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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 YH, Wauki K, Liu Q, Bressler J, Pan Y, Kashork CD, Shaffer LG, Beaudet AL - BMC Genomics (2008)

Bottom Line: GOLGA8E belongs to the golgin subfamily of coiled-coil proteins associated with the Golgi apparatus.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Baylor College of Medicine, Houston, TX 77030, USA. yjiang@bcm.tmc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Imprinting analysis of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. Northern blot analysis of CYFIP1 and NIPA2 in PWS class I and class II deletion patients using cultured lymphoblasts. The expression of CYFIP1(A)and NIPA2 (C) was comparable in the normal control and class II (CII) patient but lower in the class I (CI) patient which is consistent with haplodeficiency of CYFIPI in class I patients suggesting these two genes are not subject to imprinting. The expression of WHDC1L1 in (E), and GOLGA8E in (G) is also comparable to the normal control but the interpretation for WHDC1L1 and GOLGA8E related to imprinting is complicated by the presence of many highly similar copies in the 15q region (see text for detailed discussion). The results using a control GAPDH probe are shown in (B), (D), (F), and (H). RT-PCR expression analysis of the brain tissues from PWS patients is shown for CYFIP1 in (I), for NIPA2 in (J), for WHDC1L1 in (M), and for GOLGA8E in (N). Both transcripts were detectable in both patients but appear to be lower in PWS-2. The absence of SNRPN expression in both PWS cases is confirmed in (K) and (L) is a GADPH control for RNA input. The "+" indicates a PCR reaction carried out with reverse transcriptase and "-" is without reverse transciptase. The molecular defect of PWS-2 is most consistent with maternal UPD of chromosome 15 or a rare possibility of an imprinting mutation because of the maternal methylation pattern at the SNRPN CpG island, the absence of expression of SNRPN, and the failure to detect a deletion by array CGH (data not shown). This data is also consistent with a report by others [66].
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Figure 5: Imprinting analysis of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. Northern blot analysis of CYFIP1 and NIPA2 in PWS class I and class II deletion patients using cultured lymphoblasts. The expression of CYFIP1(A)and NIPA2 (C) was comparable in the normal control and class II (CII) patient but lower in the class I (CI) patient which is consistent with haplodeficiency of CYFIPI in class I patients suggesting these two genes are not subject to imprinting. The expression of WHDC1L1 in (E), and GOLGA8E in (G) is also comparable to the normal control but the interpretation for WHDC1L1 and GOLGA8E related to imprinting is complicated by the presence of many highly similar copies in the 15q region (see text for detailed discussion). The results using a control GAPDH probe are shown in (B), (D), (F), and (H). RT-PCR expression analysis of the brain tissues from PWS patients is shown for CYFIP1 in (I), for NIPA2 in (J), for WHDC1L1 in (M), and for GOLGA8E in (N). Both transcripts were detectable in both patients but appear to be lower in PWS-2. The absence of SNRPN expression in both PWS cases is confirmed in (K) and (L) is a GADPH control for RNA input. The "+" indicates a PCR reaction carried out with reverse transcriptase and "-" is without reverse transciptase. The molecular defect of PWS-2 is most consistent with maternal UPD of chromosome 15 or a rare possibility of an imprinting mutation because of the maternal methylation pattern at the SNRPN CpG island, the absence of expression of SNRPN, and the failure to detect a deletion by array CGH (data not shown). This data is also consistent with a report by others [66].

Mentions: To examine whether imprinted expression is associated with CYFIP1 and NIPA2, RNA from cultured lymphoblasts derived from PWS patients with class I and class II deletions was used as a template for RT-PCR analysis. The expression of GOLGA8E and WHDC1L1 was also included in the analysis, but we were aware that it is probably impossible to determine the imprinting status for GOLGA8E and WHDC1LI in humans because of the presence of highly similar copies of these two genes within the region of 15q. The expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 was readily detectable in cultured lymphoblasts from PWS class I and II patients by RT-PCR analysis (data not shown). To further examine the possibility of quantitative or leaky imprinted expression for CYFIP1, NIPA2, GOLGA8E, and WHDC1L1, we also used total RNA derived from lymphoblasts from PWS patients with class I or class II deletions, and from normal control individuals for northern blot analysis. As shown in Fig. 5A and 5H, there was no significant difference in expression between patients with paternal deletions and normal controls for CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. The decreased expression of CYFIP1 in class I deletion patients when compared to class II deletion patients is consistent with the conclusion that expression is not subject to imprinting. We then attempted to examine whether CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 display a tissue-specific imprinting pattern particularly in brain as reported for other genes in the 15q11-q13 region including UBE3A and ATP10A [63-65]. We have obtained two brain tissues (PWS-1 and PWS-2) from the University of Maryland brain bank from individuals for whom a PWS diagnosis was initially made based on the clinical description. Molecular studies on these two cases were performed in the lab after we obtained the tissues. Both cases have abnormal DNA methylation at the NotI site within the SNRPN CpG island by genomic DNA Southern blot analysis (data not shown) which supports the clinical diagnosis of PWS [9]. In addition, array CGH analysis revealed a class II deletion in the case of PWS-1 but no apparent deletion of 15q11-q13 was detected in the case of PWS-2 which supports the possibility of maternal UPD of chromosome 15 or a rare imprinting mutation in the case of PWS-2 (T. Sahoo, personal communication). A similar conclusion was also reported by others [66]. We first examined the expression of SNRPN in these two patients as shown in Fig. 5K. The results of these experiments indicated that the SNRPN transcript was not detectable by RT-PCR which is consistent with the abnormal DNA methylation found at the SNRPN CpG island and again supports the diagnosis of PWS. We then examined the expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 in these two patients and transcripts from these four genes were detected by RT-PCR as shown in Fig. 5I, J, M, and 5N. The reduced intensity of the CYFIP1 PCR products in case PWS-2 may reflect the quality of the RNAs isolated from archival tissues. Analysis of gene expression by northern blot analysis in brain tissues was attempted but was not successful due to the poor quality of the RNA isolated from autopsy brain tissues. We have also examined the expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 using RNAs derived from lymphoblast cell lines from AS patients with a class I deletion and no evidence of allele-specific expression was found (data not shown). These data together with the results from deletion analysis support the conclusion that CYFIP1 and NIPA2 are not subject to imprinting in cultured lymphoblasts and brain tissues. These results are in agreement with those reported by Chai et al. [32] but a more complicated imprinting pattern such as cell type- or developmental stage-specific imprinting cannot be completely ruled out. Because of the presence of highly homologous sequences for GOLGA8E and WHDC1L1 in the 15q region, we were not able to determine whether GOLGA8E and WHDC1L1 have imprinted or biallelic expression.


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 YH, Wauki K, Liu Q, Bressler J, Pan Y, Kashork CD, Shaffer LG, Beaudet AL - BMC Genomics (2008)

Imprinting analysis of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. Northern blot analysis of CYFIP1 and NIPA2 in PWS class I and class II deletion patients using cultured lymphoblasts. The expression of CYFIP1(A)and NIPA2 (C) was comparable in the normal control and class II (CII) patient but lower in the class I (CI) patient which is consistent with haplodeficiency of CYFIPI in class I patients suggesting these two genes are not subject to imprinting. The expression of WHDC1L1 in (E), and GOLGA8E in (G) is also comparable to the normal control but the interpretation for WHDC1L1 and GOLGA8E related to imprinting is complicated by the presence of many highly similar copies in the 15q region (see text for detailed discussion). The results using a control GAPDH probe are shown in (B), (D), (F), and (H). RT-PCR expression analysis of the brain tissues from PWS patients is shown for CYFIP1 in (I), for NIPA2 in (J), for WHDC1L1 in (M), and for GOLGA8E in (N). Both transcripts were detectable in both patients but appear to be lower in PWS-2. The absence of SNRPN expression in both PWS cases is confirmed in (K) and (L) is a GADPH control for RNA input. The "+" indicates a PCR reaction carried out with reverse transcriptase and "-" is without reverse transciptase. The molecular defect of PWS-2 is most consistent with maternal UPD of chromosome 15 or a rare possibility of an imprinting mutation because of the maternal methylation pattern at the SNRPN CpG island, the absence of expression of SNRPN, and the failure to detect a deletion by array CGH (data not shown). This data is also consistent with a report by others [66].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268926&req=5

Figure 5: Imprinting analysis of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. Northern blot analysis of CYFIP1 and NIPA2 in PWS class I and class II deletion patients using cultured lymphoblasts. The expression of CYFIP1(A)and NIPA2 (C) was comparable in the normal control and class II (CII) patient but lower in the class I (CI) patient which is consistent with haplodeficiency of CYFIPI in class I patients suggesting these two genes are not subject to imprinting. The expression of WHDC1L1 in (E), and GOLGA8E in (G) is also comparable to the normal control but the interpretation for WHDC1L1 and GOLGA8E related to imprinting is complicated by the presence of many highly similar copies in the 15q region (see text for detailed discussion). The results using a control GAPDH probe are shown in (B), (D), (F), and (H). RT-PCR expression analysis of the brain tissues from PWS patients is shown for CYFIP1 in (I), for NIPA2 in (J), for WHDC1L1 in (M), and for GOLGA8E in (N). Both transcripts were detectable in both patients but appear to be lower in PWS-2. The absence of SNRPN expression in both PWS cases is confirmed in (K) and (L) is a GADPH control for RNA input. The "+" indicates a PCR reaction carried out with reverse transcriptase and "-" is without reverse transciptase. The molecular defect of PWS-2 is most consistent with maternal UPD of chromosome 15 or a rare possibility of an imprinting mutation because of the maternal methylation pattern at the SNRPN CpG island, the absence of expression of SNRPN, and the failure to detect a deletion by array CGH (data not shown). This data is also consistent with a report by others [66].
Mentions: To examine whether imprinted expression is associated with CYFIP1 and NIPA2, RNA from cultured lymphoblasts derived from PWS patients with class I and class II deletions was used as a template for RT-PCR analysis. The expression of GOLGA8E and WHDC1L1 was also included in the analysis, but we were aware that it is probably impossible to determine the imprinting status for GOLGA8E and WHDC1LI in humans because of the presence of highly similar copies of these two genes within the region of 15q. The expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 was readily detectable in cultured lymphoblasts from PWS class I and II patients by RT-PCR analysis (data not shown). To further examine the possibility of quantitative or leaky imprinted expression for CYFIP1, NIPA2, GOLGA8E, and WHDC1L1, we also used total RNA derived from lymphoblasts from PWS patients with class I or class II deletions, and from normal control individuals for northern blot analysis. As shown in Fig. 5A and 5H, there was no significant difference in expression between patients with paternal deletions and normal controls for CYFIP1, NIPA2, GOLGA8E, and WHDC1L1. The decreased expression of CYFIP1 in class I deletion patients when compared to class II deletion patients is consistent with the conclusion that expression is not subject to imprinting. We then attempted to examine whether CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 display a tissue-specific imprinting pattern particularly in brain as reported for other genes in the 15q11-q13 region including UBE3A and ATP10A [63-65]. We have obtained two brain tissues (PWS-1 and PWS-2) from the University of Maryland brain bank from individuals for whom a PWS diagnosis was initially made based on the clinical description. Molecular studies on these two cases were performed in the lab after we obtained the tissues. Both cases have abnormal DNA methylation at the NotI site within the SNRPN CpG island by genomic DNA Southern blot analysis (data not shown) which supports the clinical diagnosis of PWS [9]. In addition, array CGH analysis revealed a class II deletion in the case of PWS-1 but no apparent deletion of 15q11-q13 was detected in the case of PWS-2 which supports the possibility of maternal UPD of chromosome 15 or a rare imprinting mutation in the case of PWS-2 (T. Sahoo, personal communication). A similar conclusion was also reported by others [66]. We first examined the expression of SNRPN in these two patients as shown in Fig. 5K. The results of these experiments indicated that the SNRPN transcript was not detectable by RT-PCR which is consistent with the abnormal DNA methylation found at the SNRPN CpG island and again supports the diagnosis of PWS. We then examined the expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 in these two patients and transcripts from these four genes were detected by RT-PCR as shown in Fig. 5I, J, M, and 5N. The reduced intensity of the CYFIP1 PCR products in case PWS-2 may reflect the quality of the RNAs isolated from archival tissues. Analysis of gene expression by northern blot analysis in brain tissues was attempted but was not successful due to the poor quality of the RNA isolated from autopsy brain tissues. We have also examined the expression of CYFIP1, NIPA2, GOLGA8E, and WHDC1L1 using RNAs derived from lymphoblast cell lines from AS patients with a class I deletion and no evidence of allele-specific expression was found (data not shown). These data together with the results from deletion analysis support the conclusion that CYFIP1 and NIPA2 are not subject to imprinting in cultured lymphoblasts and brain tissues. These results are in agreement with those reported by Chai et al. [32] but a more complicated imprinting pattern such as cell type- or developmental stage-specific imprinting cannot be completely ruled out. Because of the presence of highly homologous sequences for GOLGA8E and WHDC1L1 in the 15q region, we were not able to determine whether GOLGA8E and WHDC1L1 have imprinted or biallelic expression.

Bottom Line: GOLGA8E belongs to the golgin subfamily of coiled-coil proteins associated with the Golgi apparatus.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Baylor College of Medicine, Houston, TX 77030, USA. yjiang@bcm.tmc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus