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Clinical and molecular diagnosis of spinal muscular atrophy.
Correspondence Address:
The spinal muscular atrophies are a group of disorders characterized by flaccid limb weakness. It is necessary to differentiate these from other causes and identify the SMA variants. In classical SMA, majority of the patients shows homozygous deletion of the telomeric SMN gene (SMN1) on chromosome 5q. The availability of DNA analysis has allowed proper genetic counseling and prenatal diagnosis in the affected families. Application of newer techniques has enabled more accurate carrier detection. Our objective is to stress the variability in the clinical features and recent advances in the molecular diagnosis for SMA.
The spinal muscular atrophies (SMAs) represent a heterogeneous group of diseases with predominantly autosomal recessive inheritance, characterized by degeneration of motor neurons in the anterior horn cells of the spinal cord and the brainstem. The childhood onset SMAs are of three main types and majority of them are associated with homozygous deletion in the survival motor neuron (SMN1) gene on 5q13.[1],[2] Recurrent frameshift mutations, gene conversions, and other point mutations have also been described.[2],[3] SMA variants with gene deletions outside 5q13 further add to the genetic heterogeneity.[4],[5],[6],[7] Clinical Characteristics The childhood SMAs are an important cause of morbidity and mortality with an incidence of 1 in 6000-10,000 live births.[8] [Table I] shows the clinical features and inheritance of SMA and its variants.[9],[10] SMA with onset after 30 years of age has been described as SMA type IV. Pseudohypertrophy of calves and gluteal muscles may occur and may suggest erroneous diagnosis of Duchenne muscular dystrophy. Phenotypic variability in weakness may occur within families. Diagnosis The serum concentration of creatine kinase is usually normal but may be mildly elevated (2-4 times the upper limit of normal). On EMG studies, spontaneous discharges (fasciculations, fibrillations and positive sharp waves) may be seen at rest. In the Kugelberg-Welander variant, about 60% of patients will have abnormal spontaneous potentials. There is decreased number and increased amplitude of motor potentials.[9],[11] The motor nerve conduction velocities are usually normal, but may be slowed. Sensory nerve conduction is always normal. The muscle biopsy shows groups of small atrophic fibers adjacent to normal sized or hypertrophied fibers.[9],[12] The normal random arrangement of fiber types is replaced by 'type grouping', a sign of re-innervation. Deletions in survival motor neuron (SMN1) gene are identified in 92% of all classical SMA patients.[13] Pre-clinical diagnosis of SMA can be made from the presence of nocturnal cramps and minimal EMG abnormalities like large amplitude motor action potentials.[14] Genetics The SMN gene was identified as the SMA determining gene in 1995.[2] Each chromosome 5 carries two homologous copies of the SMN gene, one telomeric (SMN1) and one centromeric (SMN2) [Figure 1a]. Both the centromeric and telomeric copies are transcribed and give rise to identical proteins. However, homozygous deletions in only the telomeric copy (SMN1) are found in majority of SMA patients. Phenotypic variability may result from more extensive deletions, de novo point mutations, variation in centromeric copy (SMN2) number and chimeric SMN gene.[15-17] SMN2 may also be associated with disease phenotype in selected cases.[18] In addition to deletion of SMN1, point mutations may also be present [Table II].[19-21] Other genes implicated in the causation of SMA are the neuronal apoptosis inhibitory protein (NAIP) gene and the P44 gene.[22],[23] However, their role in the disease is controversial. Molecular Diagnosis The molecular diagnosis of SMN gene deletions can be carried out by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP).[2],[24] The telomeric and centromeric copies in exon 7 and exon 8 of SMN gene differ from each other by single base changes [Figure 1b] that can be identified by selective restriction enzyme digestion of DNA. For exon 7, the PCR amplified DNA product is digested with DraI restriction enzyme and visualized with ethidium bromide on agarose gel electrophoresis. This allows the SMN1 and SMN2 genes to be distinguished [Figure 2a]. Similarly for exon 8, the restriction enzyme DdeI is used ([Figure 2b]. In 510% patients, deletion of both SMN1 and SMN2 genes are not detected. In such cases, point mutations have been identified. However, detection of point mutation requires specialized techniques like SSCP (single strand conformation polymorphism) and heteroduplex analysis followed by DNA sequencing. Therefore, point mutation analysis is not routinely performed for diagnostic purposes. Absence of exon 7 of SMN1 gene in peripheral blood has become a diagnostic tool for confirmation of the disease. The test has a sensitivity of 95% and specificity is over 99%[25] if SMA is diagnosed with strictly defined criteria.[10] A patient with exon 7 deletions has 99% chances of having SMA. Carrier Detection and Prenatal Diagnosis Identification of SMA carriers is possible by the use of a quantitative PCR-based assay for the determination of SMN1 copy number.[26],[27] Alternately, restriction enzyme digested products are run on 6% denaturing polyacrylamide gel electrophoresis and quantitated by autoradiography and densitometric analysis. The ratio of SMN1 to SMN2 can be determined, which decreases in carriers.[28] Recent studies have described an SSCPbased carrier test for SMA.[29] Once deletion/mutation in the SMN1 gene has been identified, it serves as a basis for providing prenatal diagnosis to the family. On prenatal testing, the interpretation of a deletion of SMN1 in the fetal DNA is straightforward. The prenatal sampling in the first trimester of pregnancy is done routinely and mutation detection results are quite reliable. In case no surviving sib is available then also prenatal testing can diagnose presence or absence of SMA if there is strong clinical suspicion of proximal SMA in the family. For non-deletion cases, quantitative analysis after enzyme digestion or mutation detection from the DNA obtained from chorionic villus sample will be necessary.[30] The susceptibility of the SMA locus to de-novo deletions and earrangements is another concern in the prenatal prediction of SMA, especially in the families for which DNA samples from index cases are not available.[31] Preimplantation genetic diagnosis in families at risk has also been reported.[32] Genetic Counseling The correct prediction of the risk of recurrence of the disease and genetic counseling would partly depend on the molecular confirmation of the diagnosis. DNA testing for mutation detection is carried out in peripheral blood and does not involve any risk to the patients. For autosomal recessive cases (two affected children in consanguineous family), the risk of recurrence is 25% in subsequent pregnancies. In the absence of molecular diagnostic services, the empirical risk figures are given in [Table III].[33],[34] Therapy for Spinal Muscular Atrophy In recent years the study of molecular genetics has created lot of excitement over the possibility of newer drug treatments for SMA. In 92% of SMA patients, SMN1 gene is missing but they do have intact SMN2 gene. In normal circumstances, SMN2 gene produces very little amount of SMN protein. Therefore, the current strategies are based on modulating the genetic machinery to increase the amount of SMN protein from SMN2 gene.[35] Recently, a compound sodium butyrate has shown lot of promise in tissue culture and in mouse models for SMA by increasing the expression of SMN protein from SMN2 gene. The treatment also resulted in significant improvement of clinical symptoms of SMA in mice. However, safety and efficacy of the new drug in humans is still under progress.
Spinal muscular atrophies constitute an important group of neuromuscular disorders with genetic heterogeneity. In the absence of effective treatment for the disease, prenatal prediction and selective termination of affected fetus is an acceptable preventive option, especially for the more severe forms of the disease. With increasing availability of simpler molecular techniques, more families are likely to benefit from DNA analysis. Recent advances in molecular pathogenesis are also likely to help in development of novel drugs and therapeutic modalities for spinal muscular atrophy.
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