Clinical and Molecular Diagnosis of Spinal Muscular Atrophy I. Panigrahi, A. Kesari, S.R. Phadke, B. Mittal
Department of Medical Genetics Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014, India.
Summary 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.
Key words : Carrier analysis, Spinal muscular atrophy, SMN gene, SMA variants.
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Introduction 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 Correspondence to : Dr. B. Mittal, Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014, India. E-mail : [email protected]
described.2,3 SMA variants with gene deletions outside 5q13 further add to the genetic heterogeneity.4-7
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.
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Table I Clinical Features of SMA and its Variants Condition I. Classical SMA SMA Type I (Werdnig-Hoffman disease)
Onset between birth to 6 months, reduced fetal movements in utero, generalized weakness, hypotonia, areflexia, most die by 2 years of age.
SMA type II
Onset between 6-24 months of age, predominant proximal weakness, most will be able to sit, most will stand alone and some will walk, associated pes cavus, scoliosis, contractures, pseudohypertrophy of calves and gluteal muscles
SMA Type III (Kugelberg-Welander disease)
Begins after 18 months of age, proximal weakness, gait instability, slow progression, calf hypertrophy ±, facial weakness in severe cases, exaggerated physiological tremor
SMA Type IV
Late onset, in adulthood, slow progression, proximal weakness
Anterior horn cell disease with pontocerebellar hypoplasia
Floppiness at birth, brisk reflexes, jerky eye movements, abnormal EEG, multiple joint contractures, cerebellar atrophy
Spinal muscular atrophy with congenital fractures
Floppiness, IUGR, multiple congenital metaphyseal or epiphyseal long bone fractures, large joint and digital contractures, dysmorphic features
Spinal muscular atrophy with only early respiratory insufficiency
Generalized muscle weakness, acute respiratory distress in the neonatal period, distal joint contractures, diaphragmatic abnormalities
Spinal muscular atrophy with cong. heart defects
Floppiness, ASD, VSD, arthrogryposis, respiratory distress, bone fractures, CNS abnormalities
Spinal muscular atrophy with mental retardation
Early onset SMA, both SMA and MR were nonprogressive, small skull size, syndactyly
Spinal muscular atrophy (segmental)
Affects predominantly hand muscles, non-progressive, learning difficulties
Spinal muscular atrophy with congenital contractures
Severe hypotonia, muscle weakness, congenital or early-onset contractures.
Spinal muscular atrophy, distal, with upper limb predominance
Weakness and wasting (more in the upper limb) thenar muscles and first dorsal interossei, mean age of onset 17 years, in feet in 40%, reduced vibratory sense in 10% slow progression.
Spinal muscular atrophy, X-linked spinal and bulbar atrophy (Kennedy type)
Facial, bulbar and spinal proximal muscle atrophy, fasciculations, cramps tremor, gynecomastia and sexual dysfunction.
Spinal muscular atrophy, congenital non progressive, (of lower limbs)
Delayed walking, mild flexion contractures of knees, pes equinovarus, minimal involvement of jaw muscles and neck flexor
Spinal muscular atrophy with arthrogryposis
Floppiness, arthrogryposis, facial dysmorphic features, no other organ pathology
AR X-linked recessive (XLR)
Gene locus 12q23-q24 X-linked
AR = Autosomal recessive; AD = Autosomal dominant, IUGR = Intra uterine growth retardation, ASD = Atrial septal defect, VSD = Ventricular septal defect, MR = Mental retardation.
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Spinal Muscular Atrophy
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 KugelbergWelander 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) (Fig. 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 (Fig. 1b)
Table II Mutations in SMN Gene in Spinal Muscular Atrophy Patients Author/s Bussaglia et al3 Lefebvre et al2
Parsons et al36 Brahe et al37 Burglen et al38 Clermont et al39 Talbot et al40 Rochette et al41 Hahnen et al42 Gambardella et al43 Parsons et al44 Wang et al45 Wirth B19
Skordis et al20 Sossi et al21
Mutation of SMN 1
Exon (E) / Intron (I)
Type of mutation
430 del4 c 868-11del 7 c922+3del4 Y 272 C Del exon 7** 813ins/dup11 472del5 G275S E134K 618insT G279V P245L S262I Del exon 8 542delGT A2G G279C Q15X 124insT 241-242in4 c922+6T®G
E3 SJ* 16 SJ 17 E6 E7 E6 E3 E6 E3 E4 E7 E6 E6 E8 E4 E1 E7 E1 E2a E2b SJ17
Frameshift mutation Splice-site mutation Splice-site mutation Mis-sense mutation Deletion Frameshift mutation Frameshift mutation Mis-sense mutation Mis-sense mutation Frameshift mutation Mis-sense mutation Mis-sense mutation Mis-sense mutation Deletion Frameshift mutation Mis-sense mutation Mis-sense mutation Non-sense mutation Frameshift mutation Frameshift mutation Splice-site mutation
Q27ins G 425del5 W102X
E1 E3 E1
Non-sense mutation Non-sense mutation Non-sense mutation
*Splice-junction, **For more details on E7 deletion, see ref. no 20
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Fig. 1a : Diagrammatic representation of SMN locus on chromosome 5q, the three genes SMN, NAIP and P44 have centromeric and telomeric counter parts.
Fig. 1b : Difference in the nucleotide sequence between SMN1 and SMN2 genes.
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 (Fig. 2a). Similarly for exon 8, the restriction enzyme DdeI is used (Fig. 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 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 1
Fig. 2 : Deletion study of SMN genes a) draI digested PCR products of exon 7 b) DdeI digested PCR products of exon 8. Lanes 1, 2 and 3 represent SMN2 deletion, SMN1 deletion and normal control. The numbers shown on the right show size of the PCR product. Neurology India, 50, June 2002
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Risk of Recurrence of SMA Age of onset 1-3 3-5 5-15* 15-20*
Sibs 1 in 5 1 in 10 < 1 in 10 1 in 5
Offspring 1 1 1 1
in in in in
50 10 to 1 in 20 10 20
*If fresh dominant mutation, risk is 1 in 10
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 rearrangements 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
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.
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.
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Accepted for publication : 15th March, 2002.