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ABSTRACT: Several Wolf-Hirschhorn syndrome patients have been studied, mouse models for a few candidate genes have been constructed and two WHS critical regions have been postulated, but the molecular basis of the syndrome remains poorly understood. Single gene contributions to phenotypes of microdeletion syndromes have often been based on the study of patients carrying small, atypical deletions. We report a 5-year-old girl harboring an atypical 1.5Mb del4p16.3 and review seven previously published patients carrying a similar deletion. They show a variable clinical presentation and the only consistent feature is post-natal growth delay. However, four of eight patients carry a ring (4), and ring chromosomes in general are associated with growth deficiency. The Greek helmet profile is absent, although a trend towards common dysmorphic features exists. Variable expressivity and incomplete penetrance might play a role in WHS, resulting in difficult clinical diagnosis and challenge in understanding of the genotype/phenotype correlation.
Gene 07/2012; 507(1):85-91. · 2.34 Impact Factor
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Breast Cancer Research 04/2012; 2:1-1. · 5.33 Impact Factor
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ABSTRACT: Chromosome 22q13 monosomy has been described as a contiguous gene syndrome. Localized in the critical region, SHANK3 is likely to play a key role in the expression of the clinical phenotype. SHANK3 mutations have also been reported in autistic patients without a syndromic phenotype. We report on a 20-year-old woman with mental retardation carrying a de novo translocation between chromosome Xq21.33 and 22q13.33, associated with a duplication on Xq21.33 and deletion on 22q13.33. As a child her development was characterized by disturbed social interaction, stereotypic hand movements and ritualistic behavior and she was considered at one time to have autistic features. All these traits match the 22q13 deletion syndrome (Phelan-McDermid syndrome, OMIM 606232), likely due to the deletion overlapping the last two exons of the SHANK3 gene. Our patient harbors the smallest and most distal SHANK3 deletion described to date, yet resulting in the full spectrum of the Phelan-McDermid syndrome. In addition, she has hypergonadotropic hypogonadism with low estrogen level, high FSH level, and irregular menstruation. Intriguingly, chromosome translocations affecting the chromosome band Xq21 can result in premature ovarian failure.
American Journal of Medical Genetics Part A 02/2011; 155A(2):403-8. · 2.39 Impact Factor
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ABSTRACT: Chromosome 22q13 monosomy has been described as a contiguous gene syndrome. Localized in the critical region, SHANK3 is likely to play a key role in the expression of the clinical phenotype. SHANK3 mutations have also been reported in autistic patients without a syndromic phenotype. We report on a 20-year-old woman with mental retardation carrying a de novo translocation between chromosome Xq21.33 and 22q13.33, associated with a duplication on Xq21.33 and deletion on 22q13.33. As a child her development was characterized by disturbed social interaction, stereotypic hand movements and ritualistic behavior and she was considered at one time to have autistic features. All these traits match the 22q13 deletion syndrome (Phelan–McDermid syndrome, OMIM 606232), likely due to the deletion overlapping the last two exons of the SHANK3 gene. Our patient harbors the smallest and most distal SHANK3 deletion described to date, yet resulting in the full spectrum of the Phelan–McDermid syndrome. In addition, she has hypergonadotropic hypogonadism with low estrogen level, high FSH level, and irregular menstruation. Intriguingly, chromosome translocations affecting the chromosome band Xq21 can result in premature ovarian failure. © 2011 Wiley-Liss, Inc.
American Journal of Medical Genetics Part A 01/2011; 155(2):403 - 408. · 2.39 Impact Factor
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ABSTRACT: We report of a spinocerebellar ataxia (SCA)27 in a daughter and her mother whose karyotype is 46, XX t(5;13)(q31.2;q33.1). The translocation breakpoint is identical in both patients, disrupting the gene-encoding fibroblast growth factor 14 isoform b (FGF14-1b). Clinically, both show signs of SCA, although the daughter is the most affected with early onset cerebellar ataxia, microcephaly, and severe mental retardation. FGF14-1b is the predominant isoform in brain, where it interacts with the voltage gated Na channel. Fgf14(-/-) mice develop ataxia and paroxysmal dyskinesia and have cognitive deficits. One missense and one non-sense mutation in FGF14 have previously been linked to SCA27. Truncation of one allele in our patients suggests that haploinsuffiency of FGF14 can cause SCA27.
Neurogenetics 06/2009; 10(4):371-4. · 3.35 Impact Factor
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ABSTRACT: Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.
European Journal of Biochemistry 11/2001; 268(19):5066-73. · 3.58 Impact Factor
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ABSTRACT: A cDNA encoding a novel member of the helicase family, MDDX28, has been cloned from a human testis library. This apparently intronless gene was transcribed in all tissues studied. MDDX28 encodes a protein of 540 amino acids, with approximately 30% homology to other helicases over the core region, containing all the conserved DEAD-box helicase motifs. No homologue is known. MDDX28 has RNA and Mg(2+)-dependent ATPase activity. Subcellular localization studies of MDDX28 using oligoclonal antibodies raised against the protein as well as its enhanced green fluorescence protein (EGFP) demonstrated that the protein is localized in the mitochondria and the nucleus. To our knowledge, MDDX28 is the first member of the RNA helicase described with this dual location. The nuclear localization of MDDX28 depended on active RNA polymerase II transcription, suggesting that the protein could be transported to and from the nucleus. This was confirmed further in an interspecies heterokaryon assay, in which MDDX28 was seen to translocate to the nucleus and mitochondria. The mitochondrial uptake of the MDDX28-EGFP-N1 fusion protein was inhibited by carbonyl cyanide p-(trichloromethoxy)phenylhydrazone. Our results indicate that MDDX28 can be transported between the mitochondria and the nucleus.
Journal of Biological Chemistry 09/2001; 276(34):32056-63. · 4.77 Impact Factor
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ABSTRACT: This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.
Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 06/2001; Chapter 5:Unit 5.15.
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E Frengen,
P Rocca-Serra,
S Shaposhnikov,
L Taine,
J Thorsen,
C Bepoldin,
M Krekling,
D Lafon,
K K Aas,
A A El Monéim,
H Johansen,
M Longy,
H Prydz,
F Dorion-Bonnet
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ABSTRACT: Loss of heterozygosity (LOH) on the long arm of human chromosome 16 is a common genetic alteration observed in both invasive ductal and invasive lobular breast carcinomas. We have generated a high-resolution integrated map encompassing the smallest region of LOH overlap within chromosome 16q22.1 (SRO2). Southern hybridization experiments using more than 140 probes resulted in the assembly of 152 bacterial large-insert clones into a 2.8-Mb contig covering SRO2. The structure of the contig was verified by long-range mapping using total human genomic DNA, and the contig orientation was determined by fluorescence in situ hybridization. A total of 68 transcripts have been identified in the map. One of the genes residing within SRO2 is the E-cadherin gene, CDH1, which has previously been shown to be mutated in lobular breast carcinomas, resulting in loss of E-cadherin expression. In most cases of ductal carcinoma, which is the major mammary cancer type, E-cadherin is normally expressed, suggesting that other genes within 16q22.1 are involved in the development of this tumor subtype. The high-resolution map presented in this study provides a valuable resource for identification of tumor suppressor genes expected to be involved in the etiology of breast carcinomas.
Genomics 01/2001; 70(3):273-85. · 3.02 Impact Factor
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ABSTRACT: Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.
Genome Research 02/2000; 10(1):116-28. · 13.61 Impact Factor
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ABSTRACT: We report the screening of thirty-one YACs with a number of markers using polymerase chain reaction (PCR) to construct a physical map of part of human chromosome 17q21.3-q22. A contig of YACs covering about 4 Mb was constructed around the TCF11 gene at 68 cM from the most telomeric marker on the p arm, localizing TCF11 telomeric to genetic marker D17S1827. Both human and mouse P1-derived artificial chromosomes (PACs) containing TCF11 were isolated and characterized. The human heterochromatin protein 1 gene, HP1Hsbeta, and its homologue in mouse, MoMOD1, were identified centromeric to TCF11.
Genetic Analysis Biomolecular Engineering 12/1999; 15(6):217-22.
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Cytogenetics and cell genetics 02/1999; 84(1-2):97-8.
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ABSTRACT: The thyrotropin-releasing hormone (TRH) receptor (TRHR) is widely distributed throughout the central and peripheral nervous systems. In addition to its role in controlling the synthesis and secretion of thyroid-stimulating hormone and prolactin from the anterior pituitary, TRH is believed to act as a neurotransmitter as well as a neuromodulator. We have isolated genomic lambda and P1-derived artificial chromosome clones encoding the human TRHR. The gene was found to be 35 kb with three exons and two introns. A 541-bp intron 1 (-629 to -89 relative to the translation start site) is conserved between human and mouse. A large intron 2 of 31 kb disrupts the open reading frame (starting in position +790) in the sequence encoding the supposed junction between the third intracellular loop and the putative sixth transmembrane domain. A similar intron was found in chimpanzee and sheep but not in rat and mouse. Promoter analysis of upstream regions demonstrated cell type-specific reporter activation, and sequencing of 2.5 kb of the promoter revealed putative cis-acting regulatory elements for several transcription factors that may contribute to the regulation of the TRHR gene expression. Functional analysis of potential response elements for the anterior pituitary-specific transcription factor Pit-1 revealed cell type-specific binding that was competed out with a Pit-1 response element from the GH gene promoter.
Journal of Neurochemistry 02/1999; 72(1):40-50. · 4.06 Impact Factor
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ABSTRACT: Presented here are improved methodologies that enable the generation of highly redundant bacterial artificial chromosome/P1-derived artificial chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent beta-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.
Genomics 09/1998; 52(1):1-8. · 3.02 Impact Factor
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ABSTRACT: The type II cGMP-dependent protein kinase (cGK) plays a pivotal role in the regulation of intestinal fluid balance in man. Furthermore, mice carrying a null mutation for the gene encoding the type II cGK develop as dwarfs indicating that this enzyme has other less characterized roles. The present report describes the isolation and characterization of bacterial artificial chromosome (BAC)- and P1-derived artificial chromosome (PAC)-clones containing the gene encoding the human type II cGK. The gene was estimated to cover at least 125 kb and consisted of 19 exons separated by introns of various lengths. The splice junctions of the type II cGK gene corresponded well with the structure of the gene encoding human type I cGK and with the splice junctions observed in the Drosophila melanogaster DG2 gene. 5'-rapid amplification of cDNA-ends established the presence of a non-translated exon.
Biochemical and Biophysical Research Communications 04/1998; 245(1):113-9. · 2.48 Impact Factor
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ABSTRACT: We have designed a YAC circularization vector, pCIRC3, allowing enrichment of the YAC DNA by exonuclease digestion of the linear yeast chromosomes. Due to the presence of P1 replicon sequences in this vector, the circular YACs would replicate as PACs in Escherischia coli.
Genetic Analysis Biomolecular Engineering 08/1997; 14(2):55-9.
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ABSTRACT: A pooled DNA probe from P1 artificial chromosome clones (PACs) containing the human lecithin:cholesterol acyl transferase (LCAT) gene cluster was used in fluorescence in situ hybridization (FISH) experiments assigning the genes to pig chromosome 6p13. In addition, probes derived from the coding regions in the human gene cluster were used in long range mapping experiments to show that the overall structures of the human and porcine LCAT gene clusters are identical. Both the linear order and the close physical distance of five apparently unrelated genes have been maintained throughout 90 million years of divergent evolution between human and pig. The extremely dense clustering of the genes in the LCAT gene cluster suggests that this gene organization has biological significance. The conservation of the gene cluster between human and pig supports this suggestion.
Cytogenetics and cell genetics 02/1997; 76(1-2):53-7.
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ABSTRACT: Long range restriction maps of the calcium release channel (CRC) and glucosephosphate isomerase (GPI) loci have been constructed using pulsed field electrophoresis, Southern blotting and CRC- and GPI-specific probes. The maps, deduced from the restriction fragments detected by the probes, covered 1.1 and 0.3 Mb respectively and no overlap between the maps of these closely linked loci was detected. The minimal distance between the GPI and CRC loci was estimated to be at least 500 kb.
Animal Genetics 07/1995; 26(3):181-4. · 2.40 Impact Factor
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ABSTRACT: Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.
Cytogenetics and cell genetics 02/1995; 68(3-4):194-6.
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ABSTRACT: Coincidence cloning is a technique that permits the isolation of sequences common to two independent sources of complex DNA, and this method has been used to isolate a set of probes from a region of porcine Chromosome (Chr) 6 containing the loci for glucosephosphate isomerase (GPI) and the skeletal muscle calcium release channel (CRC). Porcine DNA was specifically PCR-amplified from a pig x hamster hybrid cell line containing the centromere region (p1.2-q1.2) of pig Chr 6 and other pig chromosome fragments by use of a porcine SINE specific primer with an EcoRI site in the 5'-end. Flow-sorted Chr 6 preparations were amplified with the same SINE primer, but with a SalI site in the 5'-end. The products were digested with EcoRI and SalI respectively, combined, denatured, and reannealed. The heteroduplex molecules, containing both an EcoRI and a SalI cohesive end, were selected by cloning in SalI/EcoRI-digested pUC13. Approximately 40% of the primary clones contained a single SalI/EcoRI-insert, indicating that they are coincidence clones. The average insert size was 1.4 kb. Fluorescence in situ hybridization of a pool of 34 coincidence clones to pig chromosomes showed a preferential labeling of the centromere region and of the q2.5-q2.7 region of pig Chr 6. Nineteen coincidence clones were hybridized to SINE-PCR products from flow-sorted pig Chr 6 and to pig x rodent hybrid cell lines. Eighteen clones gave positive signals correlated with the GPI/CRC content of the source DNAs.
Mammalian Genome 09/1994; 5(8):497-502. · 2.89 Impact Factor
