.8 0.04 0.06 Sample identification: PWS: PWS with an IC microdeletion (patient E in [5]); t(4;15) PWS: the PWS case reported here; PWS-triplication: intrachromosomal triplication of the PWS region [33]; t-PWS intron 2: previously reported PWS case with t(4;15)(q27;q11.2) and breakpoint in SNRPN intron 2 [26, 27]; LCL, lymphoblastoid cell line, FB, fibroblast strain. The numbers represent the ratio of target product to GAPDH control product. DNA methylation analysis some 15, there was a ~11.5 kb band in lane 4, detected To exclude alternative explanations for the phenotype, with the SB-1 probe, and a ~7 kb band in lane 6, detected such as an imprinting defect, DNA methylation analysis with probe SB-3 (Fig. 5b). The novel ~11.5 kb band arose was performed. Methylation-specific PCR of the SNURF- from the der(15) chromosome, with an NheI site on the SNRPN exon 1 region revealed a normal bi-parental chromosome 15 portion and an ApaI site on the chromo- methylation pattern (Fig. 3b). some 4 portion (Fig. 5c, upper panel). The novel band of ~7 kb arose from the der(4) chromosome, with an ApaI Mapping of the translocation breakpoint by FISH site on the chromosome 15-part and an NheI site on the We performed cytogenetic and molecular studies to char- chromosome 4-part. Taken together, these results delimit acterize the breakpoint at 15q11 in detail. Preliminary the breakpoint region to ~3.6 kb between the BsaWI and FISH analysis showed that the breakpoint in 15q11 was ApaI sites (Fig. 5a). located between D15S11 and GABRB3, which flank the SNRPN locus (data not shown). On this basis, a chromo- Breakpoint mapping at the nucleotide level some walking strategy was used across this region to nar- By DNA sequencing, we mapped the breakpoint to row down the breakpoint region. We identified two BACs, SNRPN intron 17 (position chr 15: 22803227) and to RP11-160D9 (current position 22577151-22735621 on chromosome 4 at position chr. 4:123965881 (UCSC UCSC Genome Browser, May 2004 release) and RP11- Genome Browser, May 2004) (Fig. 6). On chromosome 4, 876N20 (current position 22857334-23036552), that a long terminal repeat (LTR) retrotransposon, LTR1B, flanked the breakpoint and, thus, mapped it to a ~122 kb spans the breakpoint. On chromosome 15, we found a interval (Fig. 1b). short interspersed element (SINE), AluY, and a long inter- spersed element (LINE), L1M4, surrounding the break- Fine mapping of the breakpoint by SNRPN expression and point (Fig. 6a). Thirty-nine bp upstream of the breakpoint Southern blot analysis on chromosome 15 starts a common 26 bp core sequence To further refine the breakpoint, we carried out quantita- of Alu elements (Alu-DEIN) in an inverted orientation. tive RT-PCR and RT-PCR experiments using RNA from an This sequence is known to be involved in gene rearrange- LCL and skin fibroblasts (FB) for expression of SNRPN ments [36]. While the sequence across the breakpoint is transcripts. As shown in Figure 4 and Table 2, we found contiguous on the der(15), an extra A is inserted on the expression of SNPRN exons 2, 3, and 14 to 17, but no der(4) chromosome (Fig. 6b). Furthermore, the expression of exons 18 to 20, and concluded that the breakpoint on chromosome 4 falls in a large intron breakpoint falls within intron 17. For mapping intron 17, between exons 10 and 11 of a spliced transcript we designed a Southern blot using unique restriction sites. (BC045668). By RT-PCR, we found that this transcript is DNA cleaved with NheI and BsaWI showed a 6.4 kb band expressed in fibroblasts, but not in LCLs (data not for the t(4;15) carrier and the normal control (Fig. 5b, shown). lanes 1 and 2), indicating that the breakpoint is located downstream of the BsaWI site. Samples doubly digested Expression of upstream genes MKRN3, MAGEL2, and NDN with NheI and ApaI (Fig. 5b, lanes 3–6), revealed addi- tional bands for the translocation carrier. Besides the Expression of the three imprinted genes MKRN3, expected 10 kb band derived from the normal chromo- MAGEL2, and NDN upstream of SNRPN was tested by RT- Page 7 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 SNRPN expression a Figure 4 nalysis by RT-PCR of RNA from LCLs SNRPN expression analysis by RT-PCR of RNA from LCLs . On the left, the sizes of the PCR products are shown, and on the right, the location of the primers in SNRPN exons is listed. +RT: with reverse transcriptase; -RT: without reverse tran- scriptase; H O: no template control. All SNRPN +RT products tested were absent in the PWS control, and present in the nor- mal control. The t(4;15) cells were positive for SNURF/ SNRPN exons 2–3, 15–16 and 16–17 and negative for exons 18 through 20a. GAPDH primers were used as control for the integrity of the cDNA. PCR in t(4;15) fibroblasts and found to be indistinguish- in the PWS control [6] and t(4;15) LCLs, but were able from expression in normal control fibroblasts (data expressed in the normal control LCL (Fig. 7). not shown). Discussion Expression of C/D box snoRNAs and intron-encoded ESTs Breakpoint mapping and mechanism of the translocation When testing for the intron-encoded C/D box snoRNAs, event we were able to document expression of HBII-13 and Dissecting the PWS deletion region and identifying indi- HBII-437 and lack of expression for HBII-438A/B and vidual genes as responsible for parts of the phenotype rep- HBII-85/PWCR1 (Fig. 7). By use of a more sensitive resent a challenge because all reported smaller deletions method, quantitative real-time RT-PCR, we obtained sim- inactivate all imprinted genes on the paternally- derived ilar results for the SNRPN exons and snoRNAs tested chromosome 15. Rare reciprocal translocations, therefore, (Table 2). Two ESTs, AK094315 and AB061718 (= HBT8) provide unique insights. We here report our studies of a located in the 30 kb SNRPN intron 20 were not expressed 22 year old male with features of PWS who has a de novo Page 8 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 South Figure 5 ern blot analysis identifies breakpoint in SNRPN intron 17 Southern blot analysis identifies breakpoint in SNRPN intron 17 . a . Restriction map of the intron 17 region of the SNRPN gene on the normal chromosome 15. Black arrowheads indicate the boundaries of intron 17. The positions of the two hybridization probes (SB-1 and SB-3) are indicated by green lines. b . Lanes 1 and 2 contain double digests with NheI and BsaWI to release a fragment of 6.4 kb, lanes 3 and 4 contain double digests with NheI and ApaI to release a fragment of 10 kb. The membrane was probed with probe SB-1. The arrow indicates an additional band above the 10 kb fragment ~11.5 kb in length. The two bands are not well resolved on the rendition of this blot. This novel fragment is represented in c, upper panel. Lanes 5 and 6 contain double digests with NheI and ApaI to release a 10 kb fragment. The membrane is probed with SB-3. The arrow indicates an additional band of ~ 7 kb. This novel fragment is represented schematically in c, lower panel. c . Schematic repre- sentation of the junction fragments identified on the Southern blot in b. The upper panel represents the der(15) and the lower panel represents the der(4). Chromosome 15 material is indicated as a black line and material from chromosome 4 as a blue line. Location of restriction sites and of hybridization probes (green lines) are indicated. balanced reciprocal translocation t(4;15)(q27;q11.2). translocation that may be facilitated by genomic repeats This is the first such case where the translocation break- or other distinct molecular features. points have been identified at the DNA sequence level. The cytogenetic breakpoint designations in this individual In the present case, however, we mapped the breakpoint are identical to those in another male PWS-like case with to SNRPN intron 17 (position on chr. 15: 22803227, t(4;15)(q27;q11.2), previously reported by Kuslich and UCSC Genome Browser, May 2004) that differs from that colleagues [27] and restudied by Gallagher and colleagues in the previous case (SNRPN intron 2). Furthermore, the [28], which raised the intriguing possibility of a recurrent breakpoint in our case is novel as it does not fall into one Page 9 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Repeat sequences surrounding Figure 6 the breakpoint Repeat sequences surrounding the breakpoint . a . One hundred nucleotides on either side of the breakpoints on chro- mosome 4 and 15 contain repetitive sequences (grey lines). The Alu-DEIN sequence is located 13–39 bp upstream of the breakpoint on chromosome 15. b . Sequence across the breakpoint on the der(4) chromosome reveals an additional A inserted at the breakpoint. Arrows indicate the direction centromere to telomere. of the two previously described "breakpoint clusters" in consensus Alu element [38]. Sequence analyses of regions intron 2 and exon 20a/intron 20 (Table 3 and Table 4). directly adjacent to translocation breakpoints has shown On chromosome 4 (chr. 4 123965881), the LTR retro- presence of the 26-bp Alu core sequence at or close transposon LTR1B is spanning the breakpoint, and a short (within 20–50 bp downstream or upstream) to the sites of interspersed element (SINE), AluY, and a LINE element, recombination [36]. Therefore, this sequence might stim- L1M4, surround the breakpoint on chromosome 15 (Fig. ulate homologous and non-homologous recombination 6). Interestingly, 39 bp upstream of the breakpoint on within the core or at nearby sites and could be the mech- chromosome 15 starts a common 26-bp core sequence of anism of recombination in the t(4;15) case reported here. Alu elements (Alu-DEIN) that has been shown to be involved in gene rearrangements and has homology with The translocation is de novo, as is true for all the previously prokaryotic χ, an 8-bp sequence motif known to stimulate described cases with translocation breakpoints involving recBC mediated recombination in E. coli[37]. The core the SNPRN gene. Given the PWS-like phenotype, the sequence is identical to sequences in the left arm of the translocation was assumed to be of paternal origin. This Page 10 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Expres Figure 7 sion analysis in LCLs of snoRNAs and two ESTs in intron 20 Expression analysis in LCLs of snoRNAs and two ESTs in intron 20 . RT-PCR analysis of the C/D box snoRNAs reveals expression of HBII-13, but not of HBII-438A/B, PWCR1/HBII-85 and the two ESTs in intron 20 in the t(4;15) transloca- tion carrier. +RT: with reverse transcriptase; -RT: without reverse transcriptase; H O: no template control. assumption was confirmed by the expression studies. Wirth et al. 2001, and also in the present case. But this is Paternal origin of the translocation was formally proven not a consistent feature in classical PWS, as in a in 2 of the 5 previously reported cases [26,27]. retrospective evaluation of 90 molecularly-proven PWS cases, only 49% had the characteristic facial gestalt [39]. Karyotype – phenotype correlations Two individuals with SNRPN intron 2 breakpoints were It is apparent from the review of the previously reported described as having classical PWS, meeting the major cases and the individual reported here (Table 3 and Table clinical criteria by age 3.5 years and additional minor clin- 4) that some of these translocation cases tend to have a ical criteria [25,27]. The individuals with a breakpoint in milder, 'atypical' clinical picture, in comparison with clas- SNRPN Exon 20/Intron 20 were described as having a sical PWS. There is not a complete absence of any of the milder or atypical form of PWS (Table 3 and Table 4). The major phenotypic features (neonatal hypotonia and feed- weight gain started later than in classical PWS, at 7 and 5 ing difficulty, hyperphagia from early childhood, obesity, years, respectively, for the patients described by Schulze et cognitive compromise, hypogenitalism), but the degree of al. 1996 and Wirth et al. 2001, and at 8 years in our case. affection may be lower. None of the reported The characteristic facial features were absent in the case of translocation cases had any additional features that might Page 11 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 3: Clinical findings associated with paternally-derived de novo reciprocal translocations involving SNRPN Breakpoint in SNRPN Intron 2 Breakpoint in SNRPN Exon 20/ Intron 20 Breakpoint in SNRPN Intron 17 Sun et al. 1996 Kuslich et al. 1999 Schulze et al. 1996 Conroy et al. 1997 Wirth et al. 2001 Present case Karyotype 46, XY, t(15;19) 46, XY, t(4;15) 46, XY, t(9;15) 46, XY, t(2;15) 46, X, t(X;15) 46 XY, designations (q12;q13.41) (q27;q11.2) (q21;q12–q13) (q37.2;q11.2) (q28;q12) t(4;15)(q27;q11.2) Age of 3.5 years 3 years 3 months 29 years 4.5 years 18 years 22 years examination Major criteria (each scores one point) from [1] as revised in [2]. 1. Neonatal Floppy and Hypotonicity, Neonatal Neonatal - Reduced tone with central hypotonia lethargic in the poor sucking hypotonia (weak hypotonia, poor head control, first 6 months with reflex during cry, poor suck) (1 lethargy, poor poor suck (1 pt.) poor suck (1pt.) infancy (1pt.) pt.) suck (1 pt.) 2. Infantile feeding Failure to thrive Feeding problems Special feeding - Feeding problems, problems/ failure (1pt.) in infancy, failure techniques, but no but no failure to to thrive to thrive (1 pt.) failure to thrive thrive (1pt.) 3. Rapid weight Obesity starting at Eating behavior Periodic excessive Onset of obesity Obesity began at Late onset obesity gain between 1–6 6 months, leading to weight gain from at 1.5–2 yr with 4–5 yr with (at approx. 8 yr) years hyperphagia (1 pt.) increased weight age 7 yr excessive appetite hyperphagia and gain at age 2 yr (1 and food foraging food foraging (1 pt.) (1 pt.) pt.) 4. Characteristic Narrow bifrontal Narrow bifrontal Narrow bifrontal Narrow bifrontal -- facial features diameter, almond- diameter, almond- diameter, narrow diameter, squared shaped eyes, shaped eyes, face, small mouth, nasal tip, down-turned upslanted poor facial mimic downturned mouth (1pt.) palpebral fissures (1pt.) mouth (1 pt.) (1 pt.) 5. Hypogonadism: Undescended Undescended Hypoplastic Primary Undescended Scrotum normal, genital hypoplasia, testes (1 pt.) small testes, genitalia, penile length at amenorrhea, small testes, th pubertal deficiency hypogonadism (1 incomplete 10 %ile hypoplastic uterus hypogonadism, pt.) gonadal (1 pt.) delayed pubertal maturation with signs (1 pt.) delayed pubertal signs after age 16 yr (1 pt.) 6. Mental Developmental Developmental Mental Developmental Slight Developmental retardation, delay (1 pt.) delay (1 pt.) retardation, delay, special developmental delay, special developmental developmental school setting (1 delay, school for school setting (1 delay delay/ learning pt.) mentally retarded pt.) problems (1 pt.) children (1 pt.) Score 5 points 6 points 5 points 4 points 3 points 4 points Blank cell = no information - = absent possibly be attributed to disruption of a gene on the recip- Our sequence data mapped the breakpoint on chromo- rocal chromosome, and in no prior case had an attempt some 4 within intron 10 of a spliced polyadenylated tran- been made to identify a gene at this location. script (BC045668). This unique cDNA clone represents a Page 12 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 4: Clinical findings associated with paternally-derived de novo reciprocal translocations involving SNRPN (continued) Sun et al. 1996 Kuslich et al. 1999 Schulze et al. 1996 Conroy et al. 1997 Wirth et al. 2001 Present case Minor criteria (1/2 point each) 1. Decreased fetal Decreased fetal Decreased fetal - - Slightly reduced movement and activity (0.5 pt.) movements fetal movements infantile lethargy (0.5pt.) (0.5pt.) 2. Typical Behavior problems Temper tantrums, Aggressive Behavior problems Temper tantrums, Temper tantrums, behaviour (0.5pt.) violent outbursts, outbursts, rigid with temper violent outbursts abnormal social problems obsessive- personality, tantrums and after food behavior (0.5pt.) compulsive (0.5 perseveration severe restrictions pt.) (0.5pt.) aggressiveness (0.5 (0.5pt.) pt.) 3. Sleep Sleep disturbance, Sleep disturbance Sleep disturbance, disturbance, sleep sleep apnea (0.5pt.) amphetamine apnea (0.5pt.) treatment from age 9 ys. (0.5pt.) th rd 4. Short stature Short stature at 50–75 percentile 151 cm (3 %tile) Height 155.7 cm at rd for the family by the age of 15 (0.5pt.) (0.5pt.) 16 years < 3 age 15 years (0.5pt.) %tile (0.5 pt.) 5. - - Hypopigmentation -- Hypopigmentation (0.5 pt.) th th 6. Small hands and Hand length 25 - Normal hands, but Short 3rd finger Hands 20 %ile, /or feet for height percentile, finger small feet (< bilaterally feet 5th %ile th th %ile 10 %tile) (0.5 pt.) age length 10 (0.5pt.) (0.5pt.) 7. Narrow hands - - with straight ulnar border 8. Eye - Esotropia (0.5 pt.) Alternating Left esotropia (0.5 Esotropia (0.5pt.) abnormalities: esotropia in pt.) esotropia, myopia infancy (0.5 pt.) 9. Thick viscous Viscous saliva Thick viscous - saliva (0.5pt.) saliva (0.5 pt.) 10. Speech Articulation Poor articulation - articulation defect difficulty (0.5 pt.) (0.5pt.) 11. Skin picking Skin picking Skin picking Skin picking (0.5pt.) (0.5pt.) (0.5pt.) Score (minor only) 1.5 points 3 points 3.5 points 2.5 points 1.5 points 3.5 points Total Score 6.5 points 9 points 8.5 points 8.5 points 4.5 points 7.5 points Blank cell = no information - = absent 3764 bp mRNA from a human testis library that does not leukin 21 (IL21) transcript by 511 bp in the opposite appear to encode a protein. Its 5' end overlaps the inter- direction (UCSC Genome Browser, May 2004). It appears Page 13 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 unlikely that heterozygous disruption of this gene con- tissues [42] were found to be expressed in a normal con- tributes to the phenotype in our patient. trol LCL, but not in the t(4;15) LCL. This result suggests that these ESTs do not have their own promoter but are Translocation has no effect on imprinting center dependent on transcription from the SNRPN promoter methylation and upstream genes that is located on the other translocation derivative in To assess whether the translocation event had affected the these cells. Therefore, these ESTs most likely represent sta- allele-specific methylation pattern at the imprinting ble derivatives of large alternatively spliced non-coding center (IC), and/or to exclude a coincident imprinting SNRPN transcripts. defect, we carried out methylation studies of the SNRPN exon 1 region that revealed a normal bi-parental methyl- Conclusion ation pattern. Similar results were reported for each of the (1) Expression of the ESTs and/or C/D box snoRNAs that other five PWS individuals who had translocation break- are located downstream of the translocation breakpoint is points within the SNRPN gene. These results predict that not necessary for establishing and maintaining the pater- expression of the genes located centromeric to the SNRPN nal-specific pattern of gene expression pattern that is exon 1/ IC region, NDN, MAGEL2 and MKRN3, should controlled by the imprinting center upstream of the trans- not be affected in these individuals. By studying t(4;15) location breakpoint. fibroblasts by RT-PCR, we indeed found expression of all three genes. Previously, only MKRN3 was reported to be (2) The C/D box snoRNAs HBII-438A and PWCR1/HBII- expressed in the three PWS translocation cases in which it 85 are the only stable transcripts in this region that are dis- was studied [20,25,27]. rupted in this t(4;15) PWS individual. As PWCR1/HBII-85 sequences are highly conserved between human and In t(4;15) lymphoblasts, the SNRPN transcript was detect- mice, while no copy of HBII-438A has been found in able by RT-PCR and quantitative RT-PCR and found to mouse, we conclude that the basis of PWS pathogenesis extend all the way to exon 17. The major transcript that resides, in whole or in part, in the absence of PWCR1/ encodes the SNURF/SNRPN proteins terminates in exon HBII-85 snoRNA. SNURF/SNRPN and the centromeric 10 [20] and, therefore, should be unaffected by this genes MKRN3, NDN and MAGEL2 are unlikely to play a t(4;15) translocation. With the caveat that studies on major role in the causation of PWS-associated features. peripheral tissues, fibroblasts and lymphoblasts, may not While the function of known C/D box snoRNAs is to accurately reflect gene expression in the brain, our results guide 2'- O -ribose methylation of mainly ribosomal RNA, indicate that SNURF/SNRPN and the centromeric genes these novel imprinted snoRNAs have no known target. MKRN3, NDN and MAGEL2 are unlikely to play a prime They might be involved in a posttranscriptional role in the causation of PWS-associated features, although regulation process of a gene or genes that – if non-func- it remains an open question whether their loss or non- tional – gives rise to the PWS phenotype. functioning might contribute to the more marked pheno- typic expression that is seen in typical PWS. Competing interests The author(s) declare that they have no competing Genes downstream of the breakpoint are not expressed interests. With respect to expression of downstream transcripts, the reported results on LCLs with breakpoints in exon 20/ Authors' contributions intron 20 were consistent, whereas for the two patients BS carried out the molecular genetic studies (RT-PCR, with breakpoints in intron 2, the reported results were methylation assay, Southern blot analysis, and breakpoint conflicting for expression of downstream transcripts IPW analysis) and drafted the manuscript. MA carried out and PAR-1. In a re-evaluation of the t(4;15) case reported quantitative RT-PCR assays. EN performed the FISH anal- by Kuslich and colleagues [26], no expression of these ysis with BAC clones. DIF carried out the initial cytoge- transcripts and of the PWCR1/HBII-85 snoRNA cluster netic analysis. MR revised the clinical data and re- was detected by real-time quantitative RT-PCR [28]. examined the patient. Therefore, we focused our analysis on the snoRNAs and HRS supervised the cell culturing, cytogenetic and FISH two ESTs in intron 20. As for the intron-encoded C/D box studies. RJMG diagnosed the patient, collected the clinical snoRNAs, HBII-13 and HBII-437 were expressed, and data and obtained skin and blood samples. UF conceived HBII-438A/B and HBII-85/PWCR1 were not. HBII-52 the study design, and coordinated its progress, supervised snoRNAs were not studied, as they are not expressed in the the work of BS and MA and prepared the final manuscript. available tissues and have previously been excluded from contributing to the PWS phenotype [40,41]. The two ESTs Acknowledgements We are indebted to the family participating in this study and to Prof. in the large intron 20 that are highly expressed in brain George A. Werther who referred the patient to us, and in his letter wrote Page 14 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 "I wonder whether this translocation may involve the Prader-Willi gene". multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum Mol Genet 2001, 10:2687-2700. The work in the laboratory of UF was supported by grants from the NIH 20. Wirth J, Back E, Huttenhofer A, Nothwang HG, Lich C, Gross S, Men- (HD41623) and the Deutsche Forschungsgemeinschaft (BS – SCHU 1567/ zel C, Schinzel A, Kioschis P, Tommerup N, Ropers HH, Horsthemke 1-1). B, Buiting K: A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15. Hum Mol Genet 2001, 10:201-210. References 21. de Los Santos T, Schweizer J, Rees CA, Francke U: Small evolution- 1. Holm VA, Cassidy SB, Butler MG, Hanchett JM, Greenswag LR, Whit- arily conserved RNA, resembling C/D box small nucleolar man BY, Greenberg F: Prader-Willi syndrome: consensus diag- RNA, is transcribed from PWCR1, a novel imprinted gene in nostic criteria. Pediatrics 1993, 91:398-402. the Prader-Willi deletion region, which is highly expressed in 2. Cassidy SB: Prader-Willi syndrome. 2001:301-322. brain. Am J Hum Genet 2000, 67:1067-1082. 3. Ledbetter DH, Riccardi VM, Airhart SD, Strobel RJ, Keenan BS, Craw- 22. Cavaille J, Buiting K, Kiefmann M, Lalande M, Brannan CI, Horsthemke ford JD: Deletions of chromosome 15 as a cause of the B, Bachellerie JP, Brosius J, Huttenhofer A: Identification of brain- Prader-Willi syndrome. N Engl J Med 1981, 304:325-329. specific and imprinted small nucleolar RNA genes exhibiting 4. Buiting K, Gross S, Lich C, Gillessen-Kaesbach G, el-Maarri O, an unusual genomic organization. Proc Natl Acad Sci U S A 2000, Horsthemke B: Epimutations in Prader-Willi and Angelman 97:14311-14316. syndromes: a molecular study of 136 patients with an 23. Kiss T: Small Nucleolar RNAs: An Abundant Group of Non- imprinting defect. Am J Hum Genet 2003, 72:571-577. coding RNAs with Diverse Cellular Functions. Cell 2002, 5. Mascari MJ, Gottlieb W, Rogan PK, Butler MG, Waller DA, Armour 109:145-148. JA, Jeffreys AJ, Ladda RL, Nicholls RD: The frequency of uniparen- 24. Schulze A, Hansen C, Skakkebaek NE, Brondum-Nielsen K, Ledbeter tal disomy in Prader-Willi syndrome. Implications for molec- DH, Tommerup N: Exclusion of SNRPN as a major determi- ular diagnosis. N Engl J Med 1992, 326:1599-1607. nant of Prader-Willi syndrome by a translocation 6. Sutcliffe JS, Nakao M, Christian S, Orstavik KH, Tommerup N, Led- breakpoint. Nat Genet 1996, 12:452-454. better DH, Beaudet AL: Deletions of a differentially methylated 25. Sun Y, Nicholls RD, Butler MG, Saitoh S, Hainline BE, Palmer CG: CpG island at the SNRPN gene define a putative imprinting Breakage in the SNRPN locus in a balanced 46,XY,t(15;19) control region [see comments]. Nat Genet 1994, 8:52-58. Prader-Willi syndrome patient. Hum Mol Genet 1996, 5:517-524. 7. Buiting K, Saitoh S, Gross S, Dittrich B, Schwartz S, Nicholls RD, 26. Conroy JM, Grebe TA, Becker LA, Tsuchiya K, Nicholls RD, Buiting Horsthemke B: Inherited microdeletions in the Angelman and K, Horsthemke B, Cassidy SB, Schwartz S: Balanced translocation Prader-Willi syndromes define an imprinting centre on 46,XY,t(2;15)(q37.2;q11.2) associated with atypical Prader- human chromosome 15 [published erratum appears in Nat Willi syndrome. Am J Hum Genet 1997, 61:388-394. Genet 1995 10:249]. Nat Genet 1995, 9:395-400. 27. Kuslich CD, Kobori JA, Mohapatra G, Gregorio-King C, Donlon TA: 8. Jong MT, Carey AH, Caldwell KA, Lau MH, Handel MA, Driscoll DJ, Prader-Willi syndrome is caused by disruption of the Stewart CL, Rinchik EM, Nicholls RD: Imprinting of a RING zinc- SNRPN gene. Am J Hum Genet 1999, 64:70-76. finger encoding gene in the mouse chromosome region 28. Gallagher RC, Pils B, Albalwi M, Francke U: Evidence for the role homologous to the Prader-Willi syndrome genetic region. of PWCR1/HBII-85 C/D box small nucleolar RNAs in Prader- Hum Mol Genet 1999, 8:795-803. Willi syndrome. Am J Hum Genet 2002, 71:669-678. 9. Jong MT, Gray TA, Ji Y, Glenn CC, Saitoh S, Driscoll DJ, Nicholls RD: 29. Li L, Moore P, Ngo C, Petrovic V, White SM, Northrop E, Ioannou A novel imprinted gene, encoding a RING zinc-finger pro- PA, McKinlay Gardner RJ, Slater HR: Identification of a haplosuf- tein, and overlapping antisense transcript in the Prader-Willi ficient 3.6-Mb region in human chromosome 11q14.3-->q21. syndrome critical region. Hum Mol Genet 1999, 8:783-793. 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Proc Natl Acad Sci U S A 1981, 78:7033-7037. 6:163-167. 38. Deininger PL, Jolly DJ, Rubin CM, Friedmann T, Schmid CW: Base 17. Glenn CC, Saitoh S, Jong MT, Filbrandt MM, Surti U, Driscoll DJ, sequence studies of 300 nucleotide renatured repeated Nicholls RD: Gene structure, DNA methylation, and human DNA clones. J Mol Biol 1981, 151:17-33. imprinted expression of the human SNRPN gene. Am J Hum 39. Gunay-Aygun M, Schwartz S, Heeger S, O'Riordan MA, Cassidy SB: Genet 1996, 58:335-346. The changing purpose of Prader-Willi syndrome clinical 18. Gray TA, Saitoh S, Nicholls RD: An imprinted, mammalian bicis- diagnostic criteria and proposed revised criteria. Pediatrics tronic transcript encodes two independent proteins. Proc Natl 2001, 108:E92. Acad Sci U S A 1999, 96:5616-5621. 40. Hamabe J, Kuroki Y, Imaizumi K, Sugimoto T, Fukushima Y, 19. Runte M, Huttenhofer A, Gross S, Kiefmann M, Horsthemke B, Buit- Yamaguchi A, Izumikawa Y, Niikawa N: DNA deletion and its ing K: The IC-SNURF-SNRPN transcript serves as a host for Page 15 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 parental origin in Angelman syndrome patients. Am J Med Genet 1991, 41:64-68. 41. Runte M, Varon R, Horn D, Horsthemke B, Buiting K: Exclusion of the C/D box snoRNA gene cluster HBII-52 from a major role in Prader-Willi syndrome. Hum Genet 2005, 116:228-230. 42. Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Waka- matsu A: Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature Genetics 2004, 36:40-45. 43. Chai JH, Locke DP, Greally JM, Knoll JH, Ohta T, Dunai J, Yavor A, Eichler EE, Nicholls RD: Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons. Am J Hum Genet 2003, 73:898-925. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2350/6/18/prepub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome

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Springer Journals
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Copyright © 2005 by Schüle et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Human Genetics; Cytogenetics; Gene Function
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1471-2350
DOI
10.1186/1471-2350-6-18
pmid
15877813
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Abstract

Background: Prader-Willi syndrome (MIM #176270; PWS) is caused by lack of the paternally-derived copies, or their expression, of multiple genes in a 4 Mb region on chromosome 15q11.2. Known mechanisms include large deletions, maternal uniparental disomy or mutations involving the imprinting center. De novo balanced reciprocal translocations in 5 reported individuals had breakpoints clustering in SNRPN intron 2 or exon 20/intron 20. To further dissect the PWS phenotype and define the minimal critical region for PWS features, we have studied a 22 year old male with a milder PWS phenotype and a de novo translocation t(4;15)(q27;q11.2). Methods: We used metaphase FISH to narrow the breakpoint region and molecular analyses to map the breakpoints on both chromosomes at the nucleotide level. The expression of genes on chromosome 15 on both sides of the breakpoint was determined by RT-PCR analyses. Results: Pertinent clinical features include neonatal hypotonia with feeding difficulties, hypogonadism, short stature, late-onset obesity, learning difficulties, abnormal social behavior and marked tolerance to pain, as well as sticky saliva and narcolepsy. Relative macrocephaly and facial features are not typical for PWS. The translocation breakpoints were identified within SNRPN intron 17 and intron 10 of a spliced non-coding transcript in band 4q27. LINE and SINE sequences at the exchange points may have contributed to the translocation event. By RT-PCR of lymphoblasts and fibroblasts, we find that upstream SNURF/SNRPN exons and snoRNAs HBII-437 and HBII-13 are expressed, but the downstream snoRNAs PWCR1/HBII-85 and HBII-438A/B snoRNAs are not. Conclusion: As part of the PWCR1/HBII-85 snoRNA cluster is highly conserved between human and mice, while no copy of HBII-438 has been found in mouse, we conclude that PWCR1/HBII-85 snoRNAs is likely to play a major role in the PWS- phenotype. Page 1 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 copy snoRNA genes (HBII-436, HBII-13, and HBII-437), Background Prader-Willi syndrome (PWS) is a complex neurodevelop- and one snoRNA gene (HBII-438) present in two copies mental disorder and a classic example for genomic that are 240 kb apart [19]. Since the snoRNAs are derived imprinting in humans. The incidence is about 1 in 10– from processed spliced-out introns, their expression is 20,000, and the clinical manifestations include decreased controlled by the SNRPN promoter and is highest in fetal activity, neonatal hypotonia, neonatal feeding diffi- brain. The known function of other C/D box snoRNAs is culties, hyperphagia with obesity, hypogonadism, short to guide 2'- O – ribose methylation of ribosomal RNA or stature, small hands and feet, characteristic facial features, small nuclear RNA. This post-transcriptional modification and mild to moderate mental retardation. Diagnostic cri- is conserved throughout evolution and is thought to con- teria have been proposed [1] and revised recently [2]. fer increased stability to the small RNA molecules [23]. The modification targets of the imprinted C/D box snoR- About 70% of individuals clinically diagnosed with PWS NAs in the PWS/AS region are still unknown. have a ~4 Mb interstitial deletion at 15q11-13 of paternal origin, with clustered breakpoints (BP) at either of two Spontaneous chromosome translocations can be proximal sites (BP1 or BP2) and one on the distal site extremely valuable for assessing the contributions of indi- (BP3) (Fig. 1a). The majority of the remainder have mater- vidual loci to the phenotype of microdeletion syndromes. nal uniparental disomy 15. In about 1 % of the cases, the Five individuals with features of PWS have been reported disease is due to aberrant imprinting and gene silencing. who have balanced reciprocal translocations with break- Of these, 14% have small deletions in the imprinting points in the PWS/AS deletion region. All of them involve center (IC) region of the paternal allele that abolish the the SNRPN locus. The breakpoints are located in intron 2 expression of all imprinted paternally-expressed genes in (proximal, n = 2), disrupting the SNURF/SNRPN coding cis. In the remainder no demonstrable DNA sequence region, or in exon 20a/intron 20 (distal, n = 3) within the changes have been observed [3-7]. In Angelman syn- 3'-untranslated region of the long SNRPN transcript. One drome (AS, MIM#105830), which is usually caused by the individual with a proximal and two of three patients with same mechanisms affecting the maternal chromosome a distal breakpoint meet the diagnostic criteria for PWS 15, mutations in the maternal copy of a single gene, (score of 8 or more points) [20,24-28]. UBE3A (MIM#601623), encoding a ubiquitin ligase, are detected in about 5% of cases, whereas in PWS, no dis- Here we report the clinical, cytogenetic and molecular ease-causing mutations in a single imprinted gene have characterization of a 22 year old male with features of yet been reported. PWS who has a different de novo balanced reciprocal trans- location t(4;15)(q27;q11.2). We mapped the breakpoint Three paternally expressed genes have been identified to SNRPN intron 17 (position on chr 15: 22803227, between BP2 and SNRPN. These include MKRN3/ UCSC Genome browser May 2004) and determined the ZNF127 (MIM# 603856; Makorin 3 or Zinc finger protein expression of snoRNAs on both sides of the breakpoint in 127) [8,9], MAGEL2/NDNL1 (MIM# 605283; MAGE-like cultured fibroblasts and lymphoblasts. 2 or Necdin-like 1) [10,11], and NDN (MIM# 602117; Necdin) [12,13] (Figure 1a). The small nuclear ribonucle- Methods oprotein polypeptide N (MIM# 182279; SNRPN) gene Cytogenetic and FISH analysis Metaphase spreads obtained from short-term blood lym- was the first gene with a known function to be mapped to the PWS/AS deletion region, and is expressed from the phocyte cultures and Epstein-Barr virus (EBV)-trans- paternal chromosome only [14-17]. Multiple alternatively formed lymphoblastoid cells (LCL) were processed for spliced transcripts originate at the SNPRN promoter [18- high-resolution GTG- banding by standard methods. For 20]. The major SNRPN transcript is bi-cistronic encoding FISH studies, Bacterial Artificial Chromosomes (BACs) two mRNA species. Exons 1–3 encode a protein product were sourced from the RPCI-11 library and selected using of unknown function called SNURF (SNRPN upstream the UCSC Genome Browser, Assemblies: July 2003 and reading frame). Exons 4–10 encode SmN, a homolog of May 2004). Fluorescence labelling, hybridization proce- the SmB/B' protein that binds small nuclear RNAs dures and imaging were performed as previously involved in pre-mRNA splicing. The largest transcripts described [29]. extend over a ~460 kb genomic region and include a large 3'UTR comprising up to 148 exons [19]. DNA methylation study Genomic DNA was purified by phenol-chloroform extrac- Multiple introns downstream of the SNURF-SNRPN cod- tion from LCLs from the study subject, a normal control, ing region contain C/D box small nucleolar RNA and a PWS individual (Patient E in [6], Coriell Human (snoRNA) genes. There are two multi-copy snoRNA clus- Mutant Cell Repository # GM12134). To investigate ters (HBII-52 and PWCR1/HBII-85) [21,22], three single methylation at exon 1 of SNRPN, 50 µg DNA were used Page 2 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Map Figure 1 ping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes Mapping the t(4;15) breakpoint and expression patterns of SNRPN exons and intronic genes . a . Schematic map of human chromosome region 15q11-q13. Black and gray circles represent imprinted genes, expressed from the paternal or maternal allele, respectively. White circles designate bi-allelically expressed genes. BP1, 2, and 3 indicate the locations of the deletion breakpoint hotspots [43]. b . FISH results placed BAC RP11-160D9 highlighted in green (nucleotide position 22577151-22735621) proximal to the translocation breakpoint and RP11-876N20 highlighted in blue (position 22857334- 23036552) distal to the breakpoint. Intron 17, comprising nucleotides 22795282 to 22811656, thus is located ~ 63.4 kb down- stream of RP11-160D9 and ~ 42 kb upstream of RP11-876N20. c . On representation of the SNRPN region (not drawn to scale) boxes represent exons and ESTs, lines represent snoRNA copies. Orange boxes and lines indicate exons, ESTs or snoR- NAs tested for expression either by RT-PCR or quantitative RT-PCR. Black flash indicates the breakpoint in intron 17 of the SNRPN locus. for the bisulfite reaction and PCR with primers according DNaseI (Roche) and RT-PCR was performed using Super- to standard protocols [30,31]. PCR products were sepa- script II (Invitrogen). Primers were designed for exon-to- rated on a 3% agarose gel, stained with ethidium bro- exon amplification in an overlapping fashion – where mide, and visualized under UV illumination. possible – for SNRPN, MKRN3, MAGEL2, NDN, snoR- NAs, and two ESTs within Intron 20 of SNRPN (Table 1). Expression studies by RT-PCR and quantitative RT-PCR Total RNA was extracted from LCL and primary fibroblast For a subset of exons in the SNPRN gene and the snoRNAs cultures (FB) using RNA Stat 60. The RNA was treated with HBII-13, HBII-437, and PWCR1/HBII-85, quantitative Page 3 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 1: Primers and conditions for PCR RT-PCR Gene/Exon fwd (5'-3') rev (5'-3'); complement strand size T ann. ZNF127 GGG TTG CGG TTT TGC TAT TA TTT CTC GTG TGC TTC AAT GC 168 bp 59C MAGEL2 CTG AAG CCT GGG ACT TTC TG GGA CCT TGG CCA CAA ACT TA 225 bp 59C Necdin GAA GAA GCA CTC CAC CTT CG CCA TGA TTT GCA TCT TGG TG 164 bp 59C SNRPN Ex 1–3 ATG GAG CGG GCA AGG GAT CGC GGT ACA ACT GAC ACT CTT GG 124 bp 53C SNRPN Ex 14/16 CTG CAA ACA TAG GAG ATG ATA GTT CC CTT ATG AAA GCA CTG AGA TGA AGC C 459 bp 53C SNRPN Ex 16/17 GAA AGT GAC CTA AAG AGT GTC ATT G CTT GCA GTT GGA CAG CCG ACT C 515 bp 53C SNRPN Ex 17/18 AGA TAT CTT TAA AAT TGA GTC TTC TGT CCA TGA AGA TGC AGC ACT TTT GAA GAA 218 bp 53C SNRPN Ex 19/20a CAT TGT GCT TAT TTA CTA TTT TTG TAG ACG CTG CAG GTG GTG ACC ATG TG 150 bp 53C AK094315 TCT TCT CTA CCC TCA TTC CCA GC TCG CTA CAC CCC TTT GCT TAT G 222 bp 53C AB061718 AGG AGG GGT TCA AAG ATG C CTG GTA AAC AAA CTG GTA AAG GTG 204 bp 50C HBII-85/PWCR1 CGA TGA TGA GTC CCC CAT AAA AAC CAG TTC CGA TGA GAA CGA CG 79 bp 53C HBII-13 GGA TTT GTG ATG AGC TGT GTT TAC GGA CTT CAG AGT AAT CAC GTT G 67 bp 54C HBII-438A/B GGA TCG ATG ATG AGA ATA ATT ATT G GGA CCT CAG ATT GAC ATC TG 67 bp 53C GABRB3 TCA GGC GGC ATT GGC GAT ACC ATA AAA ACT TGA CAG GCA GAG 352 bp 52C GABRA5 AAT ATT GCC TTA ATG TTT CTA GCC TAT TCT ATT TCT TCG TGT 425 bp 48C GABRG3 GCG TAT TCA CAT AGA CAT CTT G GAT TGG TCA CTA CTG GTC TGG 188 bp 52C GAPDH TGG GCT ACA CTG AGC ACC AG GGG TGT CGC TGT TGA AGT CA 50 bp 53C Quantitative RT-PCR Gene/Exon fwd (5'-3') rev (5'-3');complement strand size T ann. SNURF Ex2 ACG AAC TAC AGA ACA GCA CGT ACC CTG CGT TTG ACT TGG ACT TCC 50 bp 60C SNURF Ex3 TTC TCA GCA GCA GCA AGT ACC T TGC CTC AGT TCA GCC TGG A 50 bp 60C HBII-437 ATC ATT ATT TCT TGA ATT GG CCC TCA CGC TCC CTT TGC 50 bp 60C SNRPN Ex 14/15 CTG CAA ACA TAG GAG ATG ATA GTT CC CAA AGA CGA TAA AAT GTT CCT TCT TG 50 bp 60C SNRPN Ex 19/20a GGA ACC ACC ATT TGT CTA TGA TCC CTG CAG GTG GTG ACC ATG TG 50 bp 60C HBII-438 ATA ATT GTC TGA GGA TGC T GAT TGA CAT CTG GAA TGA GTC 50 bp 60C HBII-85/PWCR1 TCG ATG ATG AGT CCC CCA TAA CAT TTT GTT CAG CTT TTC CAA GG 50 bp 60C PCR to generate Southern probes in intron 17 Gene/Exon fwd (5'-3') rev (5'-3');complement strand size T ann. SB 1 ACC ATC AGT GAA TGA CCT GTT GC CCC AGC CTC TTT CCT ATG TCT TG 565 bp 53C SB 3 TGG TAA ACT GAT GAG AGC ACA GCC GCC TGG GAG ACA GAA TGA GAA AC 416 bp 53C RT-PCR assays were performed with SYBR Green I™ dye in Southern blot analysis an ABI 7700 cycler (Applied Biosystems) by using Southern blot analysis was performed according to stand- standard protocols [32,33]. Primers were designed to ard methods with ExpressHyb™ solution (BD Bio- amplify products of 50 bp in length. GAPDH expression sciences). Genomic DNA from a normal individual and was used as a reference. Each sample was run at least in the t(4;15) carrier was cleaved in a double digestion with triplicate. The results were interpreted as described previ- restriction enzymes NheI and BsaWI to release a 6.4 kb ously [28]. fragment, and with NheI and ApaI to release a 10 kb frag- ment in the normal chromosome. The DNA probes were LCL RNA samples from a PWS individual with a microde- synthesized by PCR from genomic DNA and cloned into letion of the imprinting center (GM12134), a normal a pCRII T/A-vector (Invitrogen). The probes were individual, an individual with an intrachromosomal trip- designed to hybridize within intron 16 (SB-1) and lication of the PWS region on the paternally-derived chro- upstream of the ApaI restriction site (SB-3) (Table 1). mosome 15 (Patient 1 in [34], Coriell Human Mutant Cell Repository # GM12135), and fibroblast RNA from Breakpoint cloning with a PCR-based method another t(4;15) PWS individual with the breakpoint in Genomic DNA from a normal individual and the t(4;15) intron 2 of SNRPN [27,28] served as controls. carrier was cleaved in a double digestion with restriction Page 4 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 t(4;15) ca Figure 2rrier at 15 years of age t(4;15) carrier at 15 years of age . Note absence of typical PWS facial features and presence of mild truncal obesity. enzymes EcoRV and ApaI, followed by adapter ligation undescended testes were observed. During infancy, he had according to the manufacturer's instructions (BD Genome poor suck and prolonged feeding times, but his weight Walker Universal Kit) [35]. A nested PCR reaction with gain was satisfactory and he did not require tube feeding. adapter primers and sequence-specific primers was per- He was suspected to have absence seizures of about 20 formed and the amplification products were cloned into seconds duration, along with proneness to giggling, some- the pC2.1 T/A-vector (Invitrogen) after gel purification. times with eye-rolling. These episodes resolved by four The clones were sequenced from both directions with uni- years of age, and an EEG was normal. versal primers from the vector (M13) and sequence spe- cific primers. He had a left esotropia that was surgically corrected. Dur- ing childhood, sticky saliva, dry mouth, skin picking and Results a marked tolerance to pain were noted and have persisted. Clinical case report The patient (Fig. 2) was born at 41 weeks of gestation with Excessive daytime somnolence continued beyond infancy a birth weight of 8 lb. Pregnancy was uneventful, but fetal and treatment with amphetamine was started at 9 years of movements were somewhat reduced. In the newborn, age. A sleep study, at 13 years of age, was normal. In 2002, poor muscle tone, weak cry, excessive sleepiness, and a further sleep study and a multiple sleep latency tests Page 5 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 confirmed the diagnosis of narcolepsy. His daytime sleep- Developmentally he had a mild delay in comparison to iness has continued to respond to dexamphetamine. his older siblings. He attended normal school but had some difficulties due to rigid behaviours and poor peer Regarding his body weight, there was no rapid weight gain interactions. Psychological testing (WISC 111, Wide between 1 and 6 years. Around 8 years of age, his interest Range Achievement test and BASC self report) revealed an in food increased, and now he would keep eating if he had overall normal intellect. However, he had some involun- unrestricted access to favorite sweet foods. He lives with tary fluctuation in attention and significant visual percep- his parents who help to control his food intake. At 14.5 tual difficulties, e.g. deficits in visual organization, in th years, he had small hands and feet, at the 20 percentile making sense of his visual world and transcribing visual th and 5 percentile, respectively, and showed mild truncal material. These perceptual problems have had a signifi- th obesity. His head circumference of 56.7 cm was at the 98 cant effect on his learning and social life. At the age of 22, percentile. Brain MRI scan was normal. At age 16 years, his he is attending a mainstream high school requiring extra height was 155.7 cm and weight 65 kg. At the age of 22 time and assistance in completing a diploma in informa- years, his height is approximately 164 cm and his weight tion technology. He is good at dismantling computers and has increased to 90 kg (BMI = 33.5). installing hardware, and prefers working on his computer to socializing. Hyperphagia and skin picking are still a At 13 years of age, he was found to have delayed puberty challenge for him. and reduced linear growth velocity with his height falling rd Cytogenetic analysis below the 3 centile. Treatment with testosterone resulted in improved height gain and genital development. At 15 High-resolution chromosome analysis showed an appar- years of age, he had a left orchidopexy and removal of a ently balanced reciprocal translocation between the long dysplastic intra-abdominal right testis. He remains on 6 arm of chromosome 4 and the proximal long arm of chro- monthly testosterone implants because of reduced mosome 15. The breakpoints were assigned to chromo- hypothalamic function. He has never been on growth hor- some bands 4q27 and 15q11: 46, XY, t(4;15)(q27;q11) mone treatment. (Fig. 3a). Parental chromosomes were normal, indicating that the patient's translocation was de novo. ho Figure 3 a. H mio gh lo resolution G-ba gs nded ideograms and prometaphase chromosomes of the translocation derivatives and their normal a. High resolution G-banded ideograms and prometaphase chromosomes of the translocation derivatives and their normal homologs . An apparently balanced translocation t(4;15)(q27;q11) was identified with arrows indicating band location of breakpoints. b. DNA methylation analysis of CpG island of SNRPN promoter and exon 1. 1. The 174 bp PCR product is derived from the methylated maternal chromosome. 2. The 100 bp product is derived from the paternal chro- mosome. PWS: PWS control, Normal: normal control, and t(4;15) carrier; H O: no template control. The t(4;15) carrier shows the normal bi-parental methylation pattern. Page 6 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 2: SNRPN and snoRNA expression analysis with quantitative RT-PCR Amplification product PWS Normal control t-PWS (4;15) PWS triplication t-PWS intron 2 t-PWS (4;15) LCL LCL LCL LCL FB FB SNURF Ex 2 0.0001 0.53 0.54 2 0.002 0.56 SNURF Ex 3 0.0002 1.1 1.43 6.4 0.0004 0.56 HBII-437 0.00003 0.86 1.13 4.9 0.00008 0.1 SNRPN 14/15 0.0013 4.7 4.91 17.23 - - SNRPN 19/20a 0.005 1.34 0.003 4.7 0.007 0.007 HBII-438 0.03 1.5 0.02 6.2 0.07 0.07 PWCR1/HBII-85 0.03 3.7 0.02 16.8 0.04 0.06 Sample identification: PWS: PWS with an IC microdeletion (patient E in [5]); t(4;15) PWS: the PWS case reported here; PWS-triplication: intrachromosomal triplication of the PWS region [33]; t-PWS intron 2: previously reported PWS case with t(4;15)(q27;q11.2) and breakpoint in SNRPN intron 2 [26, 27]; LCL, lymphoblastoid cell line, FB, fibroblast strain. The numbers represent the ratio of target product to GAPDH control product. DNA methylation analysis some 15, there was a ~11.5 kb band in lane 4, detected To exclude alternative explanations for the phenotype, with the SB-1 probe, and a ~7 kb band in lane 6, detected such as an imprinting defect, DNA methylation analysis with probe SB-3 (Fig. 5b). The novel ~11.5 kb band arose was performed. Methylation-specific PCR of the SNURF- from the der(15) chromosome, with an NheI site on the SNRPN exon 1 region revealed a normal bi-parental chromosome 15 portion and an ApaI site on the chromo- methylation pattern (Fig. 3b). some 4 portion (Fig. 5c, upper panel). The novel band of ~7 kb arose from the der(4) chromosome, with an ApaI Mapping of the translocation breakpoint by FISH site on the chromosome 15-part and an NheI site on the We performed cytogenetic and molecular studies to char- chromosome 4-part. Taken together, these results delimit acterize the breakpoint at 15q11 in detail. Preliminary the breakpoint region to ~3.6 kb between the BsaWI and FISH analysis showed that the breakpoint in 15q11 was ApaI sites (Fig. 5a). located between D15S11 and GABRB3, which flank the SNRPN locus (data not shown). On this basis, a chromo- Breakpoint mapping at the nucleotide level some walking strategy was used across this region to nar- By DNA sequencing, we mapped the breakpoint to row down the breakpoint region. We identified two BACs, SNRPN intron 17 (position chr 15: 22803227) and to RP11-160D9 (current position 22577151-22735621 on chromosome 4 at position chr. 4:123965881 (UCSC UCSC Genome Browser, May 2004 release) and RP11- Genome Browser, May 2004) (Fig. 6). On chromosome 4, 876N20 (current position 22857334-23036552), that a long terminal repeat (LTR) retrotransposon, LTR1B, flanked the breakpoint and, thus, mapped it to a ~122 kb spans the breakpoint. On chromosome 15, we found a interval (Fig. 1b). short interspersed element (SINE), AluY, and a long inter- spersed element (LINE), L1M4, surrounding the break- Fine mapping of the breakpoint by SNRPN expression and point (Fig. 6a). Thirty-nine bp upstream of the breakpoint Southern blot analysis on chromosome 15 starts a common 26 bp core sequence To further refine the breakpoint, we carried out quantita- of Alu elements (Alu-DEIN) in an inverted orientation. tive RT-PCR and RT-PCR experiments using RNA from an This sequence is known to be involved in gene rearrange- LCL and skin fibroblasts (FB) for expression of SNRPN ments [36]. While the sequence across the breakpoint is transcripts. As shown in Figure 4 and Table 2, we found contiguous on the der(15), an extra A is inserted on the expression of SNPRN exons 2, 3, and 14 to 17, but no der(4) chromosome (Fig. 6b). Furthermore, the expression of exons 18 to 20, and concluded that the breakpoint on chromosome 4 falls in a large intron breakpoint falls within intron 17. For mapping intron 17, between exons 10 and 11 of a spliced transcript we designed a Southern blot using unique restriction sites. (BC045668). By RT-PCR, we found that this transcript is DNA cleaved with NheI and BsaWI showed a 6.4 kb band expressed in fibroblasts, but not in LCLs (data not for the t(4;15) carrier and the normal control (Fig. 5b, shown). lanes 1 and 2), indicating that the breakpoint is located downstream of the BsaWI site. Samples doubly digested Expression of upstream genes MKRN3, MAGEL2, and NDN with NheI and ApaI (Fig. 5b, lanes 3–6), revealed addi- tional bands for the translocation carrier. Besides the Expression of the three imprinted genes MKRN3, expected 10 kb band derived from the normal chromo- MAGEL2, and NDN upstream of SNRPN was tested by RT- Page 7 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 SNRPN expression a Figure 4 nalysis by RT-PCR of RNA from LCLs SNRPN expression analysis by RT-PCR of RNA from LCLs . On the left, the sizes of the PCR products are shown, and on the right, the location of the primers in SNRPN exons is listed. +RT: with reverse transcriptase; -RT: without reverse tran- scriptase; H O: no template control. All SNRPN +RT products tested were absent in the PWS control, and present in the nor- mal control. The t(4;15) cells were positive for SNURF/ SNRPN exons 2–3, 15–16 and 16–17 and negative for exons 18 through 20a. GAPDH primers were used as control for the integrity of the cDNA. PCR in t(4;15) fibroblasts and found to be indistinguish- in the PWS control [6] and t(4;15) LCLs, but were able from expression in normal control fibroblasts (data expressed in the normal control LCL (Fig. 7). not shown). Discussion Expression of C/D box snoRNAs and intron-encoded ESTs Breakpoint mapping and mechanism of the translocation When testing for the intron-encoded C/D box snoRNAs, event we were able to document expression of HBII-13 and Dissecting the PWS deletion region and identifying indi- HBII-437 and lack of expression for HBII-438A/B and vidual genes as responsible for parts of the phenotype rep- HBII-85/PWCR1 (Fig. 7). By use of a more sensitive resent a challenge because all reported smaller deletions method, quantitative real-time RT-PCR, we obtained sim- inactivate all imprinted genes on the paternally- derived ilar results for the SNRPN exons and snoRNAs tested chromosome 15. Rare reciprocal translocations, therefore, (Table 2). Two ESTs, AK094315 and AB061718 (= HBT8) provide unique insights. We here report our studies of a located in the 30 kb SNRPN intron 20 were not expressed 22 year old male with features of PWS who has a de novo Page 8 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 South Figure 5 ern blot analysis identifies breakpoint in SNRPN intron 17 Southern blot analysis identifies breakpoint in SNRPN intron 17 . a . Restriction map of the intron 17 region of the SNRPN gene on the normal chromosome 15. Black arrowheads indicate the boundaries of intron 17. The positions of the two hybridization probes (SB-1 and SB-3) are indicated by green lines. b . Lanes 1 and 2 contain double digests with NheI and BsaWI to release a fragment of 6.4 kb, lanes 3 and 4 contain double digests with NheI and ApaI to release a fragment of 10 kb. The membrane was probed with probe SB-1. The arrow indicates an additional band above the 10 kb fragment ~11.5 kb in length. The two bands are not well resolved on the rendition of this blot. This novel fragment is represented in c, upper panel. Lanes 5 and 6 contain double digests with NheI and ApaI to release a 10 kb fragment. The membrane is probed with SB-3. The arrow indicates an additional band of ~ 7 kb. This novel fragment is represented schematically in c, lower panel. c . Schematic repre- sentation of the junction fragments identified on the Southern blot in b. The upper panel represents the der(15) and the lower panel represents the der(4). Chromosome 15 material is indicated as a black line and material from chromosome 4 as a blue line. Location of restriction sites and of hybridization probes (green lines) are indicated. balanced reciprocal translocation t(4;15)(q27;q11.2). translocation that may be facilitated by genomic repeats This is the first such case where the translocation break- or other distinct molecular features. points have been identified at the DNA sequence level. The cytogenetic breakpoint designations in this individual In the present case, however, we mapped the breakpoint are identical to those in another male PWS-like case with to SNRPN intron 17 (position on chr. 15: 22803227, t(4;15)(q27;q11.2), previously reported by Kuslich and UCSC Genome Browser, May 2004) that differs from that colleagues [27] and restudied by Gallagher and colleagues in the previous case (SNRPN intron 2). Furthermore, the [28], which raised the intriguing possibility of a recurrent breakpoint in our case is novel as it does not fall into one Page 9 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Repeat sequences surrounding Figure 6 the breakpoint Repeat sequences surrounding the breakpoint . a . One hundred nucleotides on either side of the breakpoints on chro- mosome 4 and 15 contain repetitive sequences (grey lines). The Alu-DEIN sequence is located 13–39 bp upstream of the breakpoint on chromosome 15. b . Sequence across the breakpoint on the der(4) chromosome reveals an additional A inserted at the breakpoint. Arrows indicate the direction centromere to telomere. of the two previously described "breakpoint clusters" in consensus Alu element [38]. Sequence analyses of regions intron 2 and exon 20a/intron 20 (Table 3 and Table 4). directly adjacent to translocation breakpoints has shown On chromosome 4 (chr. 4 123965881), the LTR retro- presence of the 26-bp Alu core sequence at or close transposon LTR1B is spanning the breakpoint, and a short (within 20–50 bp downstream or upstream) to the sites of interspersed element (SINE), AluY, and a LINE element, recombination [36]. Therefore, this sequence might stim- L1M4, surround the breakpoint on chromosome 15 (Fig. ulate homologous and non-homologous recombination 6). Interestingly, 39 bp upstream of the breakpoint on within the core or at nearby sites and could be the mech- chromosome 15 starts a common 26-bp core sequence of anism of recombination in the t(4;15) case reported here. Alu elements (Alu-DEIN) that has been shown to be involved in gene rearrangements and has homology with The translocation is de novo, as is true for all the previously prokaryotic χ, an 8-bp sequence motif known to stimulate described cases with translocation breakpoints involving recBC mediated recombination in E. coli[37]. The core the SNPRN gene. Given the PWS-like phenotype, the sequence is identical to sequences in the left arm of the translocation was assumed to be of paternal origin. This Page 10 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Expres Figure 7 sion analysis in LCLs of snoRNAs and two ESTs in intron 20 Expression analysis in LCLs of snoRNAs and two ESTs in intron 20 . RT-PCR analysis of the C/D box snoRNAs reveals expression of HBII-13, but not of HBII-438A/B, PWCR1/HBII-85 and the two ESTs in intron 20 in the t(4;15) transloca- tion carrier. +RT: with reverse transcriptase; -RT: without reverse transcriptase; H O: no template control. assumption was confirmed by the expression studies. Wirth et al. 2001, and also in the present case. But this is Paternal origin of the translocation was formally proven not a consistent feature in classical PWS, as in a in 2 of the 5 previously reported cases [26,27]. retrospective evaluation of 90 molecularly-proven PWS cases, only 49% had the characteristic facial gestalt [39]. Karyotype – phenotype correlations Two individuals with SNRPN intron 2 breakpoints were It is apparent from the review of the previously reported described as having classical PWS, meeting the major cases and the individual reported here (Table 3 and Table clinical criteria by age 3.5 years and additional minor clin- 4) that some of these translocation cases tend to have a ical criteria [25,27]. The individuals with a breakpoint in milder, 'atypical' clinical picture, in comparison with clas- SNRPN Exon 20/Intron 20 were described as having a sical PWS. There is not a complete absence of any of the milder or atypical form of PWS (Table 3 and Table 4). The major phenotypic features (neonatal hypotonia and feed- weight gain started later than in classical PWS, at 7 and 5 ing difficulty, hyperphagia from early childhood, obesity, years, respectively, for the patients described by Schulze et cognitive compromise, hypogenitalism), but the degree of al. 1996 and Wirth et al. 2001, and at 8 years in our case. affection may be lower. None of the reported The characteristic facial features were absent in the case of translocation cases had any additional features that might Page 11 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 3: Clinical findings associated with paternally-derived de novo reciprocal translocations involving SNRPN Breakpoint in SNRPN Intron 2 Breakpoint in SNRPN Exon 20/ Intron 20 Breakpoint in SNRPN Intron 17 Sun et al. 1996 Kuslich et al. 1999 Schulze et al. 1996 Conroy et al. 1997 Wirth et al. 2001 Present case Karyotype 46, XY, t(15;19) 46, XY, t(4;15) 46, XY, t(9;15) 46, XY, t(2;15) 46, X, t(X;15) 46 XY, designations (q12;q13.41) (q27;q11.2) (q21;q12–q13) (q37.2;q11.2) (q28;q12) t(4;15)(q27;q11.2) Age of 3.5 years 3 years 3 months 29 years 4.5 years 18 years 22 years examination Major criteria (each scores one point) from [1] as revised in [2]. 1. Neonatal Floppy and Hypotonicity, Neonatal Neonatal - Reduced tone with central hypotonia lethargic in the poor sucking hypotonia (weak hypotonia, poor head control, first 6 months with reflex during cry, poor suck) (1 lethargy, poor poor suck (1 pt.) poor suck (1pt.) infancy (1pt.) pt.) suck (1 pt.) 2. Infantile feeding Failure to thrive Feeding problems Special feeding - Feeding problems, problems/ failure (1pt.) in infancy, failure techniques, but no but no failure to to thrive to thrive (1 pt.) failure to thrive thrive (1pt.) 3. Rapid weight Obesity starting at Eating behavior Periodic excessive Onset of obesity Obesity began at Late onset obesity gain between 1–6 6 months, leading to weight gain from at 1.5–2 yr with 4–5 yr with (at approx. 8 yr) years hyperphagia (1 pt.) increased weight age 7 yr excessive appetite hyperphagia and gain at age 2 yr (1 and food foraging food foraging (1 pt.) (1 pt.) pt.) 4. Characteristic Narrow bifrontal Narrow bifrontal Narrow bifrontal Narrow bifrontal -- facial features diameter, almond- diameter, almond- diameter, narrow diameter, squared shaped eyes, shaped eyes, face, small mouth, nasal tip, down-turned upslanted poor facial mimic downturned mouth (1pt.) palpebral fissures (1pt.) mouth (1 pt.) (1 pt.) 5. Hypogonadism: Undescended Undescended Hypoplastic Primary Undescended Scrotum normal, genital hypoplasia, testes (1 pt.) small testes, genitalia, penile length at amenorrhea, small testes, th pubertal deficiency hypogonadism (1 incomplete 10 %ile hypoplastic uterus hypogonadism, pt.) gonadal (1 pt.) delayed pubertal maturation with signs (1 pt.) delayed pubertal signs after age 16 yr (1 pt.) 6. Mental Developmental Developmental Mental Developmental Slight Developmental retardation, delay (1 pt.) delay (1 pt.) retardation, delay, special developmental delay, special developmental developmental school setting (1 delay, school for school setting (1 delay delay/ learning pt.) mentally retarded pt.) problems (1 pt.) children (1 pt.) Score 5 points 6 points 5 points 4 points 3 points 4 points Blank cell = no information - = absent possibly be attributed to disruption of a gene on the recip- Our sequence data mapped the breakpoint on chromo- rocal chromosome, and in no prior case had an attempt some 4 within intron 10 of a spliced polyadenylated tran- been made to identify a gene at this location. script (BC045668). This unique cDNA clone represents a Page 12 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 Table 4: Clinical findings associated with paternally-derived de novo reciprocal translocations involving SNRPN (continued) Sun et al. 1996 Kuslich et al. 1999 Schulze et al. 1996 Conroy et al. 1997 Wirth et al. 2001 Present case Minor criteria (1/2 point each) 1. Decreased fetal Decreased fetal Decreased fetal - - Slightly reduced movement and activity (0.5 pt.) movements fetal movements infantile lethargy (0.5pt.) (0.5pt.) 2. Typical Behavior problems Temper tantrums, Aggressive Behavior problems Temper tantrums, Temper tantrums, behaviour (0.5pt.) violent outbursts, outbursts, rigid with temper violent outbursts abnormal social problems obsessive- personality, tantrums and after food behavior (0.5pt.) compulsive (0.5 perseveration severe restrictions pt.) (0.5pt.) aggressiveness (0.5 (0.5pt.) pt.) 3. Sleep Sleep disturbance, Sleep disturbance Sleep disturbance, disturbance, sleep sleep apnea (0.5pt.) amphetamine apnea (0.5pt.) treatment from age 9 ys. (0.5pt.) th rd 4. Short stature Short stature at 50–75 percentile 151 cm (3 %tile) Height 155.7 cm at rd for the family by the age of 15 (0.5pt.) (0.5pt.) 16 years < 3 age 15 years (0.5pt.) %tile (0.5 pt.) 5. - - Hypopigmentation -- Hypopigmentation (0.5 pt.) th th 6. Small hands and Hand length 25 - Normal hands, but Short 3rd finger Hands 20 %ile, /or feet for height percentile, finger small feet (< bilaterally feet 5th %ile th th %ile 10 %tile) (0.5 pt.) age length 10 (0.5pt.) (0.5pt.) 7. Narrow hands - - with straight ulnar border 8. Eye - Esotropia (0.5 pt.) Alternating Left esotropia (0.5 Esotropia (0.5pt.) abnormalities: esotropia in pt.) esotropia, myopia infancy (0.5 pt.) 9. Thick viscous Viscous saliva Thick viscous - saliva (0.5pt.) saliva (0.5 pt.) 10. Speech Articulation Poor articulation - articulation defect difficulty (0.5 pt.) (0.5pt.) 11. Skin picking Skin picking Skin picking Skin picking (0.5pt.) (0.5pt.) (0.5pt.) Score (minor only) 1.5 points 3 points 3.5 points 2.5 points 1.5 points 3.5 points Total Score 6.5 points 9 points 8.5 points 8.5 points 4.5 points 7.5 points Blank cell = no information - = absent 3764 bp mRNA from a human testis library that does not leukin 21 (IL21) transcript by 511 bp in the opposite appear to encode a protein. Its 5' end overlaps the inter- direction (UCSC Genome Browser, May 2004). It appears Page 13 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 unlikely that heterozygous disruption of this gene con- tissues [42] were found to be expressed in a normal con- tributes to the phenotype in our patient. trol LCL, but not in the t(4;15) LCL. This result suggests that these ESTs do not have their own promoter but are Translocation has no effect on imprinting center dependent on transcription from the SNRPN promoter methylation and upstream genes that is located on the other translocation derivative in To assess whether the translocation event had affected the these cells. Therefore, these ESTs most likely represent sta- allele-specific methylation pattern at the imprinting ble derivatives of large alternatively spliced non-coding center (IC), and/or to exclude a coincident imprinting SNRPN transcripts. defect, we carried out methylation studies of the SNRPN exon 1 region that revealed a normal bi-parental methyl- Conclusion ation pattern. Similar results were reported for each of the (1) Expression of the ESTs and/or C/D box snoRNAs that other five PWS individuals who had translocation break- are located downstream of the translocation breakpoint is points within the SNRPN gene. These results predict that not necessary for establishing and maintaining the pater- expression of the genes located centromeric to the SNRPN nal-specific pattern of gene expression pattern that is exon 1/ IC region, NDN, MAGEL2 and MKRN3, should controlled by the imprinting center upstream of the trans- not be affected in these individuals. By studying t(4;15) location breakpoint. fibroblasts by RT-PCR, we indeed found expression of all three genes. Previously, only MKRN3 was reported to be (2) The C/D box snoRNAs HBII-438A and PWCR1/HBII- expressed in the three PWS translocation cases in which it 85 are the only stable transcripts in this region that are dis- was studied [20,25,27]. rupted in this t(4;15) PWS individual. As PWCR1/HBII-85 sequences are highly conserved between human and In t(4;15) lymphoblasts, the SNRPN transcript was detect- mice, while no copy of HBII-438A has been found in able by RT-PCR and quantitative RT-PCR and found to mouse, we conclude that the basis of PWS pathogenesis extend all the way to exon 17. The major transcript that resides, in whole or in part, in the absence of PWCR1/ encodes the SNURF/SNRPN proteins terminates in exon HBII-85 snoRNA. SNURF/SNRPN and the centromeric 10 [20] and, therefore, should be unaffected by this genes MKRN3, NDN and MAGEL2 are unlikely to play a t(4;15) translocation. With the caveat that studies on major role in the causation of PWS-associated features. peripheral tissues, fibroblasts and lymphoblasts, may not While the function of known C/D box snoRNAs is to accurately reflect gene expression in the brain, our results guide 2'- O -ribose methylation of mainly ribosomal RNA, indicate that SNURF/SNRPN and the centromeric genes these novel imprinted snoRNAs have no known target. MKRN3, NDN and MAGEL2 are unlikely to play a prime They might be involved in a posttranscriptional role in the causation of PWS-associated features, although regulation process of a gene or genes that – if non-func- it remains an open question whether their loss or non- tional – gives rise to the PWS phenotype. functioning might contribute to the more marked pheno- typic expression that is seen in typical PWS. Competing interests The author(s) declare that they have no competing Genes downstream of the breakpoint are not expressed interests. With respect to expression of downstream transcripts, the reported results on LCLs with breakpoints in exon 20/ Authors' contributions intron 20 were consistent, whereas for the two patients BS carried out the molecular genetic studies (RT-PCR, with breakpoints in intron 2, the reported results were methylation assay, Southern blot analysis, and breakpoint conflicting for expression of downstream transcripts IPW analysis) and drafted the manuscript. MA carried out and PAR-1. In a re-evaluation of the t(4;15) case reported quantitative RT-PCR assays. EN performed the FISH anal- by Kuslich and colleagues [26], no expression of these ysis with BAC clones. DIF carried out the initial cytoge- transcripts and of the PWCR1/HBII-85 snoRNA cluster netic analysis. MR revised the clinical data and re- was detected by real-time quantitative RT-PCR [28]. examined the patient. Therefore, we focused our analysis on the snoRNAs and HRS supervised the cell culturing, cytogenetic and FISH two ESTs in intron 20. As for the intron-encoded C/D box studies. RJMG diagnosed the patient, collected the clinical snoRNAs, HBII-13 and HBII-437 were expressed, and data and obtained skin and blood samples. UF conceived HBII-438A/B and HBII-85/PWCR1 were not. HBII-52 the study design, and coordinated its progress, supervised snoRNAs were not studied, as they are not expressed in the the work of BS and MA and prepared the final manuscript. available tissues and have previously been excluded from contributing to the PWS phenotype [40,41]. The two ESTs Acknowledgements We are indebted to the family participating in this study and to Prof. in the large intron 20 that are highly expressed in brain George A. Werther who referred the patient to us, and in his letter wrote Page 14 of 16 (page number not for citation purposes) BMC Medical Genetics 2005, 6:18 http://www.biomedcentral.com/1471-2350/6/18 "I wonder whether this translocation may involve the Prader-Willi gene". multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum Mol Genet 2001, 10:2687-2700. The work in the laboratory of UF was supported by grants from the NIH 20. Wirth J, Back E, Huttenhofer A, Nothwang HG, Lich C, Gross S, Men- (HD41623) and the Deutsche Forschungsgemeinschaft (BS – SCHU 1567/ zel C, Schinzel A, Kioschis P, Tommerup N, Ropers HH, Horsthemke 1-1). B, Buiting K: A translocation breakpoint cluster disrupts the newly defined 3' end of the SNURF-SNRPN transcription unit on chromosome 15. Hum Mol Genet 2001, 10:201-210. References 21. de Los Santos T, Schweizer J, Rees CA, Francke U: Small evolution- 1. 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Chai JH, Locke DP, Greally JM, Knoll JH, Ohta T, Dunai J, Yavor A, Eichler EE, Nicholls RD: Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons. Am J Hum Genet 2003, 73:898-925. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2350/6/18/prepub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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