- Research article
- Open Access
- Open Peer Review
Molecular breakpoint cloning and gene expression studies of a novel translocation t(4;15)(q27;q11.2) associated with Prader-Willi syndrome
© Schüle et al; licensee BioMed Central Ltd. 2005
- Received: 20 January 2005
- Accepted: 06 May 2005
- Published: 06 May 2005
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).
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.
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.
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.
- Long Terminal Repeat
- UCSC Genome Browser
- Translocation Breakpoint
- snoRNA Gene
- Imprint Center
Prader-Willi syndrome (PWS) is a complex neurodevelopmental disorder and a classic example for genomic imprinting in humans. The incidence is about 1 in 10–20,000, and the clinical manifestations include decreased fetal activity, neonatal hypotonia, neonatal feeding difficulties, hyperphagia with obesity, hypogonadism, short stature, small hands and feet, characteristic facial features, and mild to moderate mental retardation. Diagnostic criteria have been proposed  and revised recently .
Three paternally expressed genes have been identified between BP2 and SNRPN. These include MKRN3/ ZNF127 (MIM# 603856; Makorin 3 or Zinc finger protein 127) [8, 9], MAGEL2/NDNL1 (MIM# 605283; MAGE-like 2 or Necdin-like 1) [10, 11], and NDN (MIM# 602117; Necdin) [12, 13] (Figure 1a). The small nuclear ribonucleoprotein polypeptide N (MIM# 182279; SNRPN) gene was the first gene with a known function to be mapped to the PWS/AS deletion region, and is expressed from the paternal chromosome only [14–17]. Multiple alternatively spliced transcripts originate at the SNPRN promoter [18–20]. The major SNRPN transcript is bi-cistronic encoding two mRNA species. Exons 1–3 encode a protein product of unknown function called SNURF (SNRPN upstream reading frame). Exons 4–10 encode SmN, a homolog of the SmB/B' protein that binds small nuclear RNAs involved in pre-mRNA splicing. The largest transcripts extend over a ~460 kb genomic region and include a large 3'UTR comprising up to 148 exons .
Multiple introns downstream of the SNURF-SNRPN coding region contain C/D box small nucleolar RNA (snoRNA) genes. There are two multi-copy snoRNA clusters (HBII-52 and PWCR1/HBII-85) [21, 22], three single copy snoRNA genes (HBII-436, HBII-13, and HBII-437), and one snoRNA gene (HBII-438) present in two copies that are 240 kb apart . Since the snoRNAs are derived from processed spliced-out introns, their expression is controlled by the SNRPN promoter and is highest in brain. The known function of other C/D box snoRNAs is to guide 2'- O – ribose methylation of ribosomal RNA or small nuclear RNA. This post-transcriptional modification is conserved throughout evolution and is thought to confer increased stability to the small RNA molecules . The modification targets of the imprinted C/D box snoRNAs in the PWS/AS region are still unknown.
Spontaneous chromosome translocations can be extremely valuable for assessing the contributions of individual loci to the phenotype of microdeletion syndromes. Five individuals with features of PWS have been reported who have balanced reciprocal translocations with breakpoints in the PWS/AS deletion region. All of them involve the SNRPN locus. The breakpoints are located in intron 2 (proximal, n = 2), disrupting the SNURF/SNRPN coding region, or in exon 20a/intron 20 (distal, n = 3) within the 3'-untranslated region of the long SNRPN transcript. One individual with a proximal and two of three patients with a distal breakpoint meet the diagnostic criteria for PWS (score of 8 or more points) [20, 24–28].
Here we report the clinical, cytogenetic and molecular characterization of a 22 year old male with features of PWS who has a different de novo balanced reciprocal translocation t(4;15)(q27;q11.2). We mapped the breakpoint to SNRPN intron 17 (position on chr 15: 22803227, UCSC Genome browser May 2004) and determined the expression of snoRNAs on both sides of the breakpoint in cultured fibroblasts and lymphoblasts.
Cytogenetic and FISH analysis
Metaphase spreads obtained from short-term blood lymphocyte cultures and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells (LCL) were processed for high-resolution GTG- banding by standard methods. For FISH studies, Bacterial Artificial Chromosomes (BACs) were sourced from the RPCI-11 library and selected using the UCSC Genome Browser, Assemblies: July 2003 and May 2004). Fluorescence labelling, hybridization procedures and imaging were performed as previously described .
DNA methylation study
Genomic DNA was purified by phenol-chloroform extraction from LCLs from the study subject, a normal control, and a PWS individual (Patient E in , Coriell Human Mutant Cell Repository # GM12134). To investigate methylation at exon 1 of SNRPN, 50 μg DNA were used for the bisulfite reaction and PCR with primers according to standard protocols [30, 31]. PCR products were separated on a 3% agarose gel, stained with ethidium bromide, and visualized under UV illumination.
Expression studies by RT-PCR and quantitative RT-PCR
Primers and conditions for PCR
rev (5'-3'); complement strand
GGG TTG CGG TTT TGC TAT TA
TTT CTC GTG TGC TTC AAT GC
CTG AAG CCT GGG ACT TTC TG
GGA CCT TGG CCA CAA ACT TA
GAA GAA GCA CTC CAC CTT CG
CCA TGA TTT GCA TCT TGG TG
SNRPN Ex 1–3
ATG GAG CGG GCA AGG GAT CGC
GGT ACA ACT GAC ACT CTT GG
SNRPN Ex 14/16
CTG CAA ACA TAG GAG ATG ATA GTT CC
CTT ATG AAA GCA CTG AGA TGA AGC C
SNRPN Ex 16/17
GAA AGT GAC CTA AAG AGT GTC ATT G
CTT GCA GTT GGA CAG CCG ACT C
SNRPN Ex 17/18
AGA TAT CTT TAA AAT TGA GTC TTC TGT CCA
TGA AGA TGC AGC ACT TTT GAA GAA
SNRPN Ex 19/20a
CAT TGT GCT TAT TTA CTA TTT TTG TAG ACG
CTG CAG GTG GTG ACC ATG TG
TCT TCT CTA CCC TCA TTC CCA GC
TCG CTA CAC CCC TTT GCT TAT G
AGG AGG GGT TCA AAG ATG C
CTG GTA AAC AAA CTG GTA AAG GTG
CGA TGA TGA GTC CCC CAT AAA AAC
CAG TTC CGA TGA GAA CGA CG
GGA TTT GTG ATG AGC TGT GTT TAC
GGA CTT CAG AGT AAT CAC GTT G
GGA TCG ATG ATG AGA ATA ATT ATT G
GGA CCT CAG ATT GAC ATC TG
TCA GGC GGC ATT GGC GAT ACC
ATA AAA ACT TGA CAG GCA GAG
AAT ATT GCC TTA ATG TTT CTA
GCC TAT TCT ATT TCT TCG TGT
GCG TAT TCA CAT AGA CAT CTT G
GAT TGG TCA CTA CTG GTC TGG
TGG GCT ACA CTG AGC ACC AG
GGG TGT CGC TGT TGA AGT CA
rev (5'-3');complement strand
ACG AAC TAC AGA ACA GCA CGT ACC
CTG CGT TTG ACT TGG ACT TCC
TTC TCA GCA GCA GCA AGT ACC T
TGC CTC AGT TCA GCC TGG A
ATC ATT ATT TCT TGA ATT GG
CCC TCA CGC TCC CTT TGC
SNRPN Ex 14/15
CTG CAA ACA TAG GAG ATG ATA GTT CC
CAA AGA CGA TAA AAT GTT CCT TCT TG
SNRPN Ex 19/20a
GGA ACC ACC ATT TGT CTA TGA TCC
CTG CAG GTG GTG ACC ATG TG
ATA ATT GTC TGA GGA TGC T
GAT TGA CAT CTG GAA TGA GTC
TCG ATG ATG AGT CCC CCA TAA
CAT TTT GTT CAG CTT TTC CAA GG
PCR to generate Southern probes in intron 17
rev (5'-3');complement strand
ACC ATC AGT GAA TGA CCT GTT GC
CCC AGC CTC TTT CCT ATG TCT TG
TGG TAA ACT GAT GAG AGC ACA GCC
GCC TGG GAG ACA GAA TGA GAA AC
For a subset of exons in the SNPRN gene and the snoRNAs HBII-13, HBII-437, and PWCR1/HBII-85, quantitative RT-PCR assays were performed with SYBR Green I™ dye in an ABI 7700 cycler (Applied Biosystems) by using standard protocols [32, 33]. Primers were designed to amplify products of 50 bp in length. GAPDH expression was used as a reference. Each sample was run at least in triplicate. The results were interpreted as described previously .
LCL RNA samples from a PWS individual with a microdeletion of the imprinting center (GM12134), a normal individual, an individual with an intrachromosomal triplication of the PWS region on the paternally-derived chromosome 15 (Patient 1 in , Coriell Human Mutant Cell Repository # GM12135), and fibroblast RNA from another t(4;15) PWS individual with the breakpoint in intron 2 of SNRPN [27, 28] served as controls.
Southern blot analysis
Southern blot analysis was performed according to standard methods with ExpressHyb™ solution (BD Biosciences). Genomic DNA from a normal individual and the t(4;15) carrier was cleaved in a double digestion with restriction enzymes NheI and BsaWI to release a 6.4 kb fragment, and with NheI and ApaI to release a 10 kb fragment in the normal chromosome. The DNA probes were synthesized by PCR from genomic DNA and cloned into a pCRII T/A-vector (Invitrogen). The probes were designed to hybridize within intron 16 (SB-1) and upstream of the ApaI restriction site (SB-3) (Table 1).
Breakpoint cloning with a PCR-based method
Genomic DNA from a normal individual and the t(4;15) carrier was cleaved in a double digestion with restriction enzymes EcoRV and ApaI, followed by adapter ligation according to the manufacturer's instructions (BD Genome Walker Universal Kit) . A nested PCR reaction with adapter primers and sequence-specific primers was performed and the amplification products were cloned into the pC2.1 T/A-vector (Invitrogen) after gel purification. The clones were sequenced from both directions with universal primers from the vector (M13) and sequence specific primers.
Clinical case report
He had a left esotropia that was surgically corrected. During childhood, sticky saliva, dry mouth, skin picking and a marked tolerance to pain were noted and have persisted.
Excessive daytime somnolence continued beyond infancy and treatment with amphetamine was started at 9 years of age. A sleep study, at 13 years of age, was normal. In 2002, a further sleep study and a multiple sleep latency tests confirmed the diagnosis of narcolepsy. His daytime sleepiness has continued to respond to dexamphetamine.
Regarding his body weight, there was no rapid weight gain between 1 and 6 years. Around 8 years of age, his interest in food increased, and now he would keep eating if he had unrestricted access to favorite sweet foods. He lives with his parents who help to control his food intake. At 14.5 years, he had small hands and feet, at the 20th percentile and 5th percentile, respectively, and showed mild truncal obesity. His head circumference of 56.7 cm was at the 98th percentile. Brain MRI scan was normal. At age 16 years, his height was 155.7 cm and weight 65 kg. At the age of 22 years, his height is approximately 164 cm and his weight has increased to 90 kg (BMI = 33.5).
At 13 years of age, he was found to have delayed puberty and reduced linear growth velocity with his height falling below the 3rd centile. Treatment with testosterone resulted in improved height gain and genital development. At 15 years of age, he had a left orchidopexy and removal of a dysplastic intra-abdominal right testis. He remains on 6 monthly testosterone implants because of reduced hypothalamic function. He has never been on growth hormone treatment.
Developmentally he had a mild delay in comparison to his older siblings. He attended normal school but had some difficulties due to rigid behaviours and poor peer interactions. Psychological testing (WISC 111, Wide Range Achievement test and BASC self report) revealed an overall normal intellect. However, he had some involuntary fluctuation in attention and significant visual perceptual difficulties, e.g. deficits in visual organization, in making sense of his visual world and transcribing visual material. These perceptual problems have had a significant effect on his learning and social life. At the age of 22, he is attending a mainstream high school requiring extra time and assistance in completing a diploma in information technology. He is good at dismantling computers and installing hardware, and prefers working on his computer to socializing. Hyperphagia and skin picking are still a challenge for him.
DNA methylation analysis
To exclude alternative explanations for the phenotype, such as an imprinting defect, DNA methylation analysis was performed. Methylation-specific PCR of the SNURF-SNRPN exon 1 region revealed a normal bi-parental methylation pattern (Fig. 3b).
Mapping of the translocation breakpoint by FISH
We performed cytogenetic and molecular studies to characterize the breakpoint at 15q11 in detail. Preliminary FISH analysis showed that the breakpoint in 15q11 was located between D15S11 and GABRB3, which flank the SNRPN locus (data not shown). On this basis, a chromosome walking strategy was used across this region to narrow down the breakpoint region. We identified two BACs, RP11-160D9 (current position 22577151-22735621 on UCSC Genome Browser, May 2004 release) and RP11-876N20 (current position 22857334-23036552), that flanked the breakpoint and, thus, mapped it to a ~122 kb interval (Fig. 1b).
Fine mapping of the breakpoint by SNRPN expression and Southern blot analysis
SNRPN and snoRNA expression analysis with quantitative RT-PCR
t-PWS intron 2
SNURF Ex 2
SNURF Ex 3
Breakpoint mapping at the nucleotide level
Expression of upstream genes MKRN3, MAGEL2, and NDN
Expression of the three imprinted genes MKRN3, MAGEL2, and NDN upstream of SNRPN was tested by RT-PCR in t(4;15) fibroblasts and found to be indistinguishable from expression in normal control fibroblasts (data not shown).
Expression of C/D box snoRNAs and intron-encoded ESTs
Breakpoint mapping and mechanism of the translocation event
Dissecting the PWS deletion region and identifying individual genes as responsible for parts of the phenotype represent a challenge because all reported smaller deletions inactivate all imprinted genes on the paternally- derived chromosome 15. Rare reciprocal translocations, therefore, provide unique insights. We here report our studies of a 22 year old male with features of PWS who has a de novo balanced reciprocal translocation t(4;15)(q27;q11.2). This is the first such case where the translocation breakpoints have been identified at the DNA sequence level. The cytogenetic breakpoint designations in this individual are identical to those in another male PWS-like case with t(4;15)(q27;q11.2), previously reported by Kuslich and colleagues  and restudied by Gallagher and colleagues , which raised the intriguing possibility of a recurrent translocation that may be facilitated by genomic repeats or other distinct molecular features.
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
46, XY, t(15;19) (q12;q13.41)
46, XY, t(4;15) (q27;q11.2)
46, XY, t(9;15) (q21;q12–q13)
46, XY, t(2;15) (q37.2;q11.2)
46, X, t(X;15) (q28;q12)
46 XY, t(4;15)(q27;q11.2)
Age of examination
3 years 3 months
Major criteria (each scores one point) from  as revised in .
1. Neonatal central hypotonia
Floppy and lethargic in the first 6 months with poor suck (1pt.)
Hypotonicity, poor sucking reflex during infancy (1pt.)
Neonatal hypotonia (weak cry, poor suck) (1 pt.)
Neonatal hypotonia, lethargy, poor suck (1 pt.)
Reduced tone with poor head control, poor suck (1 pt.)
2. Infantile feeding problems/ failure to thrive
Failure to thrive (1pt.)
Feeding problems in infancy, failure to thrive (1 pt.)
Special feeding techniques, but no failure to thrive
Feeding problems, but no failure to thrive (1pt.)
3. Rapid weight gain between 1–6 years
Obesity starting at 6 months, hyperphagia (1 pt.)
Eating behavior leading to increased weight gain at age 2 yr (1 pt.)
Periodic excessive weight gain from age 7 yr
Onset of obesity at 1.5–2 yr with excessive appetite and food foraging (1 pt.)
Obesity began at 4–5 yr with hyperphagia and food foraging (1 pt.)
Late onset obesity (at approx. 8 yr)
4. Characteristic facial features
Narrow bifrontal diameter, almond-shaped eyes, down-turned mouth (1pt.)
Narrow bifrontal diameter, almond-shaped eyes, upslanted palpebral fissures (1 pt.)
Narrow bifrontal diameter, narrow face, small mouth, poor facial mimic (1pt.)
Narrow bifrontal diameter, squared nasal tip, downturned mouth (1 pt.)
5. Hypogonadism: genital hypoplasia, pubertal deficiency
Undescended testes (1 pt.)
Undescended small testes, hypogonadism (1 pt.)
Hypoplastic genitalia, incomplete gonadal maturation with delayed pubertal signs after age 16 yr (1 pt.)
Scrotum normal, penile length at 10th %ile
Primary amenorrhea, hypoplastic uterus (1 pt.)
Undescended small testes, hypogonadism, delayed pubertal signs (1 pt.)
6. Mental retardation, developmental delay
Developmental delay (1 pt.)
Developmental delay (1 pt.)
Mental retardation, developmental delay/ learning problems (1 pt.)
Developmental delay, special school setting (1 pt.)
Slight developmental delay, school for mentally retarded children (1 pt.)
Developmental delay, special school setting (1 pt.)
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
Minor criteria (1/2 point each)
1. Decreased fetal movement and infantile lethargy
Decreased fetal activity (0.5 pt.)
Decreased fetal movements (0.5pt.)
Slightly reduced fetal movements (0.5pt.)
2. Typical behaviour problems
Behavior problems (0.5pt.)
Temper tantrums, violent outbursts, obsessive-compulsive (0.5 pt.)
Aggressive outbursts, rigid personality, perseveration (0.5pt.)
Behavior problems with temper tantrums and severe aggressiveness (0.5 pt.)
Temper tantrums, violent outbursts after food restrictions (0.5pt.)
Temper tantrums, abnormal social behavior (0.5pt.)
3. Sleep disturbance, sleep apnea
Sleep disturbance, sleep apnea (0.5pt.)
Sleep disturbance (0.5pt.)
Sleep disturbance, amphetamine treatment from age 9 ys. (0.5pt.)
4. Short stature for the family by age 15 years
Short stature at the age of 15 (0.5pt.)
50–75th percentile (0.5pt.)
151 cm (3rd%tile) (0.5pt.)
Height 155.7 cm at 16 years < 3rd %tile (0.5 pt.)
Hypopigmentation (0.5 pt.)
6. Small hands and /or feet for height age
Hand length 25th percentile, finger length 10th%ile (0.5pt.)
Normal hands, but small feet (< 10th%tile) (0.5 pt.)
Short 3rd finger bilaterally
Hands 20th %ile, feet 5th %ile (0.5pt.)
7. Narrow hands with straight ulnar border
8. Eye abnormalities: esotropia, myopia
Esotropia (0.5 pt.)
Alternating esotropia in infancy (0.5 pt.)
Left esotropia (0.5 pt.)
9. Thick viscous saliva
Viscous saliva (0.5pt.)
Thick viscous saliva (0.5 pt.)
10. Speech articulation defect
Articulation difficulty (0.5 pt.)
Poor articulation (0.5pt.)
11. Skin picking
Skin picking (0.5pt.)
Skin picking (0.5pt.)
Skin picking (0.5pt.)
Score (minor only)
The translocation is de novo, as is true for all the previously described cases with translocation breakpoints involving the SNPRN gene. Given the PWS-like phenotype, the translocation was assumed to be of paternal origin. This assumption was confirmed by the expression studies. Paternal origin of the translocation was formally proven in 2 of the 5 previously reported cases [26, 27].
Karyotype – phenotype correlations
Two individuals with SNRPN intron 2 breakpoints were described as having classical PWS, meeting the major clinical criteria by age 3.5 years and additional minor clinical criteria [25, 27]. The individuals with a breakpoint in SNRPN Exon 20/Intron 20 were described as having a milder or atypical form of PWS (Table 3 and Table 4). The weight gain started later than in classical PWS, at 7 and 5 years, respectively, for the patients described by Schulze et al. 1996 and Wirth et al. 2001, and at 8 years in our case. The characteristic facial features were absent in the case of Wirth et al. 2001, and also in the present case. But this is not a consistent feature in classical PWS, as in a retrospective evaluation of 90 molecularly-proven PWS cases, only 49% had the characteristic facial gestalt .
It is apparent from the review of the previously reported cases and the individual reported here (Table 3 and Table 4) that some of these translocation cases tend to have a milder, 'atypical' clinical picture, in comparison with classical PWS. There is not a complete absence of any of the major phenotypic features (neonatal hypotonia and feeding difficulty, hyperphagia from early childhood, obesity, cognitive compromise, hypogenitalism), but the degree of affection may be lower. None of the reported translocation cases had any additional features that might possibly be attributed to disruption of a gene on the reciprocal chromosome, and in no prior case had an attempt been made to identify a gene at this location.
Our sequence data mapped the breakpoint on chromosome 4 within intron 10 of a spliced polyadenylated transcript (BC045668). This unique cDNA clone represents a 3764 bp mRNA from a human testis library that does not appear to encode a protein. Its 5' end overlaps the interleukin 21 (IL21) transcript by 511 bp in the opposite direction (UCSC Genome Browser, May 2004). It appears unlikely that heterozygous disruption of this gene contributes to the phenotype in our patient.
Translocation has no effect on imprinting center methylation and upstream genes
To assess whether the translocation event had affected the allele-specific methylation pattern at the imprinting center (IC), and/or to exclude a coincident imprinting defect, we carried out methylation studies of the SNRPN exon 1 region that revealed a normal bi-parental methylation pattern. Similar results were reported for each of the other five PWS individuals who had translocation breakpoints within the SNRPN gene. These results predict that expression of the genes located centromeric to the SNRPN exon 1/ IC region, NDN, MAGEL2 and MKRN3, should not be affected in these individuals. By studying t(4;15) fibroblasts by RT-PCR, we indeed found expression of all three genes. Previously, only MKRN3 was reported to be expressed in the three PWS translocation cases in which it was studied [20, 25, 27].
In t(4;15) lymphoblasts, the SNRPN transcript was detectable by RT-PCR and quantitative RT-PCR and found to extend all the way to exon 17. The major transcript that encodes the SNURF/SNRPN proteins terminates in exon 10  and, therefore, should be unaffected by this t(4;15) translocation. With the caveat that studies on peripheral tissues, fibroblasts and lymphoblasts, may not accurately reflect gene expression in the brain, our results indicate that SNURF/SNRPN and the centromeric genes MKRN3, NDN and MAGEL2 are unlikely to play a prime role in the causation of PWS-associated features, although it remains an open question whether their loss or non-functioning might contribute to the more marked phenotypic expression that is seen in typical PWS.
Genes downstream of the breakpoint are not expressed
With respect to expression of downstream transcripts, the reported results on LCLs with breakpoints in exon 20/intron 20 were consistent, whereas for the two patients with breakpoints in intron 2, the reported results were conflicting for expression of downstream transcripts IPW and PAR-1. In a re-evaluation of the t(4;15) case reported by Kuslich and colleagues , no expression of these transcripts and of the PWCR1/HBII-85 snoRNA cluster was detected by real-time quantitative RT-PCR .
Therefore, we focused our analysis on the snoRNAs and two ESTs in intron 20. As for the intron-encoded C/D box snoRNAs, HBII-13 and HBII-437 were expressed, and HBII-438A/B and HBII-85/PWCR1 were not. HBII-52 snoRNAs were not studied, as they are not expressed in the available tissues and have previously been excluded from contributing to the PWS phenotype [40, 41]. The two ESTs in the large intron 20 that are highly expressed in brain tissues  were found to be expressed in a normal control LCL, but not in the t(4;15) LCL. This result suggests that these ESTs do not have their own promoter but are dependent on transcription from the SNRPN promoter that is located on the other translocation derivative in these cells. Therefore, these ESTs most likely represent stable derivatives of large alternatively spliced non-coding SNRPN transcripts.
(1) Expression of the ESTs and/or C/D box snoRNAs that are located downstream of the translocation breakpoint is not necessary for establishing and maintaining the paternal-specific pattern of gene expression pattern that is controlled by the imprinting center upstream of the translocation breakpoint.
(2) The C/D box snoRNAs HBII-438A and PWCR1/HBII-85 are the only stable transcripts in this region that are disrupted in this t(4;15) PWS individual. As PWCR1/HBII-85 sequences are highly conserved between human and mice, while no copy of HBII-438A has been found in mouse, we conclude that the basis of PWS pathogenesis resides, in whole or in part, in the absence of PWCR1/HBII-85 snoRNA. SNURF/SNRPN and the centromeric genes MKRN3, NDN and MAGEL2 are unlikely to play a major role in the causation of PWS-associated features. While the function of known C/D box snoRNAs is to guide 2'- O -ribose methylation of mainly ribosomal RNA, these novel imprinted snoRNAs have no known target. They might be involved in a posttranscriptional regulation process of a gene or genes that – if non-functional – gives rise to the PWS phenotype.
We are indebted to the family participating in this study and to Prof. George A. Werther who referred the patient to us, and in his letter wrote "I wonder whether this translocation may involve the Prader-Willi gene". The work in the laboratory of UF was supported by grants from the NIH (HD41623) and the Deutsche Forschungsgemeinschaft (BS – SCHU 1567/1-1).
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