Association and Mutation Analyses of the IRF6 Gene in Families With Nonsyndromic and Syndromic Cleft Lip and/or Cleft Palate.

OBJECTIVES: (1) To detect interferon regulatory factor 6 gene (IRF6) mutations in newly recruited Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) families. (2) To test for association, in nonsyndromic cleft lip and/or cleft palate (NSCL/P) and in VWS/PPS families, the single nucleotide polymorphism (SNP) rs642961, from the IRF6 enhancer AP-2α region, alone or as haplotype with rs2235371, a coding SNP (Val274Ile). DESIGN: IRF6 mutation screening was performed by direct sequencing and genotyping of rs642961 and rs2235371 by TaqMan technology. PATIENTS: Seventy-one Swedish NSCL/P families, 24 Finnish cleft palate (CP) families, and 24 VWS/PPS families (seven newly recruited) were studied. RESULTS: Allelic and genotypic frequencies in each phenotype were compared to those of the controls, and no significant difference could be observed. IRF6 gene mutation was detected in six of the seven new VWS/PPS families. Association analysis of the entire VWS/PPS sample set revealed the A allele from rs642961 to be a risk allele. Significant association was detected in the Swedish CP subset of our NSCL/P collection where the G-C haplotype for rs642961-rs2235371 were at risk (P = .013). CONCLUSIONS: Our results do not support the previously reported association between the A allele of rs642961 and the NSCL phenotype. However, in the VWS/PPS families, the A allele was a risk allele and was, in a large majority (>80%), transmitted on the same chromosome as the IRF6 mutation.

Varicose vein recurrence and patient satisfaction 10-14 years following combined superficial and perforator vein surgery: a prospective case study.

OBJECTIVE: To assess real long-term varicose vein recurrence and patient satisfaction following surgical intervention with combined subfascial endoscopic perforator surgery (SEPS) and superficial venous surgery. METHOD: Prospective consecutive case study (C3-C4). Patients were included March 1993 to September 1998 and 83/104 legs of 80/100 patients were re-assessed 2008; 71 legs underwent duplex ultrasound scanning (DUS). RESULTS: The median follow up was 12 years (range 10-14). Twelve patients/legs had undergone additional vein surgery during follow-up. Incompetent lower leg perforators were noted in 18/71 limbs (25%). Following groin surgery 23/51 (45%) showed a duplex detected groin recurrence, neovascularization dominated 18/23. In legs where primary great saphenous vein (GSV) surgery had been performed, groin recurrence was found in 14/37 (38%). Previously unknown deep vein incompetence was detected in 14/71 legs (20%), six had axial reflux. The correlation between DUS-detected recurrence and remaining symptoms and cosmetic result was low. The overall satisfaction was high, 70/82 (85%). Patient satisfaction did not deteriorate over time (p < .557). CONCLUSION: Despite a fair number of DUS-detected recurrences, the overall long-term result, from the patients' point of view was surprisingly favorable. Technically well performed open venous surgery seems to result in a durable long-term outcome.

Deregulation of the platelet-derived growth factor B-chain gene via fusion with collagen gene COL1A1 in dermatofibrosarcoma protuberans and giant-cell fibroblastoma.

Dermatofibrosarcoma protuberans (DP), an infiltrative skin tumour of intermediate malignancy, presents specific features such as reciprocal translocations t(17;22)(q22;q13) and supernumerary ring chromosomes derived from the t(17;22). In this report, the breakpoints from translocations and rings in DP and its juvenile form, giant cell fibroblastoma (GCF), were characterised on the genomic and RNA level. These rearrangements fuse the platelet-derived growth factor B-chain (PDGFB, c-sis proto-oncogene) and the collagen type I alpha 1 (COL1A1) genes. PDGFB has transforming activity and is a potent mitogen for a number of cell types, but its role in oncogenic processes is not fully understood. COL1A1 is a major constituent of the connective tissue matrix. Neither PDGFB nor COL1A1 have so far been implicated in any tumour translocations. These gene fusions delete exon 1 of PDGFB, and release this growth factor from its normal regulation.

Early results from a randomized trial of saphenous surgery with or without subfascial endoscopic perforator surgery in patients with a venous ulcer.

BACKGROUND: The aim was to clarify the role of incompetent perforators (IPs) in venous leg ulcers. This short-term report focused on safety, patient satisfaction and the fate of IPs after subfascial endoscopic perforator surgery (SEPS), or saphenous surgery alone. METHODS: Patients aged 30-78 years with an open or recently healed venous ulcer, and with an incompetent saphenous vein and IPs, were allocated randomly to saphenous surgery alone, or in combination with SEPS. A control duplex scan was performed 6-9 months after surgery, and clinical follow-up was scheduled after 1 week, 3 and 12 months. A standard questionnaire was completed at each clinical visit. RESULTS: Seventy-five patients were enrolled; 37 had SEPS and 38 had saphenous surgery alone. SEPS prolonged the operation by a median of 15 min (P = 0.003). Duplex imaging revealed significantly more remaining IPs in the no-SEPS group (P < 0.001). Compared with the preoperative scan, significantly more legs were free from IPs in the SEPS group compared with the no-SEPS group (21 of 36 versus 7 of 37 respectively; P < 0.001). There were no other major outcome differences between the groups. CONCLUSION: There was no short-term clinical benefit from adding SEPS to saphenous surgery in patients with varicose ulcers and IPs, although SEPS reduced the number of perforators remaining after 1 year.

True long-term healing and recurrence of venous leg ulcers following SEPS combined with superficial venous surgery: a prospective study.

BACKGROUND: The role of perforator surgery remains unclear in the management of patients with leg ulcers. The aim of this study was to assess long-term healing and recurrence rates of leg ulcers following surgical intervention with combined Subfascial Endoscopic Perforator Surgery (SEPS) and superficial venous surgery. METHOD: Case series with prospective long-term follow-up of 90 consecutive patients operated on with open (CEAP C6) or healed (CEAP C5) venous ulcers in 97 legs. Popliteal vein reflux was present in 21 legs. All 97 legs were treated with SEPS and 87% had additional superficial venous surgery. Patients were follow-up for a median of 77 months (range 60-112 months) with a minimum of 5 years. RESULTS: 87% of all ulcerated legs healed. The three and five year recurrence rates were 8% and 18% respectively among survivors. In a multivariate Cox regression analysis previous vein surgery was the only factor significantly associated with recurrent ulceration (p=.004). CONCLUSION: SEPS combined with superficial venous surgery leads to healing with a low recurrence rate in patients with open and healed venous ulcers. Previous venous surgery was found to be a significant risk factor for ulcer recurrence. This result emphasizes the importance of assiduous technique for varicose vein surgery and suggests a continuing role for perforator surgery in leg ulcer patients.

Ornithine decarboxylase activity in dorsal root ganglia of regenerating frog sciatic nerve.

Ornithine decarboxylase (ODC) activity was studied in dorsal root ganglia (DRG) of regenerating frog sciatic nerve. There was a significant increase in activity two days after a crush lesion of the nerve 2.5 cm distal to the DRG. The increase reached a maximum after 7 days, then declined but remained above control levels for at least 9 days. An endoneural injection of vinblastine, a potent inhibitor of retrograde and orthograde axonal transport, between the DRG and the crush inhibited the increase in ODC. In contrast, injections of vinblastine into undamaged nerves failed to affect ODC. The increase in ODC and also the regenerative properties of the nerve could be prevented by daily i.p. injections of alpha-difluoromethyl ornithine. We suggest that a signal is formed at the site of injury in the sciatic nerve. This signal is conveyed to the DRG by retrograde axonal transport where it initiates the events leading to an increase in ODC. This increase appears to be necessary for the regeneration of sensory fibers in the frog sciatic nerve.

Familial non-syndromic cleft lip and palate--analysis of the IRF6 gene and clinical phenotypes.

The aim of this study was to characterize Swedish families with non-syndromic cleft lip and/or palate (NSCL/P) for mutations or other sequence variants in the interferon regulatory factor 6 (IRF6) gene, as well as to describe their cleft phenotypes and hypodontia. Seventeen Swedish families with at least two family members with NSCL/P were identified and clinically evaluated. Extracted DNA from blood samples was used for IRF6 mutation screening. Exonic fragments of the IRF6 gene were sequenced and chromatograms were inspected. Statistical analysis was undertaken with marker- and haplotype association tests. No disease-associated IRF6 mutation could be determined in the families analyzed. One new and seven known single nucleotide polymorphisms (SNPs) were detected. The A allele of SNP rs861019 in exon 2 and the G allele of SNP rs7552506 in intron 3 showed association with cleft lip and palate (CLP; odds ratios of 3.1 and 5.45, respectively). Hypodontia was observed more commonly in individuals affected with CL/P as compared with family members without a cleft (P < 0.01). The hypodontia most often affected the cleft area, possibly representing a secondary effect. The distribution of cleft phenotypes in 15 of the 17 families with NSCL/P differed from the mixed cleft types seen in Van der Woude syndrome (VWS), in that CLP did not occur together with an isolated cleft palate within the same family. It was concluded that mutations of the IRF6 gene are not a common cause for cleft predisposition in Swedish NSCL/P families.

Characterization of a second human clathrin heavy chain polypeptide gene (CLH-22) from chromosome 22q11.

We report cloning and characterization of the second human clathrin heavy chain polypeptide gene (CLH-22) localized to chromosome 22q11. Hence H. sapiens is the first species for which two clathrin heavy chain genes have been reported. We provide 5470 bp cDNA sequence covering the entire open reading frame of the CLH-22 gene. The predicted polypeptide is composed of 1640 amino acids. Its 6 kb transcript is expressed in all of 16 tested human tissues, suggesting it is a housekeeping gene. Skeletal muscle, testis and heart show significantly higher expression levels. Compared to the previously characterized human clathrin heavy chain gene localized on chromosome 17 (CLH-17), CLH-22 shows different transcript size and expression profile in human tissues. Northern analysis of CLH-22 suggests that several alternatively spliced transcripts exist. A presumably single, 171 bp long alternatively spliced exon has been characterized. Amino acid sequence comparison between CLH-22 and CLH-17 shows an overall identify and similarity of 84.7 and 91.1%, respectively. At the nucleic acid level, identity between open reading frames of both genes is 74.3%. Sequence comparison with previously cloned genes in other species suggests that counterparts of the CLH-17 gene have been cloned in B. taurus and R. norvegicus, whereas presumptive mammalian homologues of the CLH-22 gene are yet to be characterized. Our Northern and Southern blot analyses of meningiomas clearly suggest the CLH-22 gene may be involved in the tumor development and can be considered as a candidate for a tumor suppressor.

Cloning, expression pattern, and chromosomal assignment to 16q23 of the human gamma-adaptin gene (ADTG).

Adaptins are important components of clathrin-coated vesicles transporting ligand-receptor complexes from the plasma membrane or from the trans-Golgi network to lysosomes. Adaptins, together with medium and small subunits, form a heterotetrameric complex called an adaptor, whose role is to promote the formation of clathrin-coated pits and vesicles. We present the cloning and sequencing of the human gamma-adaptin cDNA (HGMW-approved symbol ADTG) consisting of 3723 bp with an open reading frame capable of encoding a protein of 825 amino acids, 98.9% identical to the mouse protein. Northern blot analysis of the mouse and human gamma-adaptin genes revealed a ubiquitous and abundant expression, except in human adult lung. Using a monochromosomal somatic cell hybrid panel and fluorescence in situ hybridization, we mapped this gene to human chromosome 16q23, which is syntenic with mouse chromosome 8, band D. In addition, we localized genes for two other components of the AP-1 adaptor, i.e., the medium (AP47) and small (AP19) subunits, to chromosomes 19 and 7, respectively. Expression analysis of these genes in human tissues revealed ubiquitously expressed transcripts of approximately 2.5 and 1.5 kb, respectively.

Characterization of the mouse beta-prime adaptin gene; cDNA sequence, genomic structure, and chromosomal localization.

Adaptins are important subunits of heterotetrameric complexes called adaptors, which participate in the clathrin-coated, vesicle-mediated endocytosis and intracellular receptor transport. The gene family of adaptins is divided into three classes, alpha, beta, and gamma, with further subdivision into beta- and beta-prime components. Two beta-prime adaptins, the rat AP105a and the human BAM22, have previously been characterized. The BAM22 gene is located on human Chromosome (Chr) 22q12 and can be considered a candidate meningioma tumor suppressor gene. We report here the characterization of the mouse ortholog of the BAM22 gene, and we suggest the name adtb1 for the mouse gene. Like the BAM22 gene, the adtb1 transcript is highly and ubiquitously expressed. We provide 3885-bp cDNA sequence, which entirely covers the open reading frame of the adtb1, capable of encoding a protein of 943 amino acids. The adtb1 protein is highly conserved (>96% identity) when compared with AP105a and BAM22 proteins. We also report the genomic organization of adtb1, which is similar to the BAM22 gene. The adtb1 gene has been assigned to mouse Chr 11, band 11A2, which confirms the synteny between human Chr 22q12 and mouse Chr 11.

The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is tightly linked to the muscle-type glycogen phosphorylase (PYGM) gene in 11q13. This region of the human genome contains additional disease-related loci implicated in the development of insulin-dependent diabetes mellitus, familial paraganglioma type 2, spinocerebellar ataxia type 5, Bardet-Biedl syndrome and translocation t(11;17) described in B-cell non-Hodgkin's lymphoma. We approached cloning of candidate disease genes from 11q13 by large-scale genomic sequencing. We obtained > 106 kb of sequence around the PYGM gene and established a transcriptional map that includes: (i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; (ii) the germinal center kinase (GCK, human B-lymphocyte serine/threonine protein kinase) gene; (iii) a novel human CDC25-like (HCDC25L) gene; (iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and (v) a novel ubiquitously expressed gene of unknown function (germinal center kinase- neighboring gene, GCKNG).

Cloning of a novel, anonymous gene from a megabase-range YAC and cosmid contig in the neurofibromatosis type 2/meningioma region on human chromosome 22q12.

In order to permit detailed characterization of meningioma cases showing deletions within chromosomal band 22q12 and further systematically clone genes located within this region, we established a genomic YAC and cosmid contig which encompasses a region in excess of 1000 kb of 22q12. The YAC contig consists of 6 YAC clones arranged into 5 overlapping steps covering more than 1100 kb. Two corresponding cosmid contigs consisting of 40 steps of overlapping groups of cosmids encompasses 900-1000 kb. This set of genomic clones provides a detailed physical map of this part of chromosome 22 and constitutes a basis for the isolation and characterization of genes that may be located within this chromosomal region. Employing the exon-amplification method on two cosmids from the contig, we cloned a novel, anonymous gene, pK1.3, which potentially encodes a protein of 683 amino acids with a predicted molecular weight of of 78.5 kD. Its 2.7 kb mRNA is expressed ubiquitously. We estimated the genomic size of this gene to 100-150 kb, and it is located in the immediate centromeric vicinity of the neurofibromatosis 2 (NF2) tumor suppressor gene.

Physical mapping of the NF2/meningioma region on human chromosome 22q12.

Loss of genetic information from chromosome 22 has been implicated in the development of neurofibromatosis type 2, meningioma, and several other neoplasia. Molecular studies indicate that genes within chromosomal band 22q12 may be involved in tumorigenesis. We have mapped 29 loci into 16 groups in this region, using pulsed-field gel electrophoresis, fluorescence in situ suppression hybridization, and somatic cell hybrid mapping. The region spans more than 5 Mb of genomic DNA and contains the genes for neurofibromatosis type 2 and meningioma. The order of loci presented here provides the framework for the fine mapping of this region using cosmids and yeast artificial chromosomes, and it facilitates the speedy cloning of novel genes from 22q12.

Structure of the promoter and genomic organization of the human beta'-adaptin gene (BAM22) from chromosome 22q12.

Adaptins are major structural components of heterotetrameric protein complexes called adaptors, which are essential in intracellular receptor transport via clathrin-coated vesicles. beta-adaptins constitute one of three known classes (alpha, beta, gamma) of adaptins, including beta and beta' subtypes. We previously cloned the human beta'-adaptin gene (BAM22) (GDB symbol, ADTB1) from chromosome 22q12 and proposed its involvement in the development of meningiomas. Here we describe the genomic organization of this gene, which consists of 22 exons spanning over approximately 100 kb. We also report results from point mutation screening of 7 randomly chosen exons analyzed in 110 sporadic meningiomas. As part of the genomic characterization of the BAM22 locus, we sequenced 40 kb covering exons 1-4 and 12 kb upstream from the start of gene transcription. Analysis of the sequence suggests that the BAM22 gene has a CG-rich promoter.

The genes for oncostatin M (OSM) and leukemia inhibitory factor (LIF) are tightly linked on human chromosome 22.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are members of a family of cytokines that regulate the proliferation and differentiation of a variety of cell types. In this report, cDNA probes specific for OSM and LIF were hybridized to DNA from somatic cell hybrids containing defined regions of human chromosome 22, and the gene for human OSM was found to segregate with that of LIF. Southern analysis of high-molecular-weight DNA that had been digested with rare-cutting restriction enzymes and analyzed by pulsed-field gel electrophoresis showed identical hybridization patterns with both probes. The probes also identified common cosmid clones on high-density cosmid filters prepared from chromosome 22-specific flow-sorted cosmid libraries. Restriction and Southern analyses of six cosmid clones established a contig of approximately 100 kb surrounding the genes for OSM and LIF. The OSM and LIF genes are tandemly arranged in the same transcriptional orientation separated by approximately 10 kb. The direction of gene transcription is telomeric to centromeric, with the OSM gene located upstream of the LIF gene. Our studies define a new gene cluster on chromosome 22 and provide strong evidence that OSM and LIF have resulted from duplication of a common ancestral gene.

A novel gene containing LIM domains (LIMD1) is located within the common eliminated region 1 (C3CER1) in 3p21.3.

Chromosomal deletions on 3p have been described in a large number of human tumors, suggesting the presence of a tumor suppressor gene(s). Using an experimental system, called the elimination test, we previously identified a 1 Mb segment, the common eliminated region 1 (C3CER1). C3CER1 was also covered by a PAC contig. Using the sequence of two overlapping PACs from C3CER1, we localized the human KIAA0028 cDNA, encoding the precursor of mitochondrial leucyl-tRNA synthetase. We also characterized a novel human LIM domain-containing gene (LIMD1) and its mouse ortholog (Limd1). LIM domains consist of a cysteine-rich consensus sequence containing two distinct zinc-binding subdomains, which mediate protein-protein interactions. The predicted protein sequences of the human and mouse genes reveal three LIM domains located at the C-terminal end, which indicates that they belong to the group 3 of the gene family encoding LIM motifs. We characterized the genomic structure of the human LIMD1 gene and assigned the mouse Limd1 gene to the chromosome 9F subtelomeric region. Both genes are ubiquitously expressed at the mRNA level. The LIM motif has been previously identified in many developmentally important factors from various eukaryotes. These factors have been shown to play a role in intracellular signaling, transcriptional regulation and cellular differentiation during development. The human C3CER1-located LIMD1 gene should therefore be further studied for its possible role in tumor suppression.

Perception of information, expectations and experiences among women and their partners attending a second-trimester routine ultrasound scan.

The aim of this study was to evaluate pre-scan counselling and the provision of information to parents-to-be, and their expectations before and experiences of a second trimester routine ultrasound scan. In the study, 303 pregnant women and their partners were asked to complete questionnaires before and after the scan. The main purposes of the examination were: dating, ascertaining fetal viability, and detection of multiple gestations. Although scanning for fetal malformations was not the purpose of the examination, 89% of the women and 84% of the men were concerned about this aspect. Even though it has been postulated that more women would not attend the examination if they knew it was for prenatal diagnostic purposes, the results of this study did not support this assumption. Only 57% of the women had received information at their antenatal care centers. A total of 88% of the women and 85% of the men said that they obtained sufficient information at the scan. Anxiety was low before the scan, both among women and men, with the exception of those women who had experienced problems at earlier scans. Positive feelings dominated during the scan and these feelings remained when experiences of the scan were reported by the parents-to-be after they had gone home. It is concluded that a routine second-trimester scan is a positive event for the majority of the participating women and men. In spite of this, we believe that certain measures should be taken to improve pre-scan counselling and the provision of adequate information.

The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family.

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the LARGE gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by LARGE displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse LARGE ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and in situ histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcalpha2,8NeuAcalpha2, 3Galbeta1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.

TOM1 genes map to human chromosome 22q13.1 and mouse chromosome 8C1 and encode proteins similar to the endosomal proteins HGS and STAM.

The avian tom1 (target of myb 1) gene has been previously characterized from v-myb-transformed cells. We report here cloning of the human and mouse tom1 orthologs. Both genes are expressed ubiquitously, with the highest levels in skeletal muscle, brain, and intestines, as assessed by Northern blot and mRNA in situ hybridization. The N-terminal domain of the TOM1 protein shares similarity with HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are associated with vesicular trafficking at the endosome. A putative coiled-coil domain was also detected in the central part of the TOM1 protein. This domain structure suggests that TOM1 is another member of a family of genes implicated in the trafficking regulation of growth-factor-receptor complexes that are destined for degradation in the lysosome. We also show that a human paralog of TOM1 (TOM1-like gene 1) exists. Furthermore, we provide a transcription map over a 190-kb contig of the TOM1 region. This map includes its distal neighbors HMOX1 and MCM5 and two proximal novel genes, one of which is a HMG-box-containing gene (HMG2L1), and the other of unknown function. Using a genomic PAC clone, we demonstrate that the mouse Tom1 and Hmox1 genes are part of an as yet undescribed syntenic group between mouse chromosome 8C1 and human chromosome 22q13.1.

Characterization of a new member of the human beta-adaptin gene family from chromosome 22q12, a candidate meningioma gene.

A 140 kb homozygous deletion from 22q12 in one meningioma directed us towards the cloning and characterization of a new member of the human beta-adaptin gene family (named BAM22). Adaptins are essential for the formation of clathrin coated vesicles in the course of intracellular transport of receptor-ligand complexes. The BAM22 gene is totally inactivated in the tumor with homozygous deletion. Northern blot analysis of 70 sporadic meningiomas showed specific loss of expression in 8 tumors, suggesting inactivation of BAM22. Based on this, we propose BAM22 as a second chromosome 22 locus important in meningioma development, after the neurofibromatosis type 2 gene.