Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder which presents with various clinical phenotypes ranging from severe to very mild. All forms are caused by the homozygous absence of the survival motor neuron ( SMN1 ) gene. SMN1 and a nearly identical copy ( SMN2 ) are located in a duplicated region at 5q13 and encode identical proteins. The genetic basis for the clinical variability of SMA remains unclear, but it has been suggested that the copy number of SMN2 could influence the disease severity. We have assessed the number of SMN2 genes in patients with different clinical phenotypes by fluorescence in situ hybridization (FISH) using as SMN probe a mixture of small specific DNA fragments. Gene copy number was established by FISH on interphase nuclei, but the presence of two SMN2 genes on the same chromosome could also be revealed by FISH on metaphase spreads. All patients had at least two SMN2 genes. We found two or three copies of SMN2 in severely affected type I patients, three copies in intermediately affected type II patients, generally four copies in mildly affected type III patients and four or eight copies in patients with very mild adult-onset SMA. No alterations of the genes were detected by Southern blot and sequence analysis, suggesting that all gene copies of SMN2 were intact. These data provide additional evidence that the SMN2 genes modulate the disease severity and suggest that knowledge of the gene copy number could be of some prognostic value.

SMN protein analysis in fibroblast, amniocyte and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by the homozygous absence of the telomeric copy of the survival motor neuron (SMNt) gene, due to deletion, gene conversion or point mutation. SMNt and its homologous centromeric copy (SMNc) encode the SMN protein, which is diffusely present in the cytoplasm and in dot-like structures, called gems, in the nucleus. We have studied the SMN protein in different cell cultures, including fibroblasts, amniocytes and CVS cells from SMA individuals and controls. By immunofluorescence analysis we found a marked reduction in the number of gems in fibroblasts, amniocytes and chorionic villus cells of all SMA patients and foetuses, independent of the type of the genetic defect. We also show that immunolocalisation of the SMN protein may be a useful tool for the characterisation of particular patients of uncertain molecular diagnosis.

Partial 9p monosomy in a girl with a tdic(9p23;13p11) translocation, minor anomalies, obesity, and mental retardation.

We report on a case with a partial monosomy for the regions 9p23 --> pter and 13p11 --> pter as a result of a de novo translocation (9p23;13p11). The patient, a 16-year-old girl, has mental deficiency, obesity, and minor anomalies, including trigonocephaly, hypertelorism and a short, broad neck. Cytogenetic and microsatellite marker analysis allowed us to assign the breakpoint to the chromosomal region 9p23, flanked by the markers D9S144 and D9S157. In an attempt to establish a phenotype-genotype correlation, the clinical manifestations present in our patient are compared to those with partial 9p monosomy and breakpoint in p23, referred to in the literature.

Frameshift mutation in the survival motor neuron gene in a severe case of SMA type I.

Recently, a spinal muscular atrophy (SMA) determining gene, termed survival motor neuron (SMN) gene, has been isolated from the 5q13 region and found deleted in most patients. A highly homologous copy of this gene has also been isolated and located in a centromeric position. We have analyzed 158 patients (SMA types I-IV) and found deletions of SMN exon 7 in 96.8%. Mutations other than gross deletions seem to be extremely rare. In one of the undeleted SMA type I patients, a newborn who survived for only 42 days, we detected a maternally inherited 5 bp microdeletion in exon 3, resulting in a premature stop codon. By RT-PCR and long range PCR amplification we were able to show that the deletion belongs to the SMN gene, rather than to the centromeric copy, and that the proposita had no paternal SMN gene. Analysis of the neuronal apoptosis inhibitor protein (NAIP) gene, which maps close to SMN and has been proposed as a SMA modifying gene, suggests the presence of at least one full-length copy. Haplotype analysis of closely linked polymorphic markers suggests that the proposita also lacks the maternally derived copy of the centromeric homologue of SMN supporting the hypothesis that the severity of the phenotype might depend on the reduced number of centromeric genes in addition to the frameshift mutation.

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.

Molecular and cytogenetic characterization of a recurrent unbalanced translocation (4;21)(p16.3;q22.1): relevance to the Wolf-Hirschhorn and Down syndrome critical regions.

We report on an aneuploidy syndrome due to the unbalanced segregation of a familial translocation (4;21)(p16.3;q22.1) causing a partial 4p monosomy and a partial 21q trisomy. The three affected children presented with severe failure to thrive, short stature, microcephaly, profound hypotonia, and mental retardation. The face, very similar in the three children, is characterized by frontal bossing, upslanting of the palpebral fissures, short nose, and deep set ears, giving the overall appearance of the Down syndrome. The molecular study has defined the aneuploid segment on both 4p and 21q. Most of the Down syndrome critical region was found to the trisomic, while only part of the candidate Wolf-Hirschhorn syndrome critical region was deleted, suggesting that this region is not critical for the major malformations characteristic for WHS.

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 5q13 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.

Genetic homogeneity between childhood-onset and adult-onset autosomal recessive spinal muscular atrophy.

Molecular diagnosis of childhood proximal spinal muscular atrophy has been enhanced by the discovery of the survival motor neuron (SMN) gene, which is absent or truncated in 98.6% of patients. To determine whether deletion analysis of the SMN gene may also be diagnostic for adult-onset disease, we studied six patients and found deletions in all. This finding will facilitate the diagnosis of adult-onset spinal muscular atrophy, and provides evidence for genetic homogeneity between the clinically diverse adult and childhood forms of the disease.

Identification of key recombinants in multiplex SMA families.

Recent reports have provided evidence that a major gene for autosomal recessive proximal spinal muscular atrophy (SMA) resides in a small genetic interval in bands q12-q13 of chromosome 5, a 4-cM region proximally flanked by D5S125 (EF(TG/AG)n) and distally by MAP1B/D5S112 or a 0.7-cM interval (range 0.1-2.1 cM) flanked by D5S435 proximally and MAP1B/D5S112 distally. We present the identification of key recombinants between SMA and the closest flanking DNA-markers in an analysis of Dutch and Italian SMA families. These crossovers may serve as reference points for new markers in this region and may thus be instrumental in a further refined mapping of the SMA gene. Two markers, D5S351 (I105) and D5S357 (Mfd151), could be mapped distally to SMA in the interval SMA-D5S112.

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-q13.3 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.

Highly polymorphic repeat marker within the beta-amyloid precursor protein gene.

We have identified a polymorphic compound dinucleotide repeat sequence in intron 1 of the beta-amyloid precursor protein (APP) gene on chromosome 21. Using polymerase chain reaction (PCR) amplification of the locus, designated APPivs1, we detected 13 alleles in the CEPH family members (heterozygosity = 0.69). Lod score analysis showed complete linkage of the marker to the loci D21S210 and D21221.

Presymptomatic diagnosis of SMA III by genotype analysis.

Linkage analysis and prenatal prediction in families segregating autosomal recessive spinal muscular atrophy (SMA) has become feasible since the assignment of the locus responsible for type I-III SMA to region 5q12-q13.3. We have performed a segregation study of SMA in Italian families using molecular probes and highly informative PCR-based polymorphic markers. In one family, a 7-year-old boy affected with type III SMA and an 8-year-old apparently healthy brother had identical haplotypes. These findings prompted us to reexamine the apparently unaffected child. His neurological exam was normal. However, the electromyography (EMG) showed a pattern consistent with chronic SMA. To our knowledge this is the first example of presymptomatic diagnosis of SMA based on genotype analysis.