Direct detection of novel expanded trinucleotide repeats in the human genome.

Expansion of trinucleotide repeats can give rise to genetic disease. We have developed a technique, repeat expansion detection (RED), that can identify potentially pathological repeat expansion without prior knowledge of chromosomal location. Human genomic DNA is used as a template for a two-step cycling process that generates oligonucleotide multimers when expanded trinucleotide sequences are present at the level found in myotonic dystrophy and fragile-X patients. We have identified at least one new locus exhibiting trinucleotide expansion. Analysis of three families transmitting a long CTG repeat shows that the allele in these families corresponds to a locus on chromosome 18. RED constitutes a powerful tool to identify other diseases caused by this mechanism, particularly diseases associated with anticipation.

Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome.

We have performed mRNA in situ hybridization studies and northern blot analysis in the mouse and human, respectively, to determine the normal gene expression patterns of FMR-1. Expression in the adult mouse was localized to several regions of the brain and the tubules of the testes, which are two of the major organs affected in fragile X syndrome. Universal and very strong expression was observed in early mouse embryos, with differentially decreasing expression during subsequent stages of embryonic development. The early embryonic onset and tissue specificity of FMR-1 gene expression is consistent with involvement in the fragile X phenotype, and also suggests additional organ systems in which clinical manifestations of reduced FMR-1 gene expression may occur.

Mutations in the neurofibromatosis 1 gene in sporadic malignant melanoma cell lines.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by progressive and variable involvement of tissues predominantly derived from the neural crest and a predisposition toward malignancies. The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of down-regulating ras by stimulating its intrinsic GTPase activity. We report a homozygous deletion of most of NF1 in one of eight malignant melanoma cell lines leading to loss of detectable mRNA and protein, as well as the apparent absence of protein and mRNA in another melanoma. This data suggests that NF1 can function as a tumour suppressor gene in the development or progression of malignant melanoma.

The peripheral myelin gene PMP-22/GAS-3 is duplicated in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a DNA duplication at chromosome 17p11.2. In view of the point mutation in the gene for peripheral myelin protein pmp-22/gas-3 in Trembler mice, a murine model for CMT1A, we have analysed whether this gene is altered in CMT1A. Here we show that the human homologue of the murine pmp-22 gene is located within the CMT1A DNA duplication, which is a direct repeat and does not interrupt the coding region of PMP-22. Expression of PMP-22 in CMT1A fibroblasts is similar to expression in control fibroblasts. Increased gene dosage or altered PMP-22 expression in the peripheral nervous system are therefore possible mechanisms by which PMP-22 is involved in CMT1A.

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98-HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein.

The recurrent translocation t(8;16)(p11;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ-CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ-CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

The complex pathology of trinucleotide repeats.

The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.

Human erythroid burst-forming unit: T-cell requirement for proliferation in vitro.

Human mononuclear leukocytes were fractionated into populations of null, T and B cells by immunoabsorbent column chromatography followed by E-rosette formation and purification of T cells by differential centrifugation and osmotic lysis. The unfractionated and fractionated cell populations were first separately cultured for 14 days in plasma clots in the presence of two international units erythropoietin. Typical erythroid burst-forming unit (BFU-E)-derived colonies grew in the unfractionated cell cultures but not from T- or B-cell cultures. BFU-E colonies grew in null cell cultures but most of the colonies were small and variably hemoglobinized with less than three subcolonies. When intact T cells were added to null cells and cocultured, many typical large BFU-E colonies with more than 10 well homogenized subcolonies appeared. Increasing numbers of large BFU-E colonies in null cell cultures were induced by stepwise addition of T cells but not by the addition of B cells. A conditioned medium in which T cells had been induced to divide by tetanus toxoid substituted for intact T cells in this T-cell-dependent BFU-E colony formation observed in null cells. These findings demonstrate that the BFU-E, a committeded erythroid stem cell, resides in the null cell fraction of peripheral blood, but its proliferative capacity and differentiation in vitro requires a soluble product of T cells. Such experiments now permit a new approach to the assessment of various disorders of erythropoiesis. Erythroid hypoplasia in a particular case may be due to dysfunction of the committed precursor cell or to a failure of a helper effect induced by T cells.

Clustering of cytokine genes on mouse chromosome 11.

The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17.

Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit.

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.

p53 status and the efficacy of cancer therapy in vivo.

The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53, can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.

Site-specific cleavage of human chromosome 4 mediated by triple-helix formation.

Direct physical isolation of specific DNA segments from the human genome is a necessary goal in human genetics. For testing whether triple-helix mediated enzymatic cleavage can liberate a specific segment of a human chromosome, the tip of human chromosome 4, which contains the entire candidate region for the Huntington's disease gene, was chosen as a target. A 16-base pyrimidine oligodeoxyribonucleotide was able to locate a 16-base pair purine target site within more than 10 gigabase pairs of genomic DNA and mediate the exact enzymatic cleavage at that site in more than 80 percent yield. The recognition motif is sufficiently generalizable that most cosmids should contain a sequence targetable by triple-helix formation. This method may facilitate the orchestrated dissection of human chromosomes from normal and affected individuals into megabase sized fragments and facilitate the isolation of candidate gene loci.

Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor.

Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter. Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions, and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.

A family of cAMP-binding proteins that directly activate Rap1.

cAMP (3',5' cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.

Repeat instability in the 27-39 CAG range of the HD gene in the Venezuelan kindreds: Counseling implications.

The instability of the CAG repeat size of the HD gene when transmitted intergenerationally has critical implications for genetic counseling practices. In particular, CAG repeats between 27 and 35 have been the subject of debate based on small samples. To address this issue, we analyzed allelic instability in the Venezuelan HD kindreds, the largest and most informative families ascertained for HD. We identified 647 transmissions. Our results indicate that repeats in the 27-35 CAG range are highly stable. Out of 69 transmitted alleles in this range, none expand into any penetrant ranges. Contrastingly, 14% of alleles transmitted from the incompletely penetrant range (36-39 CAGs) expand into the completely penetrant range, characterized by alleles with 40 or more CAG repeats. At least 12 of the 534 transmissions from the completely penetrant range contract into the incompletely penetrant range of 36-39 CAG repeats. In these kindreds, none of the individuals with 27-39 CAGs were symptomatic, even though they ranged in age from 11 to 82 years. We expect these findings to be helpful in updating genetic counseling practices.

Replication of twelve association studies for Huntington's disease residual age of onset in large Venezuelan kindreds.

BACKGROUND: The major determinant of age of onset in Huntington's disease is the length of the causative triplet CAG repeat. Significant variance remains, however, in residual age of onset even after repeat length is factored out. Many genetic polymorphisms have previously shown evidence of association with age of onset of Huntington's disease in several different populations. OBJECTIVE: To replicate these genetic association tests in 443 affected people from a large set of kindreds from Venezuela. METHODS: Previously tested polymorphisms were analysed in the HD gene itself (HD), the GluR6 kainate glutamate receptor (GRIK2), apolipoprotein E (APOE), the transcriptional coactivator CA150 (TCERG1), the ubiquitin carboxy-terminal hydrolase L1 (UCHL1), p53 (TP53), caspase-activated DNase (DFFB), and the NR2A and NR2B glutamate receptor subunits (GRIN2A, GRIN2B). RESULTS: The GRIN2A single-nucleotide polymorphism explains a small but considerable amount of additional variance in residual age of onset in our sample. The TCERG1 microsatellite shows a trend towards association but does not reach statistical significance, perhaps because of the uninformative nature of the polymorphism caused by extreme allele frequencies. We did not replicate the genetic association of any of the other genes. CONCLUSIONS: GRIN2A and TCERG1 may show true association with residual age of onset for Huntington's disease. The most surprising negative result is for the GRIK2 (TAA)(n) polymorphism, which has previously shown association with age of onset in four independent populations with Huntington's disease. The lack of association in the Venezuelan kindreds may be due to the extremely low frequency of the key (TAA)(16) allele in this population.

Erythroid precursors in congenital hypoplastic (Diamond-Blackfan) anemia.

To explore the etiology of congenital hypoplastic anemia (CHA) or the Diamond-Blackfan anemia, erythropoietin responsive committed erythroid precursors were enumerated by the plasma clot method. These included blood and marrow erythroid burst-forming units (BFU-E) and marrow erythroid colony-forming units (CFU-E). The peripheral blood nucleated cells of 11 patients and the marrow cells of seven of these patients were examined. Studies were repeated in several patients during relapse and after induction of remission. BFU-E were undetectable in the marrow and blood of all but one relapsed patient, and the numbers of marrow CFU-E were depressed in all relapsed patients. Blood BFU-E remained low in all of the patients in remission. No evidence was obtained for suppression of normal CFU-E or BFU-E by CHA lymphocytes. Erythropoietin dose-response curves performed in two patients revealed a 10-fold increase in erythropoietin requirement for marrow CFU-E colony growth. This marked unresponsiveness to erythropoietin was strikingly improved by steroid therapy in one patient. We suggest that CHA is the result of a qualitative and/or quantitative deficiency of BFU-E. If BFU-E are produced, they must be relatively unresponsive to erythropoietin. The abnormal BFU-E give rise to erythropoietin unresponsive CFU-E and, thence, to proerythroblasts that are, in turn, trapped in that early stage of development because of their poor erythropoietic response. Hence, red cell production is deficient. Steroids appear to improve the erythropoietin response of CHA erythroid precursors.

Determinants that contribute to cytoplasmic stability of human c-fos and beta-globin mRNAs are located at several sites in each mRNA.

We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.

Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2.

To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene.