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.     

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

Assignment of the human 8.5 H gene to chromosome 5, region 5q35

The human 8.5 H probe was isolated from a human cerebellum cDNA library with a probe corresponding to the coding region of the murine 8.5 M cDNA. This cDNA isolated from a murine cDNA library constructed from newborn cerebral hemispheres was selected because of its strong expression in embryonic neurons. Consequently the corresponding human gene could be a candidate for hereditary neurodegenerative diseases. The human 8.5 H gene was assigned by somatic hybrid analysis to chromosome 5; this chromosome contains the gene(s) for spinal muscular atrophy (SMA), a group of heritable degenerative diseases that selectively affect the anterior horn motor neuron of the spinal cord. The localization by in situ hybridation of 8.5 H on 5q35 excluded the possibility that this gene is identical to SMA. The SMA gene(s) was (were) known, from linkage analysis, to be in a region (5q11.2-q13.3) very distant from 5q35.     

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.     

Complex repetitive arrangements of gene sequence in the candidate region of the spinal muscular atrophy gene in 5q13.

Childhood-onset proximal spinal muscular atrophy (SMA) is a heritable neurological disorder, which has been mapped by genetic linkage analysis to chromosome 5q13, in the interval between markers D5S435 and D5S557. Here, we present gene sequences that have been isolated from this interval, several of which show sequence homologies to exons of beta-glucuronidase. These gene sequences are repeated several times across the candidate region and are also present on chromosome 5p. The arrangement of these repetitive gene motifs is polymorphic between individuals. The high degree of variability observed may have some influence on the expression of the genes in the region. Since SMA is not inherited as a classical autosomal recessive disease, novel genomic rearrangements arising from aberrant recombination events between the complex repeats may be associated with the phenotype observed.     

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.     

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.     

High-resolution genetic map around the spinal muscular atrophy (SMA) locus on chromosome 5.

Although autosomal recessive spinal muscular atrophy (SMA) has been mapped to chromosome 5q12-q13, there is for this region no genetic map based on highly informative markers. In this study we present the mapping of two previously reported microsatellite markers in 40 CEPH and 31 SMA pedigrees. We also describe the isolation of a new microsatellite marker at the D5S112 locus. The most likely order of markers (with recombination fractions given in parentheses) is 5cen-D5S6-(.02)-D5S125-(.04)-(JK53CA1/2,D5S11 2)-(.04)-D5S39-qter. The relative order of D5S6, D5S112, and D5S39 was confirmed by in situ hybridization. Multipoint linkage analysis in 31 SMA families indicates that the SMA locus lies in the 6-cM interval between D5S6 and JK53CA1/2, D5S112.     

The spinal muscular atrophy gene region at 5q13.1 has a paralogous chromosomal region at 6p21.3

Paralogous regions are duplicated segments of chromosomal DNA that have been acquired during the evolution of the genome. Subsequent divergent evolution of the genes within paralogous regions can lead to the formation of gene families. Here, we report the identification of a region on Chromosome (Chr) 6 at 6p21.3 that is paralogous with the Spinal Muscular Atrophy (SMA) gene region on Chr 5 at 5q13.1. Partial characterization of this region identified nine sequences all of which are highly homologous to DNA sequences of the SMA gene region at 5q13.1. These sequences include four β-glucuronidase sequences, two retrotransposon sequences, a novel cDNA, a Sequence Tagged Site (STS), and one that is homologous to exon 9 of the Neuronal Apoptosis Inhibitor Protein (NAIP) gene. The 6p21.3 paralogous SMA region may contain genes that are related to those in the SMA region at 5q13.1; however, a direct association of this region with SMA is unlikely given that no linkage of SMA with Chr 6 has been reported. Received: 12 May 1997 / Accepted: 13 November 1997     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

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.     

Genetic linkage analysis of Canadian spinal muscular atrophy kindreds using flanking microsatellite 5q13 polymorphisms

The spinal muscular atrophies (SMA) are among the most common autosomal recessive disorders. We have performed linkage analysis using both standard restriction fragment length polymorphisms (RFLPs) as well as microsatellite polymorphisms [Ca_(n)] on 49 Canadian SMA families (types 1, 2, and 3) that both flank and are linked to SMA. The closest SMA linkage was observed with the MAP1B locus (z_max=8.04, _max=0.0). Multipoint linkage analysis gave a high probability of SMA mapping between D5S6 and D5S39. Only one family (type 3) that fulfilled our diagnostic criteria for SMA showed nonlinkage with 5q13 markers. This study shows the feasibility of accurate molecular diagnosis of SMA utilizing 5q13 satellite polymorphisms.     

Integration of Genetic and Cytological Maps and Development of a Pachytene Chromosome-based Karyotype in Papaya

A significant amount of genetic and genomic resources have been developed in papaya (Carica papaya, $ {\hbox{2n = 2}} \times { = 18} $ ), including genetic linkage maps consisting of nine major and three minor linkage groups. However, the 12 genetic linkage groups have not been integrated with the nine chromosomes of papaya. Bacterial artificial chromosome (BAC) clones associated with each linkage group were recently isolated. These linkage group-specific BACs were mapped to meiotic pachytene chromosomes of papaya using fluorescence in situ hybridization (FISH). The FISH mapping results integrated the 12 linkage groups into the nine papaya chromosomes. We developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar SunUp. The chromosomal distribution of heterochromatin in the papaya genome is provided in the karyotype with the X chromosome representing the most euchromatic chromosome in the papaya genome. FISH mapping also revealed a significant amplification of sequences related to the 5S ribosomal RNA genes, which was detected in the male-specific region of the Y chromosome, but not in the corresponding region in the X chromosome.     

Maternal mosaicism for a second mutational event in a type I spinal muscular atrophy family.

Spinal muscular atrophy (SMA) is a common fatal motor-neuron disorder characterized by degeneration of the anterior horn cells of the spinal cord, which results in proximal muscle weakness. Three forms of the disease, exhibiting differing phenotypic severity, map to chromosome 5q13 in a region of unusually high genomic variability. The SMA-determining gene (SMN) is deleted or rearranged in patients with SMA of all levels of severity. A high de novo mutation rate has been estimated for SMA, based on the deletion of multicopy microsatellite markers. We present a type I SMA family in which a mutant SMA chromosome has undergone a second mutation event. Both the occurrence of three affected siblings harboring this same mutation in one generation of this family and the obligate-carrier status of their mother indicate the existence of maternal germ-line mosaicism for cells carrying the second mutation. The existence of secondary mutational events and of germ-line mosaicism has implications for the counseling of SMA families undergoing prenatal genetic analysis.     

Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays

The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557. We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1—SMA—D5S557/D5S112 interval. The D5S629/CMS-1—SMA—D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones. A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented. Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled. We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.     

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.     

Two novel microsatellite markers for prenatal prediction of spinal muscular atrophy (SMA)

Autosomal recessive spinal muscular atrophy (SMA) has been mapped to a 6-cM interval on chromosome 5q12–13.3, flanked proximally by locus D5S6 and distally by locus D5S112. In this study we describe the isolation of two new microsatellite markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cM distal to D5S6. We show by linkage analysis and the study of the recombinants in 55 SMA pedigrees that the disease lies in the 4-cM interval between EF1/2a and D5S112. Fluorescence in situ analysis of cosmids from D5S6, EF1/2a and D5S112 confirms the genetic order and relative distance of markers. The microsatellites EF1/2a and EF13/14 are the first highly polymorphic PCR based proximal markers in SMA to be described, and will be of value in prental prediction of the disorder.     

New complexities in the genetics of stuttering: significant sex-specific linkage signals

Stuttering is a speech disorder long recognized to have a genetic component. Recent linkage studies mapped a susceptibility locus for stuttering to chromosome 12 in 46 highly inbred families ascertained in Pakistan. We report here on linkage studies in 100 families of European descent ascertained in the United States, Sweden, and Israel. These families included 252 individuals exhibiting persistent stuttering, 45 individuals classified as recovered from stuttering, and 19 individuals too young to classify. Primary analyses identified moderate evidence for linkage of the broader diagnosis of "ever stuttered" (including both persistent and recovered stuttering) on chromosome 9 (LOD = 2.3 at 60 cM) and of the narrower diagnosis of persistent stuttering on chromosome 15 (LOD = 1.95 at 23 cM). In contrast, sex-specific evidence for linkage on chromosome 7 at 153 cM in the male-only data subset (LOD = 2.99) and on chromosome 21 at 34 cM in the female-only data subset (LOD = 4.5) met genomewide criteria for significance. Secondary analyses revealed a significant increase in the evidence for linkage on chromosome 12, conditional on the evidence for linkage at chromosome 7, with the location of the increased signal congruent with the previously reported signal in families ascertained in Pakistan. In addition, a region on chromosome 2 (193 cM) showed a significant increase in the evidence for linkage conditional on either chromosome 9 (positive) or chromosome 7 (negative); this chromosome 2 region has been implicated elsewhere in studies on autism, with increased evidence for linkage observed when the sample is restricted to those with delayed onset of phrase speech. Our results support the hypothesis that the genetic component to stuttering has significant sex effects.     

Refined localization of TSC1 by combined analysis of 9q34 and 16pl3 data in 14 tuberous sclerosis families

Tuberous sclerosis (TSC) is a heterogeneous trait. Since 1990, linkage studies have yielded putative TSC loci on chromosomes 9, 11, 12 and 16. Our current analysis, performed on 14 Dutch and British families, reveals only evidence for loci on chromosome 9q34 (TSC1) and chromosome 16p13 (TSC2). We have found no indication for a third locus for TSC, linked or unlinked to either of these chromosomal regions. The majority of our families shows linkage to chromosome 9. We have refined the candidate region for TSC1 to a region of approximately 5 cM between ABL and ABO.     

Evidence for asthma susceptibility genes on chromosome 11 in an African-American population

Initial genome-wide scan data provided suggestive evidence for linkage of the asthma phenotype in African-American (AA), but not Caucasian, families to chromosome 11q markers (peak at D11S1985; LOD=2). To refine this region, mapping analysis of 91 AA families (51 multiplex families and 40 asthmatic case-parent trios) was performed with an additional 17 markers flanking the initial peak linkage marker. Multipoint analyses of the 51 multiplex families yielded significant evidence of linkage with a peak non-parametric linkage score of 4.38 at marker D11S1337 (map position 68.6 cM). Furthermore, family-based association and transmission disequilibrium tests conducted on all 91 families showed significant evidence of linkage in the presence of disequilibrium for several individual markers in this region. A putative susceptibility locus was estimated to be at map position 70.8 cM with a confidence interval spanning the linkage peak. Evidence from both linkage and association analyses suggest that this region of chromosome 11 contains one or more susceptibility genes for asthma in these AA families.