Preclinical validation of a multiplex real-time assay to quantify SMN mRNA in patients with SMA.

OBJECTIVE: To determine whether survival motor neuron (SMN) expression was stable over time. METHODS: We developed a multiplex real-time reverse transcriptase (RT)-PCR assay to quantify SMN transcripts in preclinical blood samples from 42 patients with spinal muscular atrophy (SMA) drawn for three time points per patient; most blood samples were shipped to a centralized laboratory. RESULTS: We obtained a sufficient amount (9.7 +/- 5.6 microg) of good-quality total RNA, and RNAs were stable for up to a 3-year interval. This allowed RNA samples collected during a 9- to 12-month period to be analyzed in a single run, thus minimizing interexperimental variability. SMN expression was stable over time; intersample variability for baseline measures, collected during a 17-month interval, was less than 15% for 38 of 42 SMA patients analyzed. This variability was well below the 1.95-fold increase in full-length SMN (flSMN) transcripts detected in SMA fibroblasts treated with 10 mM valproic acid. CONCLUSION: Real-time quantification of SMN messenger RNA expression may be a biomarker that is amenable to multicenter SMA clinical trials.

The Delta>15 Kb deletion French Canadian founder mutation in familial hypercholesterolemia: rapid polymerase chain reaction-based diagnostic assay and prevalence in Quebec.

Approximately one in 500 individuals in Western population has autosomal dominant familial hypercholesterolemia due to mutations in the low-density lipoprotein receptor (LDLR) gene. Screening for these mutations is hampered by their large number, except in founder populations. We identified the breakpoint of the >15 kb deletion involving the LDLR gene promoter and exon 1, responsible for more than 60% of French Canadian hypercholesterolemia cases, as well as the breakpoint of the 5 kb deletion of exons 2 and 3 that accounts for an additional 5% of cases. Both deletions appear to be because of homologous recombination by unequal crossing-over between the left arms of Alu repeats. Using RepeatMasker, we determined that 55% of the LDLR gene is composed of Alu elements; thus, it is not surprising that most LDLR rearrangements involve at least one Alu. Furthermore, we developed a rapid polymerase chain reaction-based assay for the French Canadian-1 (>15 kb) and French Canadian-5 (5 kb) hypercholesterolemia alleles. Screening a representative population sample of 943 French Canadian youths whose LDL cholesterol levels were above the 50th percentile allowed us to estimate the prevalence of the >15 kb allele as 0.11% (95% confidence interval, 0.03-0.38).

The SMN genes are subject to transcriptional regulation during cellular differentiation.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of alpha-motor neurons and muscular atrophy. The causal survival motor neuron (SMN) gene maps to a complex region of chromosome 5q13 harbouring an inverted duplication. Thus, there are two SMN genes, SMN1 and SMN2, but SMN1-deficiency alone causes SMA. In this study we demonstrate, for the first time, down-regulation of SMN promoter activity during cellular differentiation. Specifically, the minimal SMN promoter is four times more active in undifferentiated embryonal carcinoma P19 cells compared to cells treated with retinoic acid (RA) to initiate neuronal differentiation. This effect is mediated by sequences contained within the minimal core promoter that we have confined to the 257 nucleotides upstream of exon 1. We have identified seven regions that are highly conserved between the mouse and human SMN core promoters and this region contains the consensus sequence for a number of transcription factors. Most notably, AhR, HNF-3 and N-Oct3 have already been shown to respond to RA treatment of EC cells, while E47, HNF-3, MAZ, N-Oct3 and Pit-1a have been implicated in embryonic, muscle or neural development. In addition, we have mapped two strong transcription initiation sites upstream of SMN exon 1. The novel -79 site identified in this study is preferentially utilized during human foetal development. Furthermore, analysis of RNA from SMA patients with deletions of the entire SMN1 gene or chimpanzees that lack SMN2 suggests that the level of transcription initiation at these sites may be different for the SMN1 and SMN2 genes. Taken together, this work provides the first demonstration of transcriptional regulation of these genes during cellular differentiation and development. Deciphering the underlying mechanisms responsible for regulating SMN transcription may provide important clues towards enhancing SMN2 gene expression, one target for the treatment of SMA.

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens.

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.

Intragenic complementation at the argininosuccinate lyase locus: reconstruction of the active site.

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus and the ASL alleles in the ASL-deficient cell strains of two complementation phenotypes have been identified. The frequent complementers, strains that participate in the majority of the complementation events, were found to be either homozygous or heterozygous for the Q286R allele, while the high-activity complementers, those strains in which complementation is associated with a high restoration of activity, were found to be either homozygous or heterozygous for the D87G allele. Direct proof of the intragenic complementation observed at the ASL locus has been obtained with the co-expression of the D87G and Q286R alleles in COS cells. A significant increase in the ASL activity was observed when the two alleles were co-expressed relative to the expression of each mutant allele alone. The increase in activity was comparable to that observed previously in the fibroblast complementation studies. The structure determinations of ASL and the homologous eye lens protein, duck delta II crystallin, have revealed that the active site of ASL is made up of residues from three different monomers. The structural mapping of the Q286 and D87 residues shows that both are located near the active site but that, in any one active site, each is contributed by a different monomer. The molecular symmetry of the ASL protein is such that when mutant monomers combine randomly, one active site will contain both mutations and at least one active site will contain no mutations at all. It is these 'native' active sites in the hybrid Q286R/D87G proteins that give rise to the partial recovery of enzymatic activity observed during intragenic complementation.

SMN(T) and NAIP mutations in Canadian families with spinal muscular atrophy (SMA): genotype/phenotype correlations with disease severity.

Childhood-onset spinal muscular atrophy (SMA) is an autosomal recessive neuropathy characterized by selective degeneration of alpha-motor neuron cells of the spinal cord. Age of onset and motor development varies greatly among patients, but the molecular basis of this variability remains unclear. The SMA locus contains two copies of a 500-kb element and deletions within the telomeric element have been shown to be the most common cause of SMA. To study the relationship between genotype and phenotype, 60 SMA families, all but two of which are of French Canadian origin, were screened for deletions in the telomeric survival motor neuron (SMN(T)) and the intact neuronal apoptosis inhibitory protein (NAIP) genes. Combining these results with those obtained for the multicopy microsatellite marker Ag1-CA (D5S1556) indicated that there are at least two types of SMA alleles. Most type I SMA patients are homozygous for large scale deletions involving the entire SMN(T) gene as well as exons 5 and 6 of the NAIP gene. The strong association between the 100-bp allele of Ag1-CA and large scale deletions in populations of diverse ethnic origin suggests that this allele marks an unstable or founder SMA chromosome. In contrast, most chronic SMA patients have at least one SMA allele with either an intragenic SMN(T) deletion or a SMN(C):SMN(T) chimeric gene which replaces the normal SMN(T) gene. The broad continuum of disease presentation in chronic SMA is most likely a consequence of the interaction between different SMA alleles.

Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number.

The survival motor neuron (SMN) transcript is encoded by two genes, SMNT and SMNC. The autosomal recessive proximal spinal muscular atrophy that maps to 5q12 is caused by mutations in the SMNT gene. The SMNT gene can be distinguished from the SMNC gene by base-pair changes in exons 7 and 8. SMNT exon 7 is not detected in approximately 95% of SMA cases due to either deletion or sequence-conversion events. Small mutations in SMNT now have been identified in some of the remaining nondeletion patients. However, there is no reliable quantitative assay for SMNT, to distinguish SMA compound heterozygotes from non-5q SMA-like cases (phenocopies) and to accurately determine carrier status. We have developed a quantitative PCR assay for the determination of SMNT and SMNC gene-copy number. This report demonstrates how risk estimates for the diagnosis and detection of SMA carriers can be modified by the accurate determination of SMNT copy number.

Cloning, characterization, and copy number of the murine survival motor neuron gene: homolog of the spinal muscular atrophy-determining gene.

Because of a 500-kb inverted duplication, there are two copies of the survival motor neuron (SMN) gene in humans, cenSMN and telSMN. Both genes produce identical ubiquitously expressed transcripts; however, only mutations in telSMN are responsible for spinal muscular atrophy (SMA), the second most common autosomal recessive childhood disease. We have cloned the murine homolog Smn and mapped the gene to Chromosome 13 within the conserved syntenic region of human chromosome 5q13. We show that the Smn transcript (1.4 kb) is expressed as early as embryonic day 7. In contrast to humans, we found no evidence of alternative splicing. The predicted amino acid sequence between mouse and human SMN is 82% identical, and a putative nuclear localization signal is conserved. FISH data indicate that the duplication of the SMA region observed in humans is not present in the mouse. We also found no evidence of multiple Smn genes using Southern blot hybridization and single-strand conformation analysis. Using these methods, we detected at least four copies of Naip exon 5 clustering distal to Smn. Finally, three biallelic markers were identified within the Smn coding region; two are silent polymorphisms, whereas the third changes a cysteine residue to a tyrosine residue in exon 7. Overall, our results indicate that Smn is single copy within the mouse genome, which should facilitate gene disruption experiments to create an animal model of SMA.

Intragenic complementation at the human argininosuccinate lyase locus. Identification of the major complementing alleles.

To determine the molecular and biochemical basis of intragenic complementation observed at the human argininosuccinate lyase (ASL) locus, we identified the ASL alleles in ASL-deficient cell strains with two unique complementation phenotypes: (i) frequent complementers, strains that participated in the majority of complementation events, and (ii) high activity complementers, strains in which complementation was associated with a relatively high level of restoration of ASL activity. Four mutations (Q286R, D87G, A398D, and a deletion of exon 13) were identified in the four strains examined. One of the two frequent complementers was homozygous, and the other heterozygous, for the Q286R allele. Similarly, one of the two high activity complementers was homozygous, and the other heterozygous, for the D87G allele. When the Q286R and D87G mutations were introduced by site-directed mutagenesis into wild-type ASL cDNA, each conferred loss of ASL activity in COS cell transfection assays. To test directly the hypothesis that intragenic complementation occurs at the ASL locus, one of the major complementation events observed previously, between strains carrying the Q286R and D87G alleles, was reconstructed in COS cell transfection assays. A partial restoration of ASL activity, comparable with the increase seen in the fibroblast complementation analysis, was observed on joint cotransfection of these two alleles. The results provide molecular confirmation of the major features of the ASL mutant complementation map, identify the Q286R and D87D alleles as the frequent and high activity complementing alleles, respectively, and provide direct proof of intragenic complementation at the ASL locus.

Molecular diagnosis of non-deletion SMA patients using quantitative PCR of SMN exon 7.

The telomeric survival motor neuron (SMN(T)) gene is a valuable molecular diagnostic tool for childhood-onset spinal muscular atrophy (SMA) as homozygous deletions of SMN(T) exon 7 (delta7SMN(T)) are present in approximately 94% of patients. In this report, we provide the first comprehensive study of 32 unrelated non-deletion SMA patients. Quantitative polymerase chain reaction (PCR) studies established that 90% had two intact copies of SMN(T) exon 7 suggesting that these patients do not have 5q SMA. Once 5q SMA is confirmed, the SMN(T) gene can be screened for subtle mutations. Using single strand conformation analysis, we identified two missense mutations (P245L and Y272C) in exon 6 of the SMN(T) gene of two SMA patients shown to have a single copy of SMN(T) exon 7. Y272 is most likely critical for SMN(T) function as it is a target for recurring mutations and is associated with type I SMA. These results emphasize the need for dosage analysis in the differential diagnosis of 5q SMA in nondeletion patients, consistent with extensive clinical heterogeneity and some genetic heterogeneity in this disease. Homozygosity or heterozygosity for a delta7SMN(T) allele confirms the diagnosis of 5q SMA with greater precision than clinical examination alone.

Clinical and genetic study of chronic (types II and III) childhood onset spinal muscular atrophy.

We have conducted a retrospective study of 63 patients affected by chronic forms of spinal muscular atrophy (SMA) to better document the natural history of this disease. Thirty-nine patients had type II and 24 type III SMA. These patients had manual muscle testing (MMT) and forced vital capacity (FVC) studies done every six to 12 months over follow up period ranging from six to 140 months. A decline in FVC was seen in both types of SMA but there was no significant change in MMT in either group. Genetic studies were also done in a subset of 17 families (23 patients) included in this study. Homozygous deletions in the telomeric survival motor neuron (SMN) and the neuronal apoptosis inhibitory protein (NAIP) genes were observed in 100% and 11.8% of the patients tested respectively.

Allelic association and deletions in autosomal recessive proximal spinal muscular atrophy: association of marker genotype with disease severity and candidate cDNAs.

The candidate region for spinal muscular atrophy (SMA) has been defined as a 750 kb interval on 5q13. In this study, we performed allelic association studies in 154 German SMA families with the multicopy markers Ag1-CA (D5S1556); C212 (D5F149S1/S2) and correlated genotype data with deletion of candidate genes. Both multicopy markers recognize 0-3 alleles pro chromosome. Deletions were detected for all copies of the markers Ag1-CA (C272) and C212 in 13 of 88 (15%) type I SMA patients and three of 48 (6%) type II patients. In all informative cases, the deletion was inherited from one parent. In two further cases (one type I and one type III SMA), de novo deletions of only one copy of Ag1-CA and C212 were found. In both cases the patients were homozygously deleted for the survival motor neuron (SMN) gene (exons 7 and 8) but only the type I SMA patient was deleted for the neuronal apoptosis inhibitory protein (NAIP) gene (exons 5 and 6). A third case (type II SMA) showed de novo deletion of SMN, but not of Ag1-CA, C212 and NAIP. Specific alleles of Ag1-CA and C212 showed significant association with SMA, particularly in type I SMA. When the number of marker copies defines genotypes, 1,1 (one allele on each chromosome) is found to be increased in type I SMA (50%) and 1,2 (one allele on one chromosome and two alleles on the other one) in type II SMA (60%). The 2,2 genotype (two alleles on each chromosome) was found in 4% of type I and II patients. By comparison, pooled normal genotype frequencies were 20, 44 and 36%, respectively. These results suggest a strong correlation between genotype and severity of disease. Based on these data we propose a model which indicates that type I SMA patients are composed of two severe alleles, type II of a mild and a severe, and type III of two mild alleles. Correlation of Ag1-CA genotype with deletion of the XS2G3/NAIP genes indicates that most patients with a deletion have a 1,1 genotype. Owing to the physical proximity of these markers, we propose that a large deletion occurs on type I SMA chromosomes that removes DNA between C212 and XS2G3/NAIP and that type II SMA results from compound heterozygosity for mild (small deletion) and severe mutations.

A novel cDNA detects homozygous microdeletions in greater than 50% of type I spinal muscular atrophy patients.

Spinal muscular atrophy (SMA) is the second most common lethal, autosomal recessive disease in Caucasians (after cystic fibrosis). Childhood SMAs are divided into three groups (type I, II and III), which are allelic variants of the same locus in a region of approximately 850 kb in chromosome 5q12-q13, containing multiple copies of a novel, chromosome 5-specific repeat as well as many atypical pseudogenes. This has hampered the identification of candidate genes. We have identified several coding sequences unique to the SMA region. A genomic fragment detected by one cDNA is homozygously deleted in 17/29 (58%) of type I SMA patients. Of 235 unaffected individuals examined, only two showed the deletion and both are carriers of SMA. Our results suggest that deletion of at least part of this novel gene is directly related to the phenotype of SMA.

Linkage disequilibrium analysis of childhood-onset spinal muscular atrophy (SMA) in the French-Canadian population.

Spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy, the most common neuromuscular disorder in childhood. The gene responsible for childhood SMA has been mapped to the q11.2-q13.3 region of chromosome 5. We have extended our linkage studies of SMA in the French-Canadian population to include microsatellite markers at the D5S125, D5S351, D5S435, JK53CA1/2 and MAP1B loci. These markers span about 4 cM of the SMA candidate region. We observed significant evidence for linkage between SMA and all the markers tested. The analysis of recombinant chromosomes provide evidence for the following genetic order: D5S125-D5S435-MAP1B-3'-JK53CA1/2 and places D5S351 proximal to JK53CA1/2. Furthermore, we confirm the current localization of the SMA gene distal to D5S435. Finally, we provide demonstration of significant linkage disequilibrium between childhood-onset SMA and four of the five marker loci, D5S125, D5S435, D5S351 and JK53CA1/2. Analysis of SMA-region haplotypes suggests that there may be a predominant SMA allele that is present on about 17% of SMA chromosomes in this sample of the French-Canadian population. We conclude that the observed linkage disequilibrium is likely due to genetic drift among regions of Quebec, consistent with this population's early history.

Linkage study of chronic childhood-onset spinal muscular atrophy (SMA): confirmation of close linkage to D5S39 in French Canadian families.

Chronic childhood-onset spinal muscular atrophy (SMA) is, after Duchenne muscular dystrophy, the most common neuromuscular disorder in childhood. Recent linkage analyses have mapped this disease to 5q12-5q14. We show that chronic SMA (Types II and III) is tightly linked to the marker locus D5S39 (Zmax = 5.47 at theta = 0.02) in eight French Canadian families. In contrast to previously published results, we do not observe close linkage between chronic SMA and D5S6 (Zmax = 0.34 at theta = 0.18) or D5S78 (Zmax = 0.25 at theta = 0.21). Last, we present a family that appears to be discordant for this localization but may represent the first example of an incompletely penetrant individual.

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec.

We have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.