Autosomal recessive cystinuria caused by genome-wide paternal uniparental isodisomy in a patient with Beckwith-Wiedemann syndrome.

Approximately 20% of Beckwith-Wiedemann syndrome (BWS) cases are caused by mosaic paternal uniparental disomy of chromosome 11 (pUPD11). Although pUPD11 is usually limited to the short arm of chromosome 11, a small minority of BWS cases show genome-wide mosaic pUPD (GWpUPD). These patients show variable clinical features depending on mosaic ratio, imprinting status of other chromosomes, and paternally inherited recessive mutations. To date, there have been no reports of a mosaic GWpUPD patient with an autosomal recessive disease caused by a paternally inherited recessive mutation. Here, we describe a patient concurrently showing the clinical features of BWS and autosomal recessive cystinuria. Genetic analyses revealed that the patient has mosaic GWpUPD and an inherited paternal homozygous mutation in SLC7A9. This is the first report indicating that a paternally inherited recessive mutation can cause an autosomal recessive disease in cases of GWpUPD mosaicism. Investigation into recessive mutations and the dysregulation of imprinting domains is critical in understanding precise clinical conditions of patients with mosaic GWpUPD.

A novel de novo point mutation of the OCT-binding site in the IGF2/H19-imprinting control region in a Beckwith-Wiedemann syndrome patient.

The IGF2/H19-imprinting control region (ICR1) functions as an insulator to methylation-sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin-specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF-binding site, but also point mutations and a small deletion of the OCT-binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT-binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT-binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT-binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.

Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis.

We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes.

A novel imprinted gene, KCNQ1DN, within the WT2 critical region of human chromosome 11p15.5 and its reduced expression in Wilms' tumors.

WT2 is defined by a maternal-specific loss of heterozygosity on human chromosome 11p15.5 in Wilms' and other embryonal tumors. Therefore, the imprinted genes in this region are candidates for involvement in Wilms' tumorigenesis. We now report a novel imprinted gene, KCNQ1DN (KCNQ1 downstream neighbor). This gene is located between p57(KIP2) and KvLQT1 (KCNQ1) of 11p15.5 within the WT2 critical region. KCNQ1DN is imprinted and expressed from the maternal allele. We examined the expression of KCNQ1DN in Wilms' tumors. Seven of eighteen (39%) samples showed no expression. In contrast, other maternal imprinted genes in this region, including p57(KIP2), IMPT1, and IPL exhibited almost normal expression in these samples, although some samples expressed IGF2 biallelically. These results suggest that KCNQ1DN existing far from the H19/IGF2 region may play some role in Wilms' tumorigenesis along with IGF2.

C11orf21, a novel gene within the Beckwith-Wiedemann syndrome region in human chromosome 11p15.5.

A novel gene, C11orf2, was identified by BLAST search in the human chromosome 11p15.5 region potentially responsible for Beckwith-Wiedemann Syndrome (BWS) and some cancers. Two cDNA clones with different sizes were obtained, which share a potential ORF of 399bp and are different in their 3' untranslated regions. This gene was revealed to be expressed exclusively in human heart and in almost no other tissues examined by northern blotting. Two transcripts of different sizes, 0.9 and 3.1kb, were identified in heart, consistent with the length of the two cDNA clones. The gene shows biallelic expression (non-imprinted) in fetal liver, although it is located in the imprinted domain of 11p15.5. C11orf21 codes a protein of 132 amino acids as proved by the expression of C11orf21-EGFP fusion protein in cultured cells. The EGFP-fusion protein expressed in cultured cells localized mainly in the cytoplasm.

Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B and C genes in adult tissues, oocytes and embryos of Xenopus laevis.

Following previous cloning and expression studies of Xenopus aldolase C (brain-type) and A (muscle-type) cDNAs, we cloned here two Xenopus aldolase B (liver-type) cDNAs (XALDB1 and XALDB2, 2447 and 1490 bp, respectively) using two different liver libraries. These cDNAs had very similar ORF with only one conservative amino acid substitution, but 3'-UTR of XALDB1 contained ca. 1 kb of unrelated reiterated sequence probably ligated during library construction as shown by genomic Southern blot analysis. In adult, aldolase B mRNA (ca. 1.8 kb) was expressed strongly in kidney, liver, stomach, intestine, moderately strongly in skin, and very weakly in all the other tissues including muscles and brain, which strongly express aldolase A and C mRNAs, respectively. In oocytes and early embryos, aldolase A and C mRNAs occurred abundantly as maternal mRNAs, but aldolase B mRNA occurred only at a residual level, and its strong expression started only after the late neurula stage, mainly in liver rudiment, pronephros, epidermis and proctodeum. Thus, active expression of the gene for aldolase B, involved in dietary fructose metabolism, starts only later during development (but before the feeding stage), albeit genes for aldolases A and C, involved in glycolysis, are expressed abundantly from early stages of embryogenesis, during which embryos develop depending on yolk as the only energy source.

Sequence-based structural features between Kvlqt1 and Tapa1 on mouse chromosome 7F4/F5 corresponding to the Beckwith-Wiedemann syndrome region on human 11p15.5: long-stretches of unusually well conserved intronic sequences of kvlqt1 between mouse and human.

Mouse chromosome 7F4/F5 is a syntenic locus of human 11p15.5 in which many imprinted genes are clustered. Transmission of aberrant human 11p15.5 or duplicated 11p causes Beckwith-Wiedemann syndrome (BWS) depending on which parent the chromosome is derived from. To analyze a syntenic mouse locus corresponding to human 11p15.5, mouse BAC contigs were constructed between Nap2 and Tapa1, in which 390 kb was sequenced between Kvlqt1 and Tapa1. An unexpected finding was that of highly conserved intronic sequences of Kvlqt1 between mouse and human, and their homologies came up to at least 160 kb because the length of this gene extended to 350 kb, suggesting the possibility of some functional constraint due to transcriptional and/or post-transcriptional regulation of this region. Many expressed sequence tags (ESTs) were mapped on this locus. Three genes, Lit1 (Kvlqt1-AS), Mtr1 and Tssc4, were identified and characterized. Lit1 is an antisense-transcript of Kvlqt1 and paternally expressed and maternally methylated throughout the developmental stage. The position where Lit1 exists corresponded to a highly conserved region between mouse and human. This transcript extends at least 60 kb from downstream to upstream of exon 10 in Kvlqt1. Tssc4 and Mtr1 carried putative open reading frames but neither was imprinted. Further characterization of this locus based on the sequence comparison between mouse and human will contribute valuable information towards resolving the mechanism of the occurrence of BWS and the associated childhood tumor.

An NsiI RFLP in the human long QT intronic transcript 1 (LIT1).

An NsiI polymorphic site has been found in the human long QT intronic transcript 1 (LIT1). In this transcript, we found a C-to-T transition, which was located between exons 10 and 11 of KVLQT1, and was confirmed by sequencing analysis. The allelic frequency of this polymorphism, was 0.82: 0.18 in Japanese individuals. Our novel polymorphism, combined with other polymorphisms, could be very useful in helping to determine whether the imprinting of LIT1 is disrupted in Beckwith-Wiedemann syndrome (BWS) or in human cancers.

Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional repressor, is involved in osteoclastogenesis.

The differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts. The cDNA encodes a protein with N-terminal POZ domain and C-terminal Krüppel-like zinc fingers. We designate this protein as osteoclast-derived zinc finger (OCZF). OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF). Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs). These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis.

Functional analysis of the p57KIP2 gene mutation in Beckwith-Wiedemann syndrome.

p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57KIP2 in patients with BWS that were transmitted from the patients' carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57KIP2 would have existed, which might have caused the disorders in BWS patients.

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells.

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

High frequency of fusion transcripts of exon 11 and exon 4/5 in AF-4 gene is observed in cord blood, as well as leukemic cells from infant leukemia patients with t(4;11)(q21;q23).

The MLL gene located on chromosome 11q23 and its translocation to the AF-4 gene located on chromosome 4q21 play a pivotal role in leukemogenesis in infancy. Studies of identical leukemic twins have provided evidence of the MLL rearrangement as a fetal event during pregnancy. We analyzed the presence and frequency of the MLL/AF-4 rearrangement in normal cord blood. Although no chimeric mRNA of MLL or AF-4 was detected in 65 cord blood samples, in-frame fusion transcripts of exon 11 and exon 4 or 5 of the AF-4 gene were detected in three of the samples by a nested polymerase chain reaction. When primers of exon 11 and exon 5 of the AF-4 gene were used, two forms of fusion transcripts (AF-4 exon 11/4 or exon 11/5) were detected in 20 of the 65 cord blood samples (31%) and also four of six leukemic cell samples with t(4;11) (67%), whereas such transcripts were not observed in any of 21 peripheral blood samples nor in fetal fibroblasts. These findings suggest that the in-frame fusion of exon 11 and exon 4 or 5 of the AF-4 gene frequently occurs in hematopoietic cells during the intrauterine period, even in a healthy fetus. Although it is unknown whether the proteins of the AF-4 fusion transcripts have some functions, the instability of the AF-4 gene may be associated with the leukemogenesis of infant leukemia.

Caenorhabditis elegans has two isozymic forms, CE-1 and CE-2, of fructose-1,6-bisphosphate aldolase which are encoded by different genes.

Two distinct types of cDNAs for fructose-1,6-bisphosphate (FBP) aldolase, Ce-1 and Ce-2, have been isolated from nematode Caenorhabditis elegans, and the respective recombinant aldolase isozymes, CE-1 and CE-2, have been purified and characterized. The Ce-1 and Ce-2 are 1282 and 1248 bp in total length, respectively, and both have an open reading frame of 1098 bp, which encodes 366 amino acid residues. The entire amino acid sequences deduced from Ce-1 and Ce-2 show a high degree of identity to one another and to those of vertebrate and invertebrate aldolases. The highest sequence diversity was found in the carboxyl-terminal region that corresponds to one of the isozyme group-specific sequences of vertebrate aldolase isozymes that play a role in determining isozyme-specific functions. Southern blot analysis suggests that CE-1 and CE-2 are encoded by different genes. Concerning general or kinetic properties, CE-2 is quite different from CE-1. CE-1 exhibits unique characteristics which are not identical to any aldolase isozymes previously reported, whereas CE-2 is similar to vertebrate aldolase C. These results suggest that CE-2 might preserve the properties of a progenitor aldolase with a moderate preference for FBP over fructose 1-phosphate (F1P) as a substrate, whereas CE-1 evolved to act as an intrinsic enzyme that exhibits a much broader substrate specificity than dose CE-2.

Molecular evolution of amphioxus fructose-1,6-bisphosphate aldolase.

The cDNA for amphioxus fructose-1,6-bisphosphate (FBP)-aldolase was isolated and its nucleotide sequence was determined. In the cDNA, there existed a probable open reading frame comprising 1080 bp; hence, 359 amino acid residues were deduced. The amino acid sequence indicates the deletion of 4 residues from N-terminus, in comparison with the sequence of FBP-aldolase isozymes from other sources. There was only one FBP-aldolase gene, and one enzyme species corresponding, in the amphioxus; this is the first report of the existence of a single FBP-aldolase species in animals. Enzymatic studies of both native and the recombinant FBP-aldolase suggest that the amphioxus enzyme belongs to an ancestral class I type which is not discovered among vertebrate aldolase isozymes.

Copy-dependent and position-independent expression of rat aldolase A gene.

In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.

The plastid aldolase gene from Chlamydomonas reinhardtii: intron/exon organization, evolution, and promoter structure.

Genomic clones encoding the plastidic fructose-1,6-bisphosphate aldolase of Chlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of the C. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5' border sequences of introns in the aldolase gene of C. reinhardtii exhibit the conserved plant consensus sequence. The 3' acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in the C. reinhardtii genome.

Structures of cDNAs encoding the muscle-type and non-muscle-type isozymes of lamprey fructose bisphosphate aldolases and the evolution of aldolase genes.

Nearly full-length cDNA clones for muscle-type and non-muscle-type aldolase mRNAs were cloned from lambda gt10 cDNA libraries constructed from skeletal muscle and liver mRNAs of lamprey (Entosphenus japonicus). The cDNA-M8 has 2,240 bp carrying an open reading frame of 1,089 bp which encodes 362 amino acids without the amino terminal methionine, while the cDNA-L3 is 1,761 bp in length and has an open reading frame of 1,092 bp, which encodes 363 amino acids without the methionine. We designated the cDNA clones M8 and L3 as the muscle-type and non-muscle-type aldolase cDNAs, respectively. The entire amino acid sequences deduced from cDNA-M8 and -L3 show a high degree of identity to one another (76%) and also to vertebrate aldolases A (74-76%), B (68-70%), and C (71-76%) and Drosophila melanogaster aldolases alpha, beta, and gamma (66-67%). Northern blot analyses using the 3'-noncoding sequences of cDNA-M8 and -L3 as hybridization probes indicated that the muscle-type mRNA is expressed mainly in the skeletal muscle, heart muscle, brain, and some other tissues, but probably not in liver, while the non-muscle-type mRNA is expressed mainly in the liver and also in brain and other tissues, except for the heart muscle. Phylogenetic analyses showed that both muscle-type and non-muscle-type aldolases of lamprey resemble one another and might share a common ancestor with vertebrate aldolases A and C, but they are not direct ancestors of vertebrate aldolases.

Expression and sequence of the only detectable aldolase in Chlamydomonas reinhardtii.

Only one aldolase can be resolved from Chlamydomonas reinhardtii by anion-exchange chromatography on DEAE-cellulose. Western blots with specific antisera against plastid and cytosol aldolase of spinach and Northern blots with the respective cDNAs from spinach indicate that only one aldolase, the plastid enzyme, is expressed. Full-length cDNA clones for the plastidic aldolase were isolated from cDNA library constructed from poly(A)+ mRNA of C. reinhardtii with plastid-aldolase-specific probes from spinach. The clones contained a 1.7-kb insert with an open reading frame for 374 amino acid residues covering the mature chloroplast protein of 347 amino acids and a N-terminal transit peptide of 27 amino acids for chloroplast import. The calculated molecular mass of the mature protein is 37.6 kDa and that of the precursor protein is 40.3 kDa. The aldolase of C. reinhardtii shows amino acid similarity of 62 to 67% with the chloroplast aldolase and of 47 to 52% with the cytosolic enzymes of higher plants, respectively. An evolutionary tree with all known class I aldolases shows separate clusters for the chloroplast and for the cytosol aldolases in higher plants and green algae. The aldolase of C. reinhardtii connects at a basal position with the chloroplast aldolases of higher plants.

Cloning of a brain-type aldolase cDNA and changes in its mRNA level during oogenesis and early embryogenesis in Xenopus laevis.

A full length cDNA clone (cXALD3) for Xenopus laevis aldolase mRNA, which exists abundantly in oocytes, was isolated from Xenopus laevis ovary cDNA library, and its nucleotide sequence was determined. The cDNA was 1.8 kb in length and encoded 363 amino acids. From the deduced amino acid sequence and the Northern blot analysis of the RNAs from several adult tissues, this clone was concluded to be a brain-type aldolase gene. The XALD3 mRNA level per egg or embryo was high during early oogenesis, but was markedly reduced during late oogenesis and was maintained at low level during early embryogenesis until it started to increase at the late neurula stage. The mRNA was also detected in testis. The characteristic change in the temporal pattern of expression and the distribution of XALD3 mRNA among different tissues suggest a possibility that brain type aldolase may play some important roles in gametogenesis and in neurulation.

A novel human homologue of a dead-box RNA helicase family.

Putative cDNA clones for a nuclear antigen that cross-reacts with anti-human aldolase A monoclonal antibody MAb1A2 were isolated from the HeLa lambda gt11 cDNA library and a candidate clone (clone 3) was analyzed. The cDNA has an open reading frame (ORF) of 1,317 bp encoding a novel RNA helicase belonging to the DEAD RNA helicase family. The ORF also contains a nuclear targeting signal and the epitope for MAb1A2. The putative RNA helicase has sequence similarity to Escherichia coli RNA helicase DEAD, mouse translation factor eIF-4A, and human p68 and p54.