Surgical management of the neck in squamous cell carcinoma of the floor of the mouth

Approximately 98% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11-13, uniparental disomy for chromosomes 15 (UPD15), or mutations affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of mutation depends upon the parent of origin. Both disorders are characterised at the molecular level by abnormal methylation of imprinted genes at 15q11-q13 including the small nuclear ribonucleoprotein N gene (SNRPN). Current diagnostic strategies include high resolution cytogenetics, fluorescence in situ hybridisation (FISH), Southern blot hybridisation, or microsatellite typing. We have developed a novel and rapid diagnostic test for PWS and AS based on differential digestion of expressed (paternally imprinted) SNRPN sequences by the methylation sensitive endonuclease NotI or repressed (maternally imprinted) SNRPN sequences by the methylation requiring nuclease McrBC, followed by PCR amplification of the SNRPN promoter. We have evaluated this test by blinded analysis of 60 characterised DNA samples (20 PWS, 20 AS, and 20 unaffected controls). SNRPN sequences could not be amplified from PWS patient DNA which had been digested with McrBC, nor from AS patient DNA which had been digested with NotI. We were able to make a correct diagnosis of PWS, AS, or unaffected in all 60 samples tested. This novel test is rapid and has a high specificity and sensitivity for deletion and UPD15 cases. These features make this new test suitable as the initial step in a molecular diagnostic strategy for PWS/AS.     

Analysis of parent of origin specific DNA methylation at SNRPN and PW71 in tissues: implication for prenatal diagnosis

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct developmental disorders caused by absence of paternal or maternal contributions of the chromosome region 15q11-q13, resulting from deletions, uniparental disomy (UPD), or rare imprinting mutations. Molecular cytogenetic diagnosis is currently performed using a combination of fluorescence in situ hybridisation (FISH), DNA polymorphism analysis, and DNA methylation analysis. Only methylation analysis will detect all three categories of PWS abnormalities, but its reliability in tissues other than peripheral blood has not been examined extensively. Therefore, we examined the methylation status at the CpG island of the small nuclear ribonucleoprotein associated polypeptide N (SNRPN) gene and at the PW71 locus using normal and abnormal lymphoblast (LB) cell lines (n = 48), amniotic fluid (AF) cell cultures (n = 25), cultured chorionic villus samples (CVS, n = 17), and fetal tissues (n = 18) by Southern blot analysis with methylation sensitive enzymes. Of these samples, 20 LB cell lines, three AF cultures, one CVS, and 15 fetal tissues had been previously diagnosed as having deletions or UPD by other molecular methods. Methylation status at SNRPN showed consistent results when compared with FISH or DNA polymorphism analysis using all cell types tested. However, the methylation pattern for PW71 was inconsistent when compared with other tests and should therefore not be used on tissues other than peripheral blood. We conclude that SNRPN, but not PW71, methylation analysis may be useful for diagnosis of PWS/AS on LB cell lines, cultured amniotic fluid, or chorionic villus samples and will allow, for the first time, prenatal diagnosis for families known to carry imprinting centre defects.     

AGO1 defines a novel locus of Arabidopsis controlling leaf development

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

Using the evidence. A need for quantity, not quality?

The gene for alpha-stimulating guanine-nucleotide binding polypeptide, Gnas, has been considered as a candidate for the imprinting effects ascribed to distal mouse Chromosome (Chr) 2. Its human homologue (GNAS1) appears, from clinical and biochemical studies of patients with Albright hereditary osteodystrophy, to be paternally imprinted. GNAS1 maps to 20q13, a region that shows linkage conservation with distal mouse Chr 2. We have mapped Gnas within the imprinting region on distal Chr 2 by linkage analysis. To establish if Gnas is imprinted, we have looked for expression differences in tissues taken from mice carrying maternal duplication/paternal deficiency for distal Chr 2 (MatDp2) and its reciprocal (PatDp2). RNA in situ hybridization revealed high levels of Gnas mRNA in glomeruli of PatDp2 embryos at late gestation and lower levels in glomeruli of MatDp2 embryos. These results strongly suggest that Gnas is maternally imprinted and suggest that the mouse gene may be imprinted in a manner opposite that predicted in human.     

Association of a redefined proximal mouse chromosome 11 imprinting region and U2afbp-rs/U2af1-rs1 expression

Mice with maternal and paternal disomy for chromosome 11 (Chr 11) show growth retarded and overgrowth phenotypes, respectively, which can be attributed to genomic imprinting. Previous studies have defined the region of Chr 11 responsible (the Chr 11 imprinting region) as lying proximal to the T30H translocation breakpoint at the borders of G-bands 11B1.2 and 11B1.3. Evidence is presented here with two new translocations, T57H and T41Ad, which sequentially reduce the size of the imprinting region and locate it proximal to the T41Ad breakpoint in G-band 11A3.2. It therefore lies close to the centromere. The imprinted gene, U2af1-rs1, is known to be located within the original region and has been regarded as a candidate for the imprinting effects. Meiotic and mitotic chromosome FISH analysis, together with U2af1-rs1 expression studies are now described which show that the gene lies within the newly defined imprinting region and that its expression levels relate to the presence/absence and number of functional paternal alleles. U2af1-rs1 therefore remains a candidate gene for the Chr 11 imprinting effects. However, another recently reported imprinted gene, Meg1/Grb10, that lies within the region is also a good candidate, as it encodes a growth factor receptor. Meg1/Grb10 maps about 15 cM from U2af1-rs1 and is separated by conserved regions showing homology with two different human chromosomes. For these reasons, and because the two human homologues of U2af1-rs1 and Meg1/Grb10 also lie on different chromosomes, it would seem likely that the two genes identify two distinct imprinting domains within the small proximal region of mouse Chr 11.     

Clinical features and molecular genetic analysis of a boy with Prader-Willi syndrome caused by an imprinting defect

Prader-Willi syndrome (PWS) is a neuroendocrine disorder caused by a non-functioning paternally derived gene(s) within the chromosome region 15q11-q13. Most cases result from microscopically visible deletions of paternal origin, or maternal uniparental disomy of chromosome 15. In both instances no recurrence has been reported. In rare cases, PWS is associated with lack of gene expression from the paternal allele due to an imprinting defect. We report the clinical features and the molecular genetic analysis of the first Danish child with PWS due to a defect of the putative imprinting centre (IC). When the imprinting mutation is inherited from a carrier father, the risk that future children will be affected is theoretically 50%. It is therefore important that these families are referred to a geneticist for counselling and further investigation. Prenatal diagnosis is currently only feasible when the mutation has been identified in the affected child.     

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.     

Why is there no diploid overdose effect in Prader-Willi syndrome due to uniparental disomy?

Due to DNA technology, it is now apparent that the mechanisms of genetic disease are more complex than the model of a gene with biallelic expression in the diploid state. If a gene is imprinted, monoallelic expression is the norm when the chromosomes of a pair are inherited normally from each parent. Uniparental disomy (UPD) is the abnormal situation where both chromosomes of a pair come from the same parent. When the chromosome contains an imprinted gene, UPD may result in nullisomy or disomy for a functional copy of that gene. If there are two imprinted loci on the same chromosome, UPD for that chromosome results in nullisomy for one imprinted gene but functional disomy for the other a "diploid overdose" (DO). This situation has been well demonstrated in the Prader-Willi syndrome (PWS) which is the nullisomic phenotype for the PWS gene(s) on chromosome 15q11-13. Chromosome 15q11-13 also contains the gene for Angelman syndrome (AS) which has a phenotype distinct from PWS. Both loci are subject to imprinting--in PWS, the imprint is on the maternal chromosome 15, in AS it is on the paternal chromosome 15. All individuals with PWS due to maternal UPD, while functionally nullisomic for the PWS locus, are functionally disomic for the AS locus--a DO situation. Assuming that biallelic expression of an imprinted gene is harmful, one would expect DO for an imprinted gene to produce a phenotypic effect. Cases of PWS due to UPD do not appear to differ from those due to deletion (hypopigmentation in deletional cases can be explained by loss of D15S12 downstream from the critical region). There is no good evidence of DO for the AS locus in PWS due to UPD. Why then was it 'necessary' in evolutionary terms to imprint the AS locus and maintain the imprint faithfully for life. A similar situation of two imprinted genes on the same chromosome occurs with IGF2 and H19 on chromosome 11p15. Maternal imprinting for IGF2 and paternal imprinting for H19 is the norm. Paternal UPD in this situation does lead to a DO effect, namely Beckwith-Wiedemann syndrome. The possibility of a DO effect needs to be considered when assessing the phenotypic spectrum of UPD for other chromosomes currently under investigation.     

Evidence for uniparental, paternal expression of the human GABAA receptor subunit genes, using microcell-mediated chromosome transfer

We have constructed mouse A9 hybrids containing a single normal human chromosome 15, via microcell-mediated chromosome transfer. Cytogenetic and DNA-polymorphic analyses identified mouse A9 hybrids that contained either a paternal or maternal human chromosome 15. Paternal specific expression of the known imprinted genes SNRPN (small nuclear ribonucleoprotein-associated polypeptide N gene) and IPW (imprinted gene in the Prader-Willi syndrome region) was maintained in the A9 hybrids. Using this system, we first demonstrated that human GABAAreceptor subunit genes, GABRB3 , GABRA5 and GABRG3 , were expressed exclusively from the paternal allele and that E6-AP (E6-associated protein or UBE3A ) was biallelically expressed. Moreover, the 5' portion of the GABRB3 gene was found to be hypermethylated on the paternal allele. Our data imply that GABAAreceptor subunit genes are imprinted and are possible candidates for Prader-Willi syndrome, and that this human monochromosomal hybrid system enables the efficient analysis of imprinted loci.     

Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies

BACKGROUND: Meiotic recombination events do not occur randomly along a chromosome, but appear to be restricted to specific regions. In addition, some regions in the genome undergo recombination more frequently in the germ cells of one sex than the other. Genomic imprinting, the process by which the two parental alleles of a gene are differentially marked, is another genetic phenomenon associated with inheritance from only one parent or the other. The mechanisms that control meiotic recombination and genomic imprinting are unknown, but both phenomena necessarily depend on the presence of some DNA signal sequences and/or on the structure of the surrounding chromatin domain. RESULTS: In the present study, we compared the frequencies of sex-specific recombination events in three chromosomal regions of the human genome that contain clustered imprinted genes. Alignment of the genetic and physical maps of the ZNF127-SNRPN-IPW-PAR-5-PAR-1 region on chromosome 15q11-q13 (associated with Prader-Willi and Angelman syndromes) and the IGF2-H19 region on chromosome 11p15.5 (associated with Beckwith-Wiedemann syndrome) shows that both regions recombine with very high frequency during male meiosis, and with very low frequency during female meiosis. A third region around the WT-1 gene on chromosome 11p13 also recombines with higher frequency during male meiosis. CONCLUSIONS: The results show that the two best-known imprinted regions in the human genome are characterized by significant differences in recombination frequency during male and female meioses. A third, less well-characterized, imprinted region shows a similar sex-specific bias. On the basis of these observations, we propose a model suggesting that the region-specific differential accessibility of DNA that leads to differential recombination rates during male and female meioses also leads to the male- and female-specific modification of the signal sequences that control genomic imprinting.     

The MAS proto-oncogene is imprinted in human breast tissue

The human MAS proto-oncogene is situated at 6q25.3-q26, a region that is homologous to mouse chromosome 17 where two parentally imprinted genes (Mas and Igf2r) have previously been identified. We investigated the imprinting status of MAS in adult lesions to establish the imprinting status of this gene in humans, as certain imprinted genes are known to have altered imprinting phenotypes in cancer. Of 14 breast samples demonstrating a MAS RT-PCR product, 4 were informative for a polymorphic marker. In all 4 cases, expression of the MAS gene was found to be mono-allelic, indicating the presence of a functional imprint at this locus in human breast tissue.     

Chromosomal localization of the human and mouse hyaluronan synthase genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17.HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Mouse U2af1-rs1 is a neomorphic imprinted gene

The mouse U2af1-rs1 gene is an endogenous imprinted gene on the proximal region of chromosome 11. This gene is transcribed exclusively from the unmethylated paternal allele, while the methylated maternal allele is silent. An analysis of genome structure of this gene revealed that the whole gene is located in an intron of the Murr1 gene. Although none of the three human U2af1-related genes have been mapped to chromosome 2, the human homolog of Murr1 is assigned to chromosome 2. The mouse Murr1 gene is transcribed biallelically, and therefore it is not imprinted in neonatal mice. Allele-specific methylation is limited to a region around U2af1-rs1 in an intron of Murr1. These results suggest that in chromosomal homology and genomic imprinting, the U2af1-rs1 gene is distinct from the genome region surrounding it. We have proposed the neomorphic origin of the U2af1-rs1 gene by retrotransposition and the particular mechanism of genomic imprinting of ectopic genes.     

Sequencing and functional analysis of the SNRPN promoter: in vitro methylation abolishes promoter activity

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.