- Research article
- Open Access
Genomic analysis of the chromosome 15q11-q13 Prader-Willi syndrome region and characterization of transcripts for GOLGA8E and WHCD1L1 from the proximal breakpoint region
© Jiang et al; licensee BioMed Central Ltd. 2008
- Received: 20 September 2007
- Accepted: 28 January 2008
- Published: 28 January 2008
Prader-Willi syndrome (PWS) is a neurobehavioral disorder characterized by neonatal hypotonia, childhood obesity, dysmorphic features, hypogonadism, mental retardation, and behavioral problems. Although PWS is most often caused by a paternal interstitial deletion of a 6-Mb region of chromosome 15q11-q13, the identity of the exact protein coding or noncoding RNAs whose deficiency produces the PWS phenotype is uncertain. There are also reports describing a PWS-like phenotype in a subset of patients with full mutations in the FMR1 (fragile X mental retardation 1) gene. Taking advantage of the human genome sequence, we have performed extensive sequence analysis and molecular studies for the PWS candidate region.
We have characterized transcripts for the first time for two UCSC Genome Browser predicted protein-coding genes, GOLGA8E (golgin subfamily a, 8E) and WHDC1L1 (WAS protein homology region containing 1-like 1) and have further characterized two previously reported genes, CYF1P1 and NIPA2; all four genes are in the region close to the proximal/centromeric deletion breakpoint (BP1). GOLGA8E belongs to the golgin subfamily of coiled-coil proteins associated with the Golgi apparatus. Six out of 16 golgin subfamily proteins in the human genome have been mapped in the chromosome 15q11-q13 and 15q24-q26 regions. We have also identified more than 38 copies of GOLGA8E-like sequence in the 15q11-q14 and 15q23-q26 regions which supports the presence of a GOLGA8E-associated low copy repeat (LCR). Analysis of the 15q11-q13 region by PFGE also revealed a polymorphic region between BP1 and BP2. WHDC1L1 is a novel gene with similarity to mouse Whdc1 (WAS protein homology region 2 domain containing 1) and human JMY protein (junction-mediating and regulatory protein). Expression analysis of cultured human cells and brain tissues from PWS patients indicates that CYFIP1 and NIPA2 are biallelically expressed. However, we were not able to determine the allele-specific expression pattern for GOLGA8E and WHDC1L1 because these two genes have highly related sequences that might also be expressed.
We have presented an updated version of a sequence-based physical map for a complex chromosomal region, and we raise the possibility of polymorphism in the genomic orientation of the BP1 to BP2 region. The identification of two new proteins GOLGA8E and WHDC1L1 encoded by genes in the 15q11-q13 region may extend our understanding of the molecular basis of PWS. In terms of copy number variation and gene organization, this is one of the most polymorphic regions of the human genome, and perhaps the single most polymorphic region of this type.
- Reverse Transcription Polymerase Chain Reaction
- Angelman Syndrome
- snoRNA Gene
- Imprint Center
- Reverse Transcription Polymerase Chain Reaction Experiment
Genomic alterations of the chromosome 15q11-q13 region are associated with two distinct genomic imprinting disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS) . PWS is characterized by neonatal hypotonia, childhood obesity, hypogonadism, moderate mental retardation, and behavioral problems. The most common molecular defect found in PWS patients is a ~6-Mb chromosomal deletion of the 15q11-q13 region on the paternal chromosome. Maternal uniparental disomy (UPD) of chromosome 15, microdeletions in a regulatory region known as the imprinting center (IC), and rare chromosome translocations have also been reported for PWS patients. It is clear from molecular studies that PWS is primarily caused by deficiency of a paternally expressed gene or genes from the 15q11-q13 region. However, it remains uncertain as to whether the major phenotypic features are caused by deficiency of one or more than one gene and whether such genes might be protein coding (e.g NDN or MAGEL2) or noncoding small nucleolar RNAs (snoRNAs) [2–4]. A 2-Mb region extending from the centromeric breakpoint 2 (BP2) to D15S10 was initially defined as the PWS candidate region . Attempts to narrow the candidate region by characterizing several rare patients with cytogenetic abnormalities have been reported by several investigators [6–8]. However, a consensus concerning a narrowed critical region has not yet been reached. The controversy may arise from the complex regulation of a large imprinted domain as evidenced by IC mutations that have been reported to disrupt the imprinting process both in humans and in mice [9–17]. Alternatively, if PWS is a contiguous gene deletion syndrome, the individual genes may only contribute to part of the phenotype . Numerous protein coding genes and non-coding transcripts have been isolated from the PWS candidate region. These include SNURF-SNRPN, NDN, MKRN3, MAGEL2, PWRN1, PWRN2, IPW, PAR-1, PAR-4, PAR-5, C15orf2, and multiple copies of different families of snoRNA genes [9, 19–27]. All these transcripts except PWRN2 are expressed from the paternal chromosome with brain tissue-specific imprinting of PWRN1 and C15orf2 and therefore were considered as legitimate PWS candidate genes. Functional studies in mutant mice have suggested that Ndn, Magel2, or the snoRNA genes may play a role in the pathogenesis of PWS [2, 28–30]. Lee et al. also determined the imprinting status of 22 transcripts located centromeric/proximal to the IC within the PWS candidate region based on an early version of the expressed sequence tag (EST) map and limited human genomic sequence. Seven of these transcripts were found to be paternally expressed but lacked protein coding potential . Chai et al. identified four protein coding genes CYFIP1, NIPA1, NIPA2, and GCP5 in the proximal breakpoint region and established the genomic organization of the region between the two proximal breakpoints (BP1 and BP2) .
There have been multiple reports over the last decade describing a subset of fragile X syndrome patients who shared overlapping clinical features with PWS. These patients were often described as Prader-Willi-like fragile X syndrome [33–38]. The shared traits include extreme obesity, dysmorphic features, mental retardation and behavior problems. These patients have typical full mutations in the FMR1 gene indicating that the primary defect for the PWS-like phenotype was dysregulation of FMR1. The specificity of the PWS-like clinical features in fragile X syndrome patients has been debated .
Chromosome 15 is one of the seven human chromosomes with a high rate of segmental duplication . Zody et al. carried out a detailed analysis of the duplication structure and history of chromosome 15 and reported that low copy repeats (LCRs), also termed segmental duplications (SDs), in chromosome 15 are largely clustered in proximal and distal 15q . There are two breakpoints (BP1 and BP2) in the centromeric region and a single common breakpoint (BP3) in the telomeric region [41–46]. The remarkable consistency of the breakpoints strongly indicates the presence of a hot spot for recombination. Indeed, large genomic LCRs derived from the duplication of the actively transcribed HERC2 gene and its pseudogenes were identified in the BP2 and BP3 regions and are believed to contribute to a certain percentage of chromosomal rearrangements between 15q11 and 15q13 . Recent reports suggest that HERC2-associated LCRs also are present within the BP1 region [32, 47]. In addition, Pujana et al. have described a second cluster of LCRs (LCR15) with golgin-like protein (GLP) genomic sequence in the 15q11-q14, 15q24, and 15q26 regions [48, 49]. Locke et al. also characterized the genomic structure and LCRs in the pericentromeric region of chromosome 15 proximal to BP1 .
Here we report an updated version of a sequence-based BAC contig covering the PWS candidate region based on the analysis of the finished version of the human genome sequence (NCBI Build 36.1), and we question whether the orientation of the BP1 to BP2 region may be polymorphic in the population. We provide further characterization of four protein coding genes, CYFIP1, NIPA2, GOLGA8E, and WHDC1L1, located at the proximal breakpoint of the PWS candidate region. GOLGA8E and WHDC1L1 are two protein coding genes from this region that are represented in genome databases but not previously discussed in the published literature. CYFIP1 and NIPA2 have been described  but we have undertaken further molecular characterization and reported new data regarding their gene structure and complex alternative splicing pattern. In addition, pulsed field gel electrophoresis (PFGE) analysis of the 15q11-q13 region has revealed a highly polymorphic region between BP1 and BP2 that is consistent with the presence of an abundance of copy number variants (CNVs) in the region as detected by genome-wide array CGH analysis [40, 51].
Sequence-based BAC contig covering the chromosome 15q11-q13 PWS candidate region
To further confirm the map position for the individual BAC clones assembled using bioinformatics tools, we performed FISH (fluorescence in situ hybridization) experiments using DNA derived from selected individual BAC clones from the region as probes. As listed in Additional file 1, the localization of 14 BAC clones to chromosome 15q was confirmed by FISH studies (data not shown). A simplified version of the BAC contig with only one BAC clone for each position is diagramed in Fig. 1C. The order of genes displayed in UCSC/Ensembl/NCBI human genome browsers is diagramed in Fig. 1B for reference and comparison [52, 56, 57]. A much more detailed description of the region including the BAC contig, STS markers, CpG islands, and the map positions of novel transcripts is summarized in Additional file 1.
Characterization of four protein-coding genes from the proximal breakpoint of 15q11-q13 PWS candidate region
There are numerous ESTs mapped to this region, but most of them are intronless and/or lack major open reading frames (ORFs), and their significance cannot be easily addressed because they may function as noncoding RNAs or contain potential artifacts from cDNA production. In order to identify protein-coding genes, only transcripts with intron-exon structure and greater than 99% sequence identity between the EST and the matched genomic sequence were considered significant and are listed in Additional file 1. Towards this goal, we have analyzed each individual EST from this region for coding potential by ORF analysis. Most of the ESTs were overlapping and did not have significant coding potential, with the exception of clones [GenBank: BC063309, AK057421, BC018097, BC048987, D38549, BC011775, AK093450, AK093463, AK093104, AF272884, and BX537997] that displayed ORFs greater than 120 amino acids in length. There were no additional protein-coding genes identified in the region between NDN and SNURF-SNRPN despite the presence of numerous ESTs with clear intron-exon structure. In contrast, the region around the proximal breakpoints appears to have a high density of protein-coding genes based on our analysis using bioinformatics tools. To rule out the possibility of artifacts in these EST clones, we first performed expression analysis by reverse transcription polymerase chain reaction (RT-PCR) using RNA templates isolated from human lymphoblasts and brain tissue. Six representative cDNA clones [Genbank: D38549, BC011775, AF272884, BX537997, BC063309, and BC048987] were particularly interesting because of their clear protein-coding potential and readily detectable expression in lymphoblasts and brain tissues by RT-PCR. Clones D38549, AF272884, BC011775, and BX537997 were identical to four genes reported by Chai et al. which correspond to CYFIP1, NIPA1, NIPA2, and GCP5, respectively, while BC063309 and BC048987 are two protein-coding genes from this region that are predicted genes in the UCSC genome browser, but have not yet been characterized in the published literature. Computational analyses of EST clones AK093450, AK093104, and AK093463 predict significant ORFs, but we were not able to detect expression in either lymphoblasts or brain tissues by RT-PCR despite multiple attempts. We were also not able to detect any significant conservation when the sequences were compared to those available for multiple species, which raised a question concerning the functional significance of these EST transcripts. There are numerous additional ESTs within the region which show clear intron-exon structure and perfect sequence identity with the genomic sequence but do not contain significant ORFs, and their biological relevance remains to be defined. This analysis also confirmed the transcripts previously reported [31, 58], but numerous additional ESTs without protein coding potential have since been deposited in the databases and mapped within the interval.
Intron-exon structure of GOLAG8E
The genome distribution of Golgin subfamily genes in human and mouse
Intron-exon structure of WHDC1L1
GOLGA8E associated LCR in 15q and other chromosomal regions
BLASTN searching of human genome sequence databases using the GOLGA8E cDNA as bait revealed numerous sequences with significant similarity across the 15q11-q14 and 15q24-q26 regions and sequence identities range from 91–97% at the nucleotide level as summarized in Additional file 3. There are also additional copies of sequence with sequence identity ranging from 50–89% that may represent different golgin subfamily genes in the 15q region (data not shown). BLAST searching also identified 6 BAC clones from chromosome 16 [GenBank: AC141597, AC141255, AC141254, AC141247, and AC140902], one BAC clone from chromosome 8 [GenBank: AC132916], and one BAC clone from chromosome 18 (AC136349) with sequence identity ranging from 90–97% compared to GOLGA8E. Careful sequence analysis revealed that the copy at genomic location chr15q11:20986537–20999858 bp (NCBI Build 36.1) proximal to the CYFIP1 and NIPA2 genes showed the highest identity to the genomic GOLGA8E DNA sequence providing evidence that the copy at this genomic position is actively transcribed. The rest of the copies distributed within the 15q11-q14, 15q24-q26, and other chromosomal regions are likely to be part of LCRs but the possibility of additional transcribed copies in other regions or an error in map position cannot be completely ruled out. There are five additional golgin subfamily genes mapped within the 15q13-q14 and 15q24-q26 regions. They are GOLGA, GOLGA6, GOLGA8A, GOLGA8B, and GOLGA8G. The map position and genomic location of these golgin subfamily genes in NCBI Build 36.1 are shown in Table 2. These results further support the initial characterization of LCR15 by Pujana et al. suggesting the presence of a new cluster of golgin like protein (GLP or GOLGA6) sequence-associated LCRs in this region [48, 49]. We have provided further evidence that multiple actively transcribed golgin subfamily genes are associated with LCRs in the 15q region. The distribution of golgin subfamily genes associated with LCRs in the 15q11-q14 region suggests that these LCRs may contribute to the frequent long-range chromosomal rearrangements between the 15q11 region and the 15q24-q26 region reported in the literature .
Mapping of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 to the proximal deletion breakpoint of the PWS/AS common deletion interval
Imprinting analysis for CYFIP1 and NIPA2
DNA methylation of CpG islands of CYFIP1 and NIPA2
Allele-specific differential methylation has been found consistently for multiple loci within the 15q11-q13 region where the paternal allele was usually unmethylated while the maternal allele was methylated. As described in the previous section, CYFIP1 and NIPA2 are localized within the common deletion interval described for PWS/AS patients but most likely lie at the most centromeric end of an imprinted domain within 15q11-q13. We have identified typical CpG islands in the 5'-region of the CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 genes by computational analysis, and a detailed map of methylation-sensitive restriction enzyme sites was deduced (data not shown). To examine whether allele-specific differential DNA methylation was associated with the CYF1P1 CpG island, a DNA probe generated by PCR amplification of genomic DNA was used for Southern blot analysis after digestion with Bss HII in combination with Hin dIII. No allele-specific methylation was found after digestion of genomic DNA derived from any tissues examined, including several sub-regions of the brain and various cell lines, and both alleles appear to be completely unmethylated (data not shown). A similar result was also obtained for the NIPA2 CpG island where both alleles are also unmethylated (data not shown). However, we were not able to generate unique probes for genomic DNA Southern analysis for the GOLGA8E and WHDC1L1 CpG islands because of associated LCRs in the region.
Pulse Field Gel Electrophoresis (PFGE) analysis of 15q11-q13 region
CYFIP1, NIPA2, and GOLGA8E are members of different protein families
The functions of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 in mammals are unknown. To determine whether the CYFIP1 and NIPA2 proteins contain any functionally relevant or conserved protein motifs or domains, we carried out protein database searching (BLASTP) and protein motif and structural analysis (RPSBLAST) . No conserved domain or motif was identified from this analysis. However, the protein structural analysis did reveal the presence of 5 and 8 transmembrane domains, respectively, for CYF1P1 and NIPA2. To examine the cross-species conservation for the CYFIP1 and NIPA2 proteins, the cDNA sequences and their predicted amino acid sequences were compared to multiple nucleotide and protein databases using the BLASTN and BLASTP programs . Multiple significant sequence matches from other spcies including mouse, Drosophila, C. elegans, pig, Fugu, Arabidopsis, and Anopheles gambiae were identified and are diagramed in Additional file 4 for CYFIP1 and NIPA2, respectively. A human protein PIR121 was also identified that shows high similarity to CYFIP1 and was named CYFIP2 . In addition, a variety of hypothetical proteins from C. elegans, Drosophila, and many other species were also identified for CYFIP2. GOLGA8E has significant similarity to many golgin subfamilies at the nucleotide and amino acid levels (Table 2). However, the protein structural analysis did not reveal a characteristic coiled-coil domain for GOLGA8E. There are total of 16 golgin subfamily genes found in the human genome and 6 of them are mapped to the proximal 15q region (Table 2). GOLGA8E has much higher similarity to the subfamily mapped in the 15q region than other subfamily genes in other chromosomal loci. In addition, there is no immediate evidence of evolutionary conservation in the mouse and other species for GOLGA8E despite the fact that several of its other family members appear to be highly conserved in mice (Table 2).
In an attempt to identify novel transcripts from the PWS candidate region, we have constructed a sequence-based BAC contig covering the ~2-Mb PWS candidate region. When compared to the previously published BAC/YAC contig covering the region of interest [32, 41, 44, 47, 73], we believe that this version of the BAC contig contributes significantly to the understanding of the physical map of the 15q11-q13 PWS candidate region. The current version of the map reported here is largely consistent with the maps reported by several other groups using the array CGH technique [32, 41, 42, 47]. There are two sequence gaps remaining in the region around the proximal common PWS/AS deletion breakpoints (BP1 and BP2) in the sequence assembly available in the public domain. There are still three sequence gaps within the 6-Mb region, and closing these gaps has been challenging because of the extremely high sequence identity between different copies of LCRs. Makoff and Flomen recently reported closure of these gaps by an extensive bioinformatic approach . The orientation of the contig drawn from this analysis differs from the contig displayed in the UCSC genome browser (NCBI Build 36.1)  in which BAC 289D12 was positioned centromeric to BAC 26F2 but is consistent with multiple reports [41, 47, 62]. The apparent discrepancy between the two genomic contigs may reflect an artifact introduced by using computational methodology because of the high sequence identity among the different LCRs or other aspects of the complex genome organization in the region. The structure depicted in the genome browsers is an attempt to select one unique order and copy number from data derived from two different chromosomes from multiple individuals, while the chromosomes may have a polymorphic gene order and copy number. This hypothesis is supported by a report that 9% of individuals in the general population have an inversion of the 15q11-q13 region , by the results from PFGE analysis reported in this study, as well as by the array CGH technique reported in the Database of Genomic Variants . Our experience in utilizing the human genome sequence also emphasizes the importance of evaluating human genome sequence data in detail for a particular genomic region of interest before making any conclusion.
We have characterized transcripts for the first time for two protein-coding genes GOLGA8E and WHDC1L1 in addition to four other genes CYFIP1, NIPA1, NIPA2, and GCP5 previously described  from a region close to the proximal breakpoint (BP1) of the PWS candidate region. Our results for CYFIP1 and NIPA2 are largely in agreement with the previous report . However, we have provided additional information regarding the genomic structure, alternative splicing, and expression pattern for both genes. These findings would be very important for understanding the function of CYFIP1 and its interaction with FMRP and Rac1 because isoforms 4 and 5 apparently lack the N-terminal FMRP and Rac1 interacting domain [76, 77]. GOLGA8E and WHDC1L1 are associated with a large number of LCRs within the 15q11-q14 and 15q24-q26 regions. The function of GOLGA8E and WHDC1L1 is unknown. GOLGA8E belongs to the golgin subfamily which was originally identified as a group of Golgi-localized autoantigens recognized by sera from patients with a variety of autoimmune conditions . There are l6 putative golgin subfamily genes in the human genome, but some of them have not yet been confirmed experimentally. These golgin subfamily members are differentiated mostly on the basis of molecular weight [78, 79]. The function of most of golgin subfamily genes remains unclear, but a role for golgins in association with a GTPase protein complex in the organization and regulation of Golgi membrane trafficking has been suggested . The presence of 6 out 16 golgin subfamily genes in the 15q11-q13 and 15q24-q26 regions is of interest relative to the possibility of actively transcribed genes with LCRs and the occurrence of LCR-mediated chromosomal rearrangements in the region. The golgin subfamily genes in 15q share higher similarity to GOLGA8E than other subfamily members on other chromosomes. Thus, golgin subfamily genes in 15q may have a common ancestry, and the SDs in the region may have played a role in the formation of new gene family members through evolution.
Although it is possible that CYFIP1 and NIPA2 as well as GOLGA8E and WHDC1L1 may all be deleted in class I PWS patients, these four genes do not appear to be strong candidates for a primary role in the PWS phenotype. Our expression analysis using RNA isolated from the cultured lymphoblasts of PWS patients carrying class I deletions and brain tissues from patients with maternal UPD 15 suggest that CYFIP1 and NIPA2 are not subject to genomic imprinting. The possibility of cell type or developmental stage-specific imprinting cannot be completely ruled out but is less likely. In addition, a possibility that the paternal allele is more active was suggested by a recent study of the expression profile of CYFIP1, NIPA1, NIPA2, and GCP5 in class I and class II deletion patients using real time RT-PCR methods, but more study is needed to make a definitive conclusion . It is impossible to analyze genomic imprinting for GOLGA8E and WHDC1L1 with high confidence because of multiple copies, some of which are distant from the BP1-BP2 region. Several recent reports comparing patients with class I and class II deletions have suggested that there are phenotypic differences between the two groups [81–83], while another investigator did not observe such distinctions . Butler et al. recently found that PWS patients with class I deletions have a more severe phenotype than those with either a class II deletion or maternal UPD using a variety of psychological and behavioral tests . Similarly, AS patients with class I deletions showed complete absence of vocalization while some AS patients with class II deletions were able to pronounce syllabic sounds [42, 85]. These studies suggest that the deletion of biallelically expressed genes located between BP1 and BP2 such as CYFIP1 may have an impact on the degree of impairment particularly in communication and behavior observed for both PWS and AS patients.
Mapping of the CYFIP1 gene encoding an FMRP interacting protein within the PWS candidate region is of particular relevance for the analysis of the Prader-Willi-like phenotype observed in fragile X patients with full mutations in FMR1. The significance of the overlapping clinical features between PWS and fragile X syndrome remains uncertain, and it is very difficult to determine whether there is any intrinsic link underlying these overlapping clinical features solely from a clinical standpoint. Several recent studies report that autistic spectrum disorder is more prevalent in PWS patients than was previously thought [86–88]. The identification of CYFIP1 in the PWS candidate region may provide a molecular framework for further investigation, particularly in the area of shared autistic behavior between fragile X and Prader-Willi syndromes. Indeed, alteration of CYFIP1 in PWS-like fragile X syndrome patients was recently reported which provides a molecular basis for the clinical observation . Altered expression of CYFIP1 has also been reported both in autism patients with maternal duplication of 15q11-q13 and in fragile X syndrome patients with autistic features .
Characterization of additional GOLGA8E LCRs around the BP1, BP3, and BP4 regions has added another layer of complexity in an already complicated region. The presence of an additional LCR (LCR15) other than HERC2-associated LCRs was first suggested by Pujana et al.  with the suggestion that golgin-like sequences are associated with LCR15. With the finished version of the human genome sequence, we were able to characterize the genomic organization and copy number of these LCRs in substantial detail. Like the HERC2-associated LCRs, this new class of LCR is associated with an actively transcribed copy of GOLGA8E. The GOLGA8E-associated LCRs have wider genomic distribution and are found in the 15q11, q13, q24, and q26, and other chromosomal regions while HERC2-associated LCRs are found in 15q11 and q13 and other chromosomes . The finding of GOLGA8E-associated LCRs in the 15q11, q13, and q24-q26 regions suggests that these LCRs could be responsible for longer chromosomal rearrangements observed between the 15q11 and q24-q26 regions . The degree of polymorphism in the BP1 and BP2 regions revealed by PFGE analysis is consistent with the data from multiple genome-wide analyses of CNVs by array CGH displayed in the Database of Genomic Variants or in other recent reports [40, 47, 50, 74]. There are 14 different genomic variants listed in the region proximal to BP2 and most of these variants are SDs, but there are two inversion variants between BP1 and BP2, and one inversion variant between BP2 and BP3 was previously reported . Murthy et al. recently reported a 250-kb heterozygous microdeletion covering the CYFIP1 and NIPA1 genes in the region between BP1 and BP2 in a male patient with mental retardation . The interpretation of the finding is complicated because the same deletion was also present in the patient's father who may have had some mild phenotypic abnormalities. Butler et al. reported a PWS case with a typical class II deletion who also had a small duplication between BP1 and BP2, and the duplication was present in the healthy father and his brother . We have observed that BAC 289D12 in the BP1 region detects a very frequent CNV in the cases referred for clinical array CGH analysis at Baylor College of Medicine (Cheung and Chaw, personal communication). In most of the cases, similar duplications were also found in one of the healthy parents and/or normal siblings. Clearly this is a highly polymorphic region. Very extensive investigations in normal individuals and in extended family members of patients with copy number gains or losses will be needed to determine if variations in this region sometimes cause abnormal phenotypes. Serious genotype/phenotype analysis for this region will require intensive copy number analysis at high resolution such as PFGE or fiber FISH to determine not only copy number but also orientation (regarding inversions), and perhaps even expression and epigenetic analysis.
We have constructed and characterized a sequence-based BAC contig covering the PWS candidate region using human genome sequence databases. We have characterized transcripts for the first time for two protein-coding genes, GOLGA8E (golgin subfamily a, 8E) and WHDC1L1 (WAS protein homology region containing 1-like 1) and have further characterized two previously reported genes, CYF1P1 and NIPA2; all four genes are in the region close to the proximal/centromeric deletion breakpoint (BP1). GOLGA8E belongs to the golgin subfamily of coiled-coil domain proteins associated with the Golgi apparatus. WHDC1L1 is a novel gene with similarity to mouse Whdc1 (WAS protein homology region 2 domain containing 1) and human JMY protein (junction-mediating and regulatory protein). Biallelic expression of CYFIP1 and NIPA2 in cultured lymphoblasts and brain tissues analyzed suggests that they are unlikely to be major contributors to the pathogenesis of PWS, but haploinsufficiency may contribute to a more prominent behavioral phenotype seen in PWS and Angelman syndrome (AS) patients with class I deletions as compared to those with class II deletions. We have also characterized a new class of GOLGA8E-asssociated LCR in the 15q11-q13 and q24-q26 region. This class of LCR together with HERC2-assocaited LCRs may contribute to the frequent chromosomal rearrangements of 15q11-q13 and q24-q26 reported in the literature. This is one of the most polymorphic regions of the human genome in terms of copy number variation and gene organization, and perhaps the single most polymorphic region of this type.
RNA was isolated from cultured human lymphocytes and human brain tissue, using Trizol reagent (GIBCO-BRL) according to the manufacturer's instructions.
For analysis of transcription, 3–5 ug total RNA treated with DNaseI was used for the reverse transcription reaction to synthesize single-stranded cDNA primed with random primers (GIBCO-BRL). The subsequent PCR reactions were performed using gene-specific primers as listed below.
Human northern blot analysis: A multiple human tissue blot was purchased from Clonetech (BD Bioscience) and hybridized with labeled probes following the manufacturer's recommendation. Mouse northern blot analysis: For each sample, 15 ug total RNA was pretreated with DNaseI and resolved on a 1.2% agarose gel in 10 mM NaPO4 buffer (pH 6.8) following glyoxal/DMSO denaturation using standard procedures. RNA was visualized by ethidium bromide staining and transferred to Hybond N+ membrane (Amersham Biosciences Corp.). Hybridization and washing conditions were the same as described below for genomic DNA Southern analysis.
BAC DNA isolation, Genomic Southern blotting, and DNA methylation analysis
BAC DNA was isolated using a Qiagen Plasmid Maxi Kit (Qiagen Inc.) with some modifications of the original protocol. Five ug of BAC DNA was digested with Eco RI and separated on a 0.8% agarose gel prior to transfer to Hybond N+ membrane (Amersham Biosciences Corp.). For DNA methylation analysis by Southern blotting, genomic DNA was first isolated from lymphoblasts by phenol-chloroform extraction, and was then digested with Sac II and Eco RI in combination. The hybridization buffer was prepared as described , and the final washing condition for the membranes was a 20 minute rinse in a 0.2 × SSC and 1% SDS buffer.
Metaphase chromosomes were prepared using standard protocols as described previously  and FISH was performed as previously described . BAC DNA probes were labeled with digoxigenin and were detected by a standard protocol. DAPI counterstain was applied, and cells were viewed with a Zeiss Axiophot fluorescent microscope (Carl Zeiss Inc.) equipped with both single-band pass filters and a triple-band pass filter. Digital images were captured by a Power Macintosh G3 system and MacProbe version 4.0 or 4.3 (Perceptive Scientific Instruments). The chromosome 15 centromere Cep (satellite III) probe was labeled with spectrum green (Cytocell, Rainbow Scientific).
High-molecular weight DNA was isolated and embedded in agarose plugs from peripheral blood samples and/or Epstein-Barr virus-transformed lymphoblastoid cells established from patients and their parents. DNA in plugs was digested by Not I (New England Biolabs, MA, USA). Separation of DNA fragments was achieved using a CHEF MAPPER (BioRAD) for 27 h with pulse time 86.54 s ramp at 6 V/cm. After treatment with 0.25 N HCl for 30 min and 0.4 N NaOH for 40 min, gels were blotted onto a nylon membrane. Radioactive probe labeling and hybridization were performed using the same protocol as described for genomic DNA Southern analysis.
PCR Primer Sequences and Conditions
The sequences of the primers used for RT-PCR analysis of CYFIP1 were as follows: CYFIP1-2F: 5'-GTGCTGGATTTCTGCTACCATCTA-3' and CYFIP1-2R: 5'-GTCACAACTGGATTTAGTGGAAGC-3'. The cycling conditions were denaturation at 94°C for 10 min, followed by 35 cycles of 94°C for 45 sec, annealing at 56°C for 45 sec, and extension at 72°C for 45 sec with a final 5-min extension at 72°C. The PCR product was also used for northern analysis of CYFIP1 transcription. The sequences of the primers used for generating the probe for DNA methylation analysis of the CYFIP1 CpG island were as follows using the same cycling conditions as above: KICGF: 5'-ACAGACACCTGTCTTAACGCAGGA-3' and KICGR: 5'-TCTTGGAGAGAGGAGTTTTGGGCT-3'. The primers used for PFGE analysis were as follows. The P1 probe was generated using primers KICGF and KICGR. The P3 probe was generated by amplification with primers H3F: 5'-GACTTTCCACCTAACTCACTCAC-3' and H3R: 5'-GCAGCTGAGTGCCATATAATGTTG-3'. Probe P4 was generated using primers ATPCGF: 5'-AAGTAGTCTGTGGTCTGGCCCTTG-3' and ATPCGR: 5'-CAGACCTGGCTCAACTGGATAACG-3'. The P5 probe was generated by amplification with primers HP2F: 5'-TGAAATGCTCTTGCGTGGTTAGGA-3' and HP2R: 5'-AAATAAGGCATGCCCTCAGAGACA-3'. Analysis of SNRPN expression was carried out using the following oligonucleotides: SNRPN forward: 5'-CACCAGGCATTAGAGGTCCAC-3' and SNRPN reverse: 5'-GCAGAATGAGGGAACAAAAACCT-3'. The cycling conditions were denaturation at 94°C for 1 min, annealing at 63°C for 1 min., and extension at 72°C for 1 min .
The PCR primers used for amplification of WHDC1L1 were as follows: forward: 5'-TCG GAA GTG AAA GAA CTC AGA AGG-3'; reverse: 5'-AGA CTG AGG ATC ATT TTG TGG AGG-3'. The PCR conditions were denaturation at 94°C for 45 sec, annealing at 63°C for 45 sec., and extension at 72°C for 1 min.
The sequences of the PCR primers used for the analysis of GOLGA8E were as follows: forward: 5'-AGC GTA CTA CAG TTG GAG CAG CAA-3'; reverse: 5'-ATC TCC TTC TTC TTG GCA GCC AAG-3'. The PCR conditions were denaturation at 94°C for 45 sec, annealing at 63°C for 45 sec., and extension at 72°C for 45 sec.
The authors are grateful to Dr. David L. Nelson for his critical review of the manuscript and Dr. Pawel Stankiewicz for discussion. We also thank Hong Li and Diane Dicks for technical assistance in performing tissue culture studies and Trilochan Sahoo for sharing unpublished data regarding the analysis of molecular defects of PWS brain tissues by the array CGH method. The brain tissues used in this study were provided by NIH University of Maryland Brain Tissue Bank. The DN34 probe was kindly provided by Dr. Daniel Driscoll, University of Florida, Gainesville, Florida. This study was supported by grants from CAN (Cure Autism Now), NAAR (National Alliance for Autism Research), fragile X syndrome foundation (FRAXA), by Prader-Willi syndrome and Angelman syndrome research foundation to Y-H Jiang, by NIH training grant T32-DK07664 to J. Bressler, and by NIH grant R01 HD3728 to A.L. Beaudet. Y-H J is also supported by Baylor College of Medicine Molecular Medicine Scholar Program (T32 HL66991-05).
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