OBJECTIVETo introduce the application of denaturing high-performance liquid chromatography (DHPLC) in the diagnosis of childhood type spinal muscular atrophy (SMA).

METHODSExon 7 and flanking area of survival motor neuron (SMN) gene were amplified by PCR in 1 standard sample, 25 normal individuals and 25 patients with SMA. The PCR products were then directly loaded onto the DHPLC system after denaturing and annealing. Different DNA segments were separated by changing the concentration of buffer A relative to that of buffer B.

RESULTSDifferent DNA segments were separable on the DHPLC chromatogram. Three peaks including SMN1/SMN2 heteroduplex peak, SMN2 homoduplex peak and SMN1 homoduplex peak were detected in 23 out of 25 normal individuals. Only SMN1 homoduplex peak was detected in 2 normal individuals and the standard sample, indicating the deletion of SMN2 On the contrary, only the SMN2 homoduplex peak was detected in 22 out of 25 patients with SMA, indicating deletion of SMN1. The three peaks as those of normal individuals were detected in the other 3 patients, indicating no SMN1 or SMN2 deletion.

CONCLUSIONAs a new technology for diagnosing SMA, DHPLC is sensitive, accurate, rapid and convenient.

Chromatography, High Pressure Liquid ; methods ; Exons ; genetics ; Humans ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Polymerase Chain Reaction ; Reproducibility of Results ; SMN Complex Proteins ; genetics ; Sensitivity and Specificity ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein

INTRODUCTIONSpinal muscular atrophy (SMA) is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The survival motor neuron (SMN) protein is encoded by 2 genes, SMN1 and SMN2. The most frequent mutation is the biallelic deletion of exon 7 of the SMN1 gene. In SMA, SMN2 cannot compensate for the loss of SMN1, due to the exclusion of exon 7. The aim of our study was to estimate the frequency of the common SMN1 exon 7 deletion in patients referred to our centre for carrier detection and prenatal diagnosis.

MATERIALS AND METHODSWe performed the detection of exon 7 deletion of the SMN1 gene for the affected patients and fetuses suspected to have SMA.

RESULTSOf 243 families, 195 were classified as SMA type I, 30 as type II, and 18 as type III according to their family histories. The analysis of exon 7 deletion among living affected children showed that 94% of the patients with SMA type I, 95% with type II families and 100% with type III had homozygous deletions. Of the prenatal diagnoses, 21 (22.8%) of the 92 fetuses were found to be affected and these pregnancies were terminated.

CONCLUSIONSThe homozygosity frequency for the deletion of SMN1 exon 7 for all 3 types was (94%), similar to those of Western Europe, China, Japan and Kuwait.

DNA ; genetics ; Exons ; Female ; Gene Deletion ; Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Iran ; epidemiology ; Male ; Muscular Atrophy, Spinal ; diagnosis ; epidemiology ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Pregnancy ; Prenatal Diagnosis ; methods ; Prevalence ; Prognosis ; Retrospective Studies ; SMN Complex Proteins ; genetics ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein

OBJECTIVETo perform prenatal diagnosis for 5 pregnant women who had given birth to children with spinal muscular atrophy (SMA).

METHODSThirty to forty mililiters of amniotic fluid was obtained by amniocentesis under ultrasonic monitoring. DNA was extracted directly from sediment of amniotic fluid. Short tandem repeat (STR) profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Two methods, PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR, were used to analyze exon 7 of SMN gene from amniotic DNA.

RESULTSComparing the 16 STR sites of each fetus with those of his/her parents, there was no or little contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, part of the PCR product (189 bp) from amniotic DNA of fetus A, C, or D remained intact after digestion with Dra I, while the PCR product from amniotic DNA of fetus B or E was completely digested by Dra I. In allele-specific PCR, exon 7 of both SMN1 and SMN2 gene could be seen when amniotic DNA of fetuses A, C, or D was analyzed, while only exon 7 of SMN2 could be seen when amniotic DNA of fetuses B or E was analyzed.

CONCLUSIONHomozygous deletion of SMN1 is not detected in fetuses A, C, and D, predicting that the risk of developing SMA after birth would be extremely low. Homozygous deletion of SMN1 was present in fetuses B and E suggesting high risk of developing SMA after birth.

Exons ; genetics ; Family Health ; Female ; Homozygote ; Humans ; Male ; Microsatellite Repeats ; genetics ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Pregnancy ; Prenatal Diagnosis ; methods ; SMN Complex Proteins ; genetics ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein

OBJECTIVETo detect the correlation between the clinical phenotype of spinal muscular atrophy (SMA) and survival motor neuron gene (SMN2) copy number.

METHODSThe SMN2 gene copy numbers of 57 different types of SMA were detected by real-time fluorescence quantitative PCR method with TaqMan technique.

RESULTSAverage SMN2 copy number was 1.017 +/- 0.090, 2.019+/- 0.080, 3.104+/- 0.170 in predicting one, two, three copy numbers, respectively, and CV was 8.9%, 3.9%, 5.4%, respectively. Average SMN2 copy number was 1.926+/- 0.460, 2.508+/- 0.460, 2.876+/- 0.270, in type I, II and III SMA, respectively. The SMN2 gene copy number in type II and III SMA were higher than that of type I SMA (P < 0.01). The SMN2 gene copy number in type III SMA was higher than that of type II SMA (P < 0.01). 85.72% of type I SMA patients usually had 2 SMN2 copies; 40% and 60% of type II SMA patients had 2 and 3 SMN2 copies, respectively; 82% of type III SMA patients had 3 SMN2 copies.

CONCLUSIONThere is significant correlation between the change of SMA clinical phenotype and SMN2 cope number. The distributions of the SMN2 gene copy number are various in different types of SMA patients. All types of SMA patients have at least one copy SMN2. The SMN2 gene copy numbers in type II, III SMA are higher than that of type I. All of these findings suggest that the severity of SMA patients depend on the change of the SMN2 copy numbers.

Gene Dosage ; Genetic Predisposition to Disease ; genetics ; Humans ; Muscular Atrophy, Spinal ; genetics ; pathology ; Phenotype ; Polymerase Chain Reaction ; SMN Complex Proteins ; genetics ; Survival of Motor Neuron 2 Protein

INTRODUCTIONChildhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran.

MATERIALS AND METHODSMolecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR.

RESULTSThe homozygous-deletion frequency rate for the telomeric copy of SMN exons 7 and 8 in all types of SMA was 97%. Moreover, exons 5 and 6 of NAIP gene were deleted in approximately 83% of all SMA types. Three deletion haplotypes were constructed by using SMN and NAIP genotypes. Haplotype A, in which both genes are deleted, was seen in approximately 83% of SMA types I and II but not type III. It was also found predominantly in phenotypically severe group with an early age of onset (i.e., less than 6-month-old). We also report 34 of our prenatal diagnosis.

CONCLUSIONSTo our knowledge, the present study is the first one giving detailed information on SMN and NAIP deletion rates in Iranian SMA patients. Our results show that the frequency of SMN1 homozygous deletions in Iran is in agreement with previous studies in other countries. The molecular analysis of SMA-related gene deletion/s will be a useful tool for pre- and postnatal diagnostic.

Age of Onset ; Child, Preschool ; Cyclic AMP Response Element-Binding Protein ; genetics ; Female ; Gene Deletion ; Humans ; Iran ; Male ; Muscular Atrophy, Spinal ; etiology ; genetics ; physiopathology ; Nerve Tissue Proteins ; genetics ; Neuronal Apoptosis-Inhibitory Protein ; genetics ; RNA-Binding Proteins ; genetics ; SMN Complex Proteins ; Survival of Motor Neuron 1 Protein

OBJECTIVETo perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family.

METHODSDeletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation.

RESULTSIn SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father.

CONCLUSIONA novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.

Base Sequence ; Child, Preschool ; DNA Mutational Analysis ; Exons ; genetics ; Humans ; Male ; Molecular Sequence Data ; Muscular Atrophy, Spinal ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; SMN Complex Proteins ; genetics ; Spinal Muscular Atrophies of Childhood ; genetics ; Survival of Motor Neuron 1 Protein ; genetics ; snRNP Core Proteins ; genetics

OBJECTIVETo establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA).

METHODSTwenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children.

RESULTSTwenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP.

CONCLUSIONPCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.

Child ; Exons ; genetics ; Female ; Gene Deletion ; Genetic Counseling ; Humans ; Male ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Pregnancy ; Prenatal Diagnosis ; SMN Complex Proteins ; genetics ; Spinal Muscular Atrophies of Childhood ; diagnosis ; genetics ; Survival of Motor Neuron 1 Protein ; genetics

OBJECTIVETo study the genetic basis in the patients with clinical diagnosis of spinal muscular atrophy(SMA) but without survival motor neuron telomeric copy (SMN-T) deletion; the relationship between the SMN-C (centromeric) copies and the phenotype; and the distribution of SMN-C and SMN-T copies in the SMA patients, the carriers and the controls.

METHODSQuantitative PCR analysis of SMN-T and SMN-C copies were carried out in 45 patients, 25 consanguineous and 33 control individuals. The patients were identified by clinical manifestation and muscular pathology. Two internal standards of SMN-T and cystic fibrosis transmembrane conductance regulator (CFTR) were constructed. Nonradioactive and nonfluorescence-labelling competitive PCR were used. The numbers of SMN-T and SMN-C copies were determined by calculating the ratios of SMN-T/CFTR and SMN-C/CFTR.

RESULTSQuantitation of SMN-T gene copies in SMA patients revealed that nine cases of type I-III were homozygously deleted. Two cases of type III had only one copy and four cases of type III had two copies. SMA IV and other type cases had two copies. Nine cases of consanguineous individuals had one copy, but other 16 had two copies. All of the normal individuals had two copies. Analysis of SMN-C copies showed that SMA I had < or = 2 copies, II-III had < or = 3 copies, SMA IV and others had 0-3 copies, the consanguineous individuals and normal individuals had 0-3 copies.

CONCLUSIONThe number of copies determined by PCR quantitative assay of SMN-T is in accordance with the result of PCR qualitative assay of homozygous deletion. Quantitative assay of the number of copies can find out the cases and the carriers of heterozygous deletion. The SMA phenotype is related to the number of copies of SMN-C; the smaller the number of copies the patient has, the severer the patient's phenotype will be. The pathogenesis of SMA IV and other types of SMA may not relate to SMN gene.

Cyclic AMP Response Element-Binding Protein ; Gene Dosage ; Humans ; Muscular Atrophy, Spinal ; genetics ; Nerve Tissue Proteins ; genetics ; RNA-Binding Proteins ; SMN Complex Proteins

Cyclic AMP Response Element-Binding Protein ; DNA ; analysis ; genetics ; Female ; Humans ; Infant ; Male ; Muscular Atrophy, Spinal ; genetics ; Nerve Tissue Proteins ; genetics ; Polymerase Chain Reaction ; RNA-Binding Proteins ; SMN Complex Proteins

Chromatography, High Pressure Liquid ; methods ; Cyclic AMP Response Element-Binding Protein ; genetics ; Female ; Humans ; Male ; Nerve Tissue Proteins ; genetics ; Prenatal Diagnosis ; methods ; RNA-Binding Proteins ; genetics ; SMN Complex Proteins ; Sequence Analysis, DNA ; Spinal Muscular Atrophies of Childhood ; diagnosis ; genetics