Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.     

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.     

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     

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.     

Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.     

Analysis of deletional damage in SMN1, SMN2, and NAIP genes in patients with spinal muscular atrophy in the northwestern region of Russia

Polymerase chain reaction with subsequent SSCP (single-strand DNA conformational polymorphism) and restriction (BselI restriction endonuclease) analyses were used to type the DNA samples of affected individuals and their relatives from 23 Russian families with high risk of spinal muscular atrophy (SMA) residing in the northwestern region of Russia. Deletions of exon 7 of the SMN gene were found in 96% of the individuals examined. The frequency of homozygous deletion of exons 7 and 8 of the SMN1 gene was 65%. The frequency of homozygous isolated deletion of the SMN1 gene exon 7 among the SMA patients was 4.3%. Homozygous deletion of exon 5 of the NAIP gene was found in 22% of SMA patients. In SMA patients, a total of seven deletion types involving the SMN1, NAIP, and SMN2 genes were detected. Deletion of exons 7 and 8 of the SMN1 gene was the most common mutation associated with SMA in patients from the northwestern Russia.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number.

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

Detection of spinal muscular atrophy carriers by nested polymerase chain reaction of single sperm cells

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a carrier frequency of approximately 1 in 40. Approximately 95% of patients have homozygous deletions of exon 7 and/or 8 of the SMN1 gene. Carrier testing for SMA is relatively complex and requires quantitative polymerase chain reaction (PCR) of genomic DNA to determine SMN1 copy number. The purpose of this study was to assess the feasibility of carrier testing for SMA in males, by nested PCR analysis of SMN1 deletions in single sperm cells. A nested PCR method was developed to amplify SMN1 exon 7 in single cells. Restriction enzyme digestion with DraI was used to differentiate between the highly homologous SMN1 and SMN2 genes. Single sperm cells from five known SMA carriers and six noncarriers were analyzed. Among the five carriers, a total of 132 single sperm cells were analyzed and SMN1 exon 7 deletion was detected in 68 cells (51.5%). In contrast, among the six noncarriers, a total of 136 single sperm cells were analyzed. Of these, an apparent SMN1 exon 7 deletion was detected in four sperm cells. This was interpreted as an allele dropout (ADO) rate of 2.9%. We conclude that nested PCR of SMN1 exon 7 is an accurate and reproducible method for detection of SMA male carriers with a SMN1 deletion.     

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3' end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.     

Restriction site polymorphisms in the genes encoding new members of group 3 outer membrane protein family of Brucella spp

Thirty-seven Brucella reference and field strains representing all the species and their biovars were analysed by PCR-RFLP to determine the degree of variation in the genes encoding the new members of group 3 outer membrane protein (Omp) family. Analysis of the omp22 and omp25c/omp25d genes indicated that the restriction patterns were identical for all species and biovars with all restriction enzymes tested, except for Brucella ovis that showed a short 30 bp deletion close to omp22 gene, and for B. abortus biovar 6 and B. ovis that lacked a DdeI site and a HinfI site, respectively, in the omp25c/omp25d genes. Analysis of PCR products of the omp31b gene digested with 20 restriction enzymes revealed that this gene has a greater level of DNA polymorphism than the other genes encoding the new members of group 3 Omp family. A deletion of 232bp was detected in fourteen B melitensis strains from different hosts and from different geographic origins, confirming that this feature is indeed a hallmark of B. melitensis. PCR-RFLP analysis of omp31b with DdeI allowed us to identify species-specific markers for B. abortus, B. melitensis, and B. ovis. Finally, by PCR analysis, Southern blot hybridization and DNA sequencing we showed that a large deletion of 15 kb, comprising the entire omp25b gene and 21 more genes, is present in all B. ovis strains, thus confirming previous observations from other authors.     

Analysis of Deletions in SMN1, SMN2, and NAIPGenes in Spinal Muscular Atrophy Patients from the Northwestern Region of Russia

Polymerase chain reaction with subsequent SSCP (single-strand DNA conformational polymorphism) and restriction (BselI restriction endonuclease) analyses were used to type the DNA samples of affected individuals and their relatives from 23 Russian families with high risk of spinal muscular atrophy (SMA) residing in the northwestern region of Russia. Deletions of exon 7 of the SMN1gene were found in 96% of the individuals examined. The frequency of homozygous deletion of exons 7 and 8 of the SMN1gene was 65%. The frequency of homozygous isolated deletion of the SMN1gene exon 7 among the SMA patients was 4.3%. Homozygous deletion of exon 5 of the NAIPgene was found in 22% of SMA patients. In SMA patients, a total of seven deletion types involving the SMN1, NAIP, and SMN2genes were detected. Deletion of exons 7 and 8 of the SMN1gene was the most common mutation associated with SMA in patients from the northwestern Russia.     

De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder. We have identified de novo rearrangements in 7 (approximately 2%) index patients from 340 informative SMA families. In each, the rearrangements resulted in the absence of the telomeric copy of the survival motor neuron (SMN) gene (telSMN), in two cases accompanied by the loss of the neuronal apoptosis-inhibitory protein gene . Haplotype analysis revealed unequal recombination in four cases, with loss of markers Ag1-CA and C212, which are near the 5' ends of the SMN genes. In one case, an interchromosomal rearrangement involving both the SMN genes and a regrouping of Ag1-CA and C212 alleles must have occurred, suggesting either interchromosomal gene conversion or double recombination. In two cases, no such rearrangement was observed, but loss of telSMN plus Ag1-CA and C212 alleles in one case suggested intrachromosomal deletion or gene conversion. In six of the seven cases, the de novo rearrangement had occurred during paternal meiosis. Direct detection of de novo SMA mutations by molecular genetic means has allowed us to estimate for the first time the mutation rate for a recessive disorder in humans. The sex-averaged rate of 1.1 x 10(-4), arrived at in a proband-based approach, compares well with the rate of 0.9 x 10(-4) expected under a mutation-selection equilibrium for SMA. These findings have important implications for genetic counseling and prenatal diagnosis in that they emphasize the relevance of indirect genotype analysis in combination with direct SMN-gene deletion testing in SMA families.     

Prenatal diagnosis of spinal muscular atrophy in Macedonian families

Spinal muscular atrophy (SMA) is the second most common lethal autosomal recessive disorder of childhood, affecting approximately 1 in 6,000-10,000 births, with a carrier frequency of 1 in 40-60. There is no effective cure or treatment for this disease. Thus, the availability of prenatal testing is important. The aim of this study was to establish an efficient and rapid method for prenatal diagnosis of SMA and genetic counseling in families with risk for having a child with SMA. In this paper we present the results from prenatal diagnosis in Macedonian SMA families using direct analysis of fetal DNA. The probands of these families were previously found to be homozygous for a deletion of exons 7 and 8 of SMN1 gene. DNA obtained from chorionic villas samples and amniocytes was analyzed for deletions in SMN gene. SMN exon 7 and 8 deletion analysis was performed by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Of the 12 prenatal diagnoses, DNA analysis showed normal results in eight fetuses. Four of the fetuses were homozygote for a deletion of exons 7 and 8 of SMN1. After genetic counseling, the parents of the eight normal fetuses decided to continue the pregnancy, while in the four families with affected fetuses, the pregnancy was terminated. The results were confirmed after birth.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and ^CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s). Received: 13 November 1996 / Accepted: 13 May 1997     

Genetic testing and risk assessment for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases, affecting approximately 1 in 10,000 live births, and with a carrier frequency of approximately 1 in 50. Because of gene deletion or conversion, SMN1 exon 7 is homozygously absent in approximately 94% of patients with clinically typical SMA. Approximately 30 small intragenic SMN1 mutations have also been described. These mutations are present in many of the approximately 6% of SMA patients who do not lack both copies of SMN1, whereas SMA of other patients without a homozygous absence of SMN1 is unrelated to SMN1. A commonly used polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) assay can be used to detect a homozygous absence of SMN1 exon 7. SMN gene dosage analyses, which can determine the copy numbers of SMN1 and SMN2 (an SMN1 homolog and a modifier for SMA), have been developed for SMA carrier testing and to confirm that SMN1 is heterozygously absent in symptomatic individuals who do not lack both copies of SMN1. In conjunction with SMN gene dosage analysis, linkage analysis remains an important component of SMA genetic testing in certain circumstances. Genetic risk assessment is an essential and integral component of SMA genetic testing and impacts genetic counseling both before and after genetic testing is performed. Comprehensive SMA genetic testing, comprising PCR-RFLP assay, SMN gene dosage analysis, and linkage analysis, combined with appropriate genetic risk assessment and genetic counseling, offers the most complete evaluation of SMA patients and their families at this time. New technologies, such as haploid analysis techniques, may be widely available in the future.     

Cloning and regulation of gene expression of EcoRV restriction- modification system

A number of recombinant plasmids, containing EcoRV restriction-modification genes have been constructed. Individual genes of this system were introduced into plasmids of various incompatibility groups. Promoter regions of genes encoding methylase and restrictase have been cloned and studied. With the use of specialized vector pVE8 it was shown that the efficiency of the endonuclease gene promoter is comparable with early lambda phage promoters and produced about 70% of PL efficiency. The efficiency of the methylase gene promoter region was twice less than the efficiency of the restriction endonuclease gene promoter. Plasmid with restriction endonuclease gene promoter located downstream in relation to the additional regulatable phage lambda promoter PL has been obtained. It enabled us to construct strains 30-40 fold overproducing this enzyme under conditions of inactivation of the temperature sensitive phage repressor c1857. This construction directs the production of a high level (10%) of the total cellular soluble proteins) of the EcoRV restriction enzyme. The factors that influenced the level of enzyme synthesis under induction are discussed.     

Feline chlamydiosis in Italy: PCR amplification and analysis of the ompA and groEL-homolog genes

The ompA and groEL-homolog genes of Chlamydia psittaci were amplified from feline conjunctival swabs. The PCR products from positive samples for ompA and groEL genes were digested with the restriction enzyme Alu I and Hind III, respectively. In addition, the PCR products from both genes were sequenced. The results confirm the high homogeneity of the ompA gene of feline C. psittaci. The RFLP analysis of groEL-homolog gene could be useful to typing the strains of feline C. psittaci.     

Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant

The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E. coli PpcI genomic DNA. This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16. Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only. This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme. When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2. The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme. Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2.     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.