A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy

The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter. We have identified a recombination event within this interval on a type-I SMA chromosome. The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613. While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely. This new proximal boundary would reduce the minimal region harboring the SMA locus from approximately 1.1 Mb to approximately 600 kb.     

Refined linkage map of chromosome 5 in the region of the spinal muscular atrophy gene

The genetic map in the region of human chromosome 5 that harbors the gene for autosomal recessive forms of spinal muscular atrophy (SMA) has been refined by a multilocus linkage study in 50 SMA-segregating families. Among six markers spanning 8 cM for combined sexes, four were shown to be tightly linked to the SMA locus. Multipoint linkage analysis was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for the SMA locus is between blocks AFM114ye7 (D5S465)/EF5.15 (D5S125) and MAP-1B/JK53 (D5S112) at a sex-combined genetic distance of 2.4 and 1.7 cM, respectively. Thus the SMA gene lies in the 4-cM region between these two blocks. This information is of primary importance for designing strategies for isolating the SMA gene.     

Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region

The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene.     

Rearrangement of the human CDC5L gene by a t(6;19)(p21;q13.1) in a patient with multicystic renal dysplasia

Alpha-2-adrenergic receptor (alpha2-AR) agonists potently inhibit the activity of noradrenergic neurons of the locus coeruleus (LC), an effect that may be mediated by the A- and/ or C-subtypes of alpha2-AR (alpha2A- and alpha2C-AR). To gain insight into the functional significance of these alpha2-AR subtypes in the LC, we have examined their ultrastructural localization by using subtype-specific antibodies. We recently demonstrated that alpha2A-ARs are localized prominently in axon terminals and catecholaminergic dendrites in the LC. In the present study, we sought to identify the subcellular substrates underlying alpha2C-AR actions in the LC by analyzing the ultrastructural distribution of alpha2C-AR immunoreactivity (alpha2C-AR-IR) in sections that were dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Alpha-2C-AR-IR was predominantly localized in dendrites, most of which also contained immunolabeling for TH. Within such dendrites, alpha2C-AR-IR was associated with the plasma membrane and occasionally Golgi cisternae and tubulovesicles. The vast majority of dendrites containing alpha2C-AR-IR received asymmetric (excitatory) contacts from unlabeled axon terminals that often contained dense core vesicles. Alpha-2C-AR-IR was observed in some unmyelinated axons and astrocytic processes that were apposed to TH-immunoreactive dendrites but was rarely associated with axon terminals. These results provide the first ultrastructural evidence that alpha2C-ARs (1) are localized postsynaptically in catecholaminergic neurons of the LC and (2) may be strategically situated to modulate the activation of LC neurons by excitatory inputs.     

Familial eosinophilia maps to the cytokine gene cluster on human chromosomal region 5q31-q33

Familial eosinophilia (FE) is an autosomal dominant disorder characterized by peripheral hypereosinophilia of unidentifiable cause with or without other organ involvement. To localize the gene for FE, we performed a genomewide search in a large U.S. kindred, using 312 different polymorphic markers. Seventeen affected subjects, 28 unaffected bloodline relatives, and 8 spouses were genotyped. The initial linkage results from the genome scan provided evidence for linkage on chromosome 5q31-q33. Additional genotyping of genetic markers located in this specific region demonstrated significant evidence that the FE locus is situated between the chromosome 5q markers D5S642 and D5S816 (multipoint LOD score of 6.49). Notably, this region contains the cytokine gene cluster, which includes three genes-namely, those for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)-whose products play important roles in the development and proliferation of eosinophils. These three cytokine genes were screened for potential disease-specific mutations by resequencing of a subgroup of individuals from the present kindred. No functional sequence polymorphisms were found within the promoter, the exons, or the introns of any of these genes or within the IL-3/GM-CSF enhancer, suggesting that the primary defect in FE is not caused by a mutation in any one of these genes but, rather, is caused by another gene in the area.     

A new non-random chromosomal translocation t(3;6)(q27;p21.3) associated with BCL6 rearrangement in two patients with non-Hodgkin's lymphoma

We describe two cases of non-Hodgkin's lymphoma associated with t(3;6)(q27;p21.3) and BCL6 rearrangement. The first case was in a 78-year old woman, whose performance status (PS) was 1, the serum lactate dehydrogenase (LDH) level was elevated, and the Ann Arbor stage was IIIA with no extra nodal lymphomatous site. The pathological diagnosis from a biopsy of the inguinal lymph node was 'malignant lymphoma (ML), follicular, small cleaved' according to the Working Formulation. Complete remission was achieved. Although she had relapse in 1992, remission was obtained again. The second case was in a 62-year old man, whose PS was 1, the serum LDH was normal, and Ann Arbor stage was IVA with the involvement of the small intestine. Histological diagnosis of the cervical lymph node was 'ML, diffuse, large cell'. Complete remission was obtained without relapse. The 3q27 translocations, found in 20-30% of non-Hodgkin's lymphoma, are unique in having multiple chromosomal translocation partners. Chromosome band 6p21.3 is one of these partner sites that may be the site of a novel gene. The two cases presented here show that this translocation is a non-random chromosomal change involving 3q27 and BCL6. Since t(3;6) was the sole karyotypic abnormality in one case, this translocation may play a role in lymphomagenesis.     

Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p

An autosomal dominant distal form of spinal muscular atrophy mainly affecting the upper limbs with a mean age of onset of 17 years has been identified in a large Bulgarian family. Linkage of the above family to the spinal muscular atrophy type I, II and III locus on chromosome 5 has been excluded. In an attempt to map this disease gene we have analysed individuals of this family, with more than 140 microsatellite polymorphic markers of the human genome. A maximum lod score of 5.99 at theta = 0.007 has been obtained with locus D7S795. We have thus mapped the gene for this hereditary form of distal spinal muscular atrophy to chromosome 7p.     

YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.     

Cloning and expression of primate Daxx cDNAs and mapping of the human gene to chromosome 6p21.3 in the MHC region

Murine Daxx, a protein that binds to Fas and enhances Fas-mediated apoptosis through a signal transduction pathway involving the Jun amino-terminal kinase, was recently described. Here we report the cloning of human and monkey Daxx cDNAs, the widespread expression of Daxx mRNA in human tissues, and the mapping of the human Daxx gene to 6p21.3 in the major histocompatibility complex (MHC) region. The location of the Daxx gene, which is implicated in the pathway for deletion of autoreactive lymphocytes, in the MHC region may shed light on the genetic basis of autoimmune diseases.     

The gnd gene encoding a novel 6-phosphogluconate dehydrogenase and its adjacent region of Actinobacillus actinomycetemcomitans chromosomal DNA

Integrin alpha IIb beta 3 is necessary for platelet thrombus formation by virtue of its ability to bind fibrinogen and von Willebrand factor in a regulated fashion and to mediate platelet aggregation. Moreover, alpha IIb beta 3 transduces inward signals in response to ligand binding which promote cytoskeletal reorganization and other post-ligand binding events. Anchorage and signaling through alpha IIb beta 3 appear to be controlled by interactions of intracellular proteins with the cytoplasmic tails of alpha IIb and/or beta 3. The cytoplasmic tails and associated cytoskeletal components may function as a scaffold or nucleation site for the assembly of enzymes, substrates and adaptor molecules into a signaling complex. Identification of the components of this signaling complex and characterization of their dynamic interactions should lead to insights into the molecular basis of platelet activation defects and may provide a rationale for the development of new anti-thrombotic drugs.     

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.     

Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.     

Analysis of a viral integration event in a CG-rich region at the 1p36 human chromosomal site

The preinsertion site of an adenovirus-5/simian virus 40 recombinant construct (Ad5/SV40) has been cloned and sequenced. Our data suggest that viral integration has occurred in a genomic region which has been the target of multiple events of Alu element retropositions within a TAA minisatellite. Extensive homologies between the left viral end and the host cellular DNA were also observed. The compositional similarity between Adenoviridae and the region of viral integration is consistent with the observed insertion of exogenous DNA in isochores of similar composition [G. Bernardi, Annu. Rev. Genet. 23 (1989) 637-661].     

The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins

Spinal muscular atrophy (SMA), one of the most common fatal autosomal recessive diseases, is characterized by degeneration of motor neurons and muscular atrophy. The SMA disease gene, termed Survival of Motor Neurons (SMN), is deleted or mutated in over 98% of SMA patients. The function of the SMN protein is unknown. We found that SMN is tightly associated with a novel protein, SIP1, and together they form a specific complex with several spliceosomal snRNP proteins. SMN interacts directly with several of the snRNP Sm core proteins, including B, D1-3, and E. Interestingly, SIP1 has significant sequence similarity with Brr1, a yeast protein critical for snRNP biogenesis. These findings suggest a role for SMN and SIP1 in spliceosomal snRNP biogenesis and function and provide a likely molecular mechanism for the cause of SMA.     

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes-survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.     

The gene encoding the catalytic subunit C alpha of cAMP-dependent protein kinase (locus PRKACA) localizes to human chromosome region 19p13.1

OBJECTIVE: The importance of portal hypertensive gastropathy, as a potentially bleeding lesion in cirrhotics with portal hypertension, has recently been appreciated. Histologically, dilation of the mucosal and submucosal vessels of the stomach is noted in this entity. The possibility of nitric oxide acting as a mediator for this mucosal vascular dilation has not been explored. METHODS: We determined, in a group of 10 male cirrhotic patients with esophageal varices and endoscopic changes consistent with severe portal hypertensive gastropathy (Group A), the gastric mucosal nitric oxide synthase activity. This was determined by measuring the rate of conversion of [3H]-arginine to [3H]-citrulline. Serum levels of nitrates and nitrites, the end products of nitric oxide, were also measured. The results were compared with those of a group of 10 male controls with no liver disease (Group B). RESULTS: Gastric mucosal constitutive and inducible nitric oxide synthase levels were significantly higher in group A (125.4 +/- 4.3 and 259.7 +/- 5.5 pmol/mg protein/minute, respectively) than in group B (88 +/- 8.6 and 130.8 +/- 6.6 pmol/mg protein/minute, respectively) ( p < 0.002 and < 0.0001, respectively). Serum nitrate/nitrite levels were 30.1 +/- 3.2 nmol/ml in group A and 15.5 +/- 0.09 nmol/ml in group B (p < 0.001). CONCLUSIONS: We conclude that the significantly increased gastric mucosal nitric oxide synthase activity, in patients with portal hypertensive gastropathy, suggests an important role for nitric oxide in the pathogenesis of this mucosal lesion.     

The human skeletal muscle adenine nucleotide translocator gene maps to chromosome 4q35 in the region of the facioscapulohumeral muscular dystrophy locus

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed mutagenesis in order to investigate the importance of elements that may influence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the viral RNA (the 3' PPT). An exact copy of this PPT is located in the middle of the genome (the internal PPT). Upstream from the internal PPT there is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was, however, less harmful than changing the polypurine nature of the PPT. Changing a conserved T on the 3' side of the PPT to a C did not affect the phenotype.