Mapping of the bovine spinal muscular atrophy locus to Chromosome 24

A hereditary form of spinal muscular atrophy (SMA) caused by an autosomal recessive gene has been reported for American Brown-Swiss cattle and in advanced backcrosses between American Brown-Swiss and many European brown cattle breeds. Bovine SMA (bovSMA) bears remarkable resemblance to the human SMA (SMA1). Affected homozygous calves also show progressive symmetric weakness and neurogenic atrophy of proximal muscles. The condition is characterized by severe muscle atrophy, quadriparesis, and sternal recumbency as result of neurogenic atrophy. We report on the localization of the gene causing bovSMA within a genomic interval between the microsatellite marker URB031 and the telomeric end of bovine Chromosome (Chr) 24 (BTA24). Linkage analysis of a complex pedigree of German Braunvieh cattle revealed a recombination fraction of 0.06 and a three-point lod score of 11.82. The results of linkage and haplotyping analysis enable a marker-assisted selection against bovSMA based on four microsatellite markers most telomeric on BTA24 to a moderate accuracy of 89-94%. So far, this region is not orthologous to any human chromosome segments responsible for twelve distinct disease phenotypes of autosomal neuropathies. Our results indicate the apoptosis-inhibiting protein BCL2 as the most promising positional candidate gene causing bovSMA. Our findings offer an attractive animal model for a better understanding of human forms of SMA and for a probable anti-apoptotic synergy of SMN-BCL2 aggregates in mammals.     

Different entities of proximal spinal muscular atrophy within one family

 The molecular analysis of the survival motor neuron (SMN) gene and several closely flanking polymorphic markers in an atypical pedigree with four patients suffering from spinal muscular atrophy (SMA) over two generations has raised new aspects concerning the etiology and the molecular spectrum of autosomal recessive SMA. Three patients in two generations show homozygous deletions of exons 7 and 8 of the telomeric copy of SMN (telSMN), thus confirming the presence of autosomal recessive SMA, with localisation on chromosome 5q12. The fourth SMA patient with mild neurogenic atrophy (confirmed by muscle biopsy and electromyography) shows no homozygous deletion of telSMN but carries a heterozygous deletion of telSMN, as can be deduced from her two affected homozygously deleted children. No intragenic mutation has been identified in the remaining telSMN. In addition, she shares only one SMA chromosome with her affected brother, is haploidentical with two healthy brothers, and has a 31-year-old healthy son, who has inherited an SMN-deleted paternal chromosome and the SMN non-deleted maternal chromosome. These results suggest that this patient either has a neurogenic atrophy of a different origin or exhibits an unusual heterozygous manifestation of SMA 5q12. Interestingly, the two haploidentical telSMN-deleted affected sibs in the second generation show a strikingly discordant clinical picture indicating that, in addition to telSMN mutations, other factors influence the phenotype of SMA in the reported pedigree. Received: 20 March 1997 / Accepted: 4 June 1997     

Linkage analysis in spinal muscular atrophy, by six closely flanking markers on chromosome 5

The proximal spinal muscular atrophies (SMA) represent the second most common autosomal recessive disorder, after cystic fibrosis. The gene responsible for chronic SMA has recently been mapped to chromosome 5q by using genetic linkage studies. Among six markers mapping to this region, five were shown to be linked with the SMA locus in 39 chronic SMA families each containing at least two affected individuals. Multilocus analysis by the method of location score was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between loci D5S6 and D5S39. The genetic distances between these two markers are estimated to be 6.4 cM in males and 11.9 cM in females. Since meiosis were informative with D5S39 and D5S6 in 92% and 87% of SMA families, respectively, it is hoped that the present study will contribute to the calculation of genetic risk in SMA families.     

Fine-mapping and candidate gene analysis of bovine spinal muscular atrophy

Bovine spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease, has been mapped at moderate resolution to the distal part of Chromosome 24. In this article we confirm this location and fine-map the SMA locus to an interval of approximately 0.8 cM at the very distal end of BTA24. Despite remarkable similarity to human SMA, the causative gene SMN can be excluded in bovine SMA. However, the interval where the disease now has been mapped contains BCL2, like SMN an antiapoptotic factor, and shown to bind to SMN. Moreover, knockout mice lacking the BCL2 gene show rapid motor neuron degeneration with early postnatal onset, as observed in bovine SMA. A comparative cattle/human map of the distal end of BTA24, based on the emerging bovine genome sequencing data, shows conserved synteny to HSA18 with hints of a segmental duplication and pericentic inversion just after the last available bovine marker DIK4971. This synteny lets us conclude that SMA is in immediate vicinity of the telomere. Candidate gene analysis of BCL2, however, excludes most of this gene, except its promoter region, and draws attention to the neighboring gene VPS4B, part of the endosomal protein-sorting machinery ESCRT-III which is involved in several neurodegenerative diseases. Stefan Krebs and Ivica Medugorac contributed equally to this work and agreed to be considered as first authors.     

In situ hybridization of two markers closely flanking the spinal muscular atrophy gene to 5q12----q13.3

In order to refine the physical location of the p105-153Ra and M4 probes which closely flank the spinal muscular atrophy gene (SMA) on human chromosome 5q, in situ hybridization has been carried out on prometaphase chromosomes. Our results demonstrate that the disease gene is located between the 5q12----q13.1 and 5q13.3 bands. The present study will hopefully contribute to microdissection of the chromosomal region of the SMA gene.     

Genome-wide expression analysis of a spinal muscular atrophy model: towards discovery of new drug targets

Spinal Muscular Atrophy is a recessive genetic disease and affects lower motor neurones and muscle tissue. A single gene is disrupted in SMA: SMN1 activity is abolished but a second copy of the gene (SMN2) provides limited activity. While the SMN protein has been shown to function in the assembly of RNA-protein complexes, it is unclear how the overall reduction in SMN activity specifically results in the neuromuscular phenotypes. Similar to humans, reduced smn activity in the fly causes earliest phenotypes in neuromuscular tissues. To uncover the effects of reduced SMN activity, we have studied gene expression in control and diseased fly tissues using whole genome micro-arrays. A number of gene expression changes are recovered and independently validated. Identified genes show trends in their predicted function: several are consistent with the function of SMN, in addition some uncover novel pathways. This and subsequent genetic analysis in the fly indicates some of the identified genes could be taken for further studies as potential drug targets for SMA and other neuromuscular disorders.     

Fine-mapping of the spinal muscular atrophy locus to a region flanked by MAP1B and D5S6.

The microtubule-associated protein 1B (MAP1B) locus has been mapped in close proximity to spinal muscular atrophy (SMA) on chromosome 5q13. We have identified a second microsatellite within a MAP1B intron, which increases the heterozygosity of this locus to 94%. Two unambiguous recombination events establish MAP1B as a closely linked, distal flanking marker for the disease locus, while a third recombinant establishes D5S6 as the proximal flanking marker. The combination of key recombinants and linkage analysis place the SMA gene in an approximately 2-cM interval between loci D5S6 and MAP1B. Physical mapping and cloning locate MAP1B within 250 kb of locus D5S112. The identification and characterization of a highly polymorphic gene locus tightly linked to SMA will facilitate isolation of the disease gene, evaluation of heterogeneity, and development of a prenatal test for SMA.     

Deletions in the SMN gene in infantile and adult spinal muscular atrophy patients from the same family

Recently, a gene determining spinal muscular atrophy (SMA), termed survival motor neuron (SMN) gene, has been isolated from the 5g13 region. This gene has been found to be deleted in most patients with childhood-onset SMA. We have studied the SMN gene in a clinically heterogeneous family, including one patient affected by infantile chronic SMA and three subjects with mild adult-onset muscle weakness. Deletions in the SMN gene were detected in all of these patients, indicating that the childhood and adult SMAs are genetically homogeneous in this family. Genotyping of the family members established that the three mildly affected individuals were homozygous for the same haplotype from the SMA region, whereas the more severely affected patient was heterozygous with one different haplotype.     

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 1.1 Mb to approximately 600 kb.     

Deletion analysis of the SMN and NAIP genes in Kuwaiti patients with spinal muscular atrophy

Two genes are known to be involved in spinal muscular atrophy (SMA), namely, SMN (survival motor neuron) and NAIP (neuronal apoptosis inhibitory protein). Deletion analysis of these genes has been reported for many ethnic groups. We have extended this analysis to include 15 Arabic patients (11 unrelated cases of type I, which represent practically all of the patients diagnosed within the last 2 years in Kuwait, and 4 type-II cases from a single kinship). Also, 41 healthy relatives (parents and sibs) and 44 control individuals of Arabic origin were analyzed. The homozygous deletions of exons 7 and 8 of the SMN gene were found in all SMA patients studied. Exon 5 of NAIP was homozygously absent in all type-I patients, but was retained in type-II cases. Among members of SMA families, one mother was found to be homozygously deleted for NAIP. All of the control individuals had both normal SMN and NAIP. Our results are in agreement with the general consensus that the incidence of NAIP deletion is higher in the more severe SMA cases. Furthermore, they suggest that SMA type-I chromosomes, with the dual deletion of the SMN and NAIP genes, are more common in Arabs than in patients of other ethnic origin. Received: 23 April 1996 / Revised: 17 June 1996     

Apparent gene conversions involving the SMN gene in the region of the spinal muscular atrophy locus on chromosome 5.

The survival motor neuron (SMN) gene has been described as a determining gene for spinal muscular atrophy (SMA). SMN has a closely flanking, nearly identical copy (cBCD541). Gene and copy gene can be discriminated by sequence differences in exons 7 and 8. The large majority of SMA patients show homozygous deletions of at least exons 7 and 8 of the SMN gene. A minority of patients show absence of SMN exon 7 but retention of exon 8. This is explained by results of our present analysis of 13 such patients providing evidence for apparent gene-conversion events between SMN and the centromeric copy gene. Instead of applying a separate analysis for absence or presence of SMN exons 7 and 8, we used a contiguous PCR from intron 6 to exon 8. In every case we found a chimeric gene with a fusion of exon 7 of the copy gene and exon 8 of SMN and absence of a normal SMN gene. Similar events, including the fusion counterpart, were observed in a group of controls, although in the presence of a normal SMN gene. Chimeric genes as the result of fusions of parts of SMN and cBCD541 apparently are far from rare and may partly explain the frequently observed SMN deletions in SMA patients.     

Synaptic defects in the spinal and neuromuscular circuitry in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ～28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3-5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy.     

Analysis of the SMN and NAIP genes in slovak spinal muscular atrophy patients

We identified homozygous absence of exon 7 of the telomeric copy of the survival motor neuron gene (telSMN) in 88.4% (38/43) of spinal muscular atrophy (SMA) patients from Slovakia. Additional deletions within the neuronal apoptosis inhibitory protein (NAIP) gene were found in 38.5% of type I, 12.5% of type II and never in type III SMA patients. Neither the SMN nor the NAIP gene was deleted in 81 healthy relatives and 25 controls tested. In one family, pseudodominant inheritance was identified. Both the type III SMA father and type II SMA son carried the homozygous deletion of the telSMN gene. One SMA I patient showed an SMN hybrid gene, probably created by intrachromosomal deletion. In two haploidentical type II SMA sibs, the telSMN exon 7 was absent on one chromosome, while the other carried an A-->G transition 96 bp upstream of exon 7 of the telSMN gene, a potential disease-causing mutation in these patients.     

A new genus and two new species of Pennellidae (Copepoda: Siphonostomatoida) and an analysis of evolution within the family

Summary A new pennellid genus,Exopenna, is established on the basis of two specimens of a new species,E. crimmeni, recovered from a deep-sea cod,Antimora, caught in the North East Atlantic. It is ectoparasitic and is diagnosed by the combination of a straight body, tightly coiled egg sacs and flattened, plate-like antennary processes. Another new species,Peniculus elongatus, is described from Australian waters and new records ofP. fistula andLernaeenicus ramosus further increase the known pennellid fauna of that region. A preliminary analysis of the phylogenetic relationships of the pennellid genera is undertaken and a partially resolved cladogram produced which identifies the main generic groupings. ac]19850522     

Microarray analysis of gene expression by skeletal muscle of three mouse models of Kennedy disease/spinal bulbar muscular atrophy

Background

Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR.

Methodology/Principal Findings

We performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntington''s Disease, and to those common to muscle atrophy from diverse causes.

Conclusions/Significance

By comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.     

Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein

Cytoplasmic assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) is a central process in eukaryotic gene expression. A large macromolecular complex containing the survival of motor neurons (SMN) protein is required for proper snRNP assembly in vivo. Defects in SMN function lead to a human neuromuscular disorder, spinal muscular atrophy (SMA). SMN protein localizes to both nuclear and cytoplasmic compartments, and a reduction in nuclear levels of SMN is correlated with the disease. The mechanism of SMN nuclear import, however, is unknown. Using digitonin-permeabilized cells, we show that SMN import depends on the presence of Sm snRNPs. Conversely, import of labeled U1 snRNPs was SMN complex dependent. Thus, import of SMN and U snRNPs are coupled in vitro. Furthermore, we identify nuclear import defects in SMA patient-derived SMN mutants, uncovering a potential mechanism for SMN dysfunction.     

Proximal spinal muscular atrophy (SMA) types II and III in the same sibship are not caused by different alleles at the SMA locus on 5q.

Proximal spinal muscular atrophy (SMA) is a group of progressive muscular diseases recently mapped to chromosome 5q. SMA is usually classified into types I-III, and there are cases of two types of SMA in the same sibship. Becker and others later proposed that these sibships might be due to the existence of several alleles at the same locus predisposing to the different forms of the disease. In a sample of four sibships in which both SMA type II and SMA type III occur, this hypothesis was clearly rejected for the SMA locus on 5q, by using information on the segregation of linked markers (P less than .001). Thus the difference between SMA type II and SMA type III is not due to different alleles at the SMA locus on 5q. This finding is suggestive of an involvement of other factors, genetic or environmental, in the determination of disease severity in SMA.     

A sublocus of the multicopy microsatellite marker CMS1 maps proximal to spinal muscular atrophy (SMA) as shown by recombinant analysis

The critical region containing the spinal muscular atrophy (SMA) gene is flanked by the 5q11–q13 markers, D5S435 and D5S557, as determined by linkage analysis. Here we present the results of an analysis of a Dutch SMA family with the multicopy microsatellite marker CMS1. A crossover is revealed in the critical SMA region. We conclude that at least one of the CMS1 subloci maps proximal to the SMA gene. This reduces the minimal SMA region from approximately 1.4 Mb to 600–700 kb.