Original Article Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients Rashna S. Dastur, Pradnya S. Gaitonde, Satish V. Khadilkar*, Vrajesh P. Udani**, Jayshree J. Nadkarni Department of Neuropathology and Applied Biology, Medical Research Centre, *Department of Neurology, Bombay Hospital, Mumbai, **Department of Neurology, P. D. Hinduja National Hospital and Medical Research Centre, Mumbai, India Background: Spinal muscular atrophy (SMA) is an Introduction autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting Spinal muscular atrophies (SMA) are a clinically and genetically in progressive muscle weakness and atrophy. Aims: The heterogeneous group of disorders characterized by primary molecular analysis of two marker genes for spinal degeneration of anterior horn cells of the spinal cord and brain muscular atrophy (SMA) i.e., the survival motor neuron gene stem nuclei causing progressive muscle weakness.[1] (SMN) and the neuronal apoptosis inhibitory protein gene SMA is one of the most common lethal autosomal recessive (NAIP) was conducted in 39 Indian patients with clinical disorder, with the childhood onset form of SMA between 1 in symptoms of SMA. Out of these, 28 showed homozygous 6000 and 1 in 10,000 live births.[2,3] The three types of SMAs deletions and the phenotypic features of these SMA patients present with diverse symptoms and differ in age of onset, mode of were compared with the corresponding genotypes. inheritance, distribution of muscle weakness and progression of Settings: A tertiary care teaching Hospital. Design: This is symptoms.[2] a prospective hospital based study. Materials and SMA of all types is associated with homozygous mutations in Methods: Polymerase chain reaction (PCR) combined with the Survival Motor Neuron gene (SMN1 gene) located at the restriction fragment length polymorphism (RFLP) was used telomeric end of chromosome region 5q13.[4,5] A large inverted to detect the deletion of exon 7 and exon 8 of SMN1 gene, repeat at the centromeric region of 5q13 consists of homologous as well as multiplex PCR for exon 5 and 13 of NAIP gene. gene SMN2. SMN1 and SMN2 differ in exons 7, 8 and 6 by only Results: Exons 7 and 8 of SMN and NAIP (exon 5) were one base pair each and in five introns.[2,6] homozygously deleted in 73% of SMA I and 27% of SMA II The presence of a C ->T transition at codon 280 within exon 7 patients. SMN exon 7 and 8 deletions without NAIP of SMN2 causes disruption of an exonic splice enhancer.[7] This deletions were seen in 27% of type I SMA and 46% of SMA leads to abnormal splicing which produces unstable truncated type II patients. Two patients of type III SMA showed single [8] deletion of SMN exon 7 along with 27% of SMA type II protein. SMN2 does produce a small amount of functional full- patients. Conclusion: With the advent of molecular biology length protein but the majority of the full-length protein is [9] techniques, SMN gene deletion studies have become the produced by SMN1. first line of investigation for confirmation of a clinical Another gene, the neuronal apoptosis inhibitory protein gene diagnosis of SMA. The findings of homozygous deletions (NAIP), located on the same chromosomal region of 5 q 13, was of exons 7 and/or 8 of SMN1 gene confirms the diagnosis found to be deleted in SMA patients.[1,10] The NAIP has been of SMA, even in patients with atypical clinical features. postulated to have neuroprotective effect and acts as a negative Deletions of NAIP gene were mainly seen in severely regulator of motor neuron apoptosis.[10] It was suggested that the affected patients, hence is useful for predicting the absence of the full-length NAIP gene is necessary but not sufficient prognosis. to produce SMA.[10] The purpose of this study was to confirm the clinical diagnosis Key words: Neuronal apoptotic inhibitory protein gene, spinal muscular atrophy, survival motor neuron gene of SMA patients and to correlate the frequency of deletions within SMN and NAIP genes. The study mainly concentrates on the J. J. Nadkarni Department of Neuropathology and Applied Biology, Medical Research Centre, Bombay Hospital Trust, New Marine Lines, Mumbai - 400 020, India. E-mail: [email protected] Neurology India | September 2006 | Vol 54 | Issue 3 255 CMYK255 Dastur et al.: SMN and NAIP gene deletions in SMA pattern of gene deletions seen in three different types of SMA The nucleotide in exon 8 which differs between SMN 1 and patients and their correlation with the clinical severity. It does not SMN 2 genes is G>A. The A in the SMN 2 gene (located at attempt to determine the prevalence of deletions in clinically position 1155 of the cDNA) creates a Dde I site which will digest suspected SMA patients. the amplified DNA into two products of 122 bp and 78 bp.[6,11] The exon 8 of SMN1 gene (200 bp) does not contain any Dde Materials and Methods I, site hence it will not be cut and will remain as 200 bp.[2] A total of 49 blood samples received between the period of NAIP exon 5 September 2004 and December 2005, all from the Indian For NAIP, exon 5 and 13 (exon 13 taken as positive control), a population, were studied for SMN1 and NAIP gene deletions. multiplex reaction was carried out for 30 cycles of 94oC for 1 min, The 39 patients included in the study were strongly suspected of 60oC for 1 min and 72oC for 1 min using primers 1864 and 1863 SMA after a full clinical evaluation. Ten samples from non-SMA for exon 5 and primers 1258 and 1343 for exon 13.[10,12] patients were also run as controls. PCR products from both SMN gene products and NAIP were run on 2% agarose high fidelity gel (Sigma) stained with ethidium Detection of SMN1 and NAIP gene deletions by PCR­ bromide in 1x TBE buffer and visualised under UV RFLP method transilluminator and gel documentation system (Vilber Lourmat Genomic DNA was extracted using sigma NA 2020 kit from - France). peripheral blood (collected in EDTA bulbs). Polymerase chain SMA was classically subdivided into three types based on age of reaction (PCR) amplification of SMN exons 7 and 8 and NAIP onset and clinical severity. The standard ISMAC (International exons 5 and 13 was carried out in a total volume of 50 µl using SMA consortium) classification for SMA given in Table 1 was Hot Star Taq Master Mix (QIAGEN) for SMN (7 and 8) gene used for this study.[13] and QIAGEN multiplex PCR kit for NAIP exons 5 and 13. The Hot Star Taq used in all the PCR reactions was activated at 95oC Results for 15 mins before the cycles commenced. Molecular analysis using PCR-RFLP assay, on 39 clinically SMN gene suspected cases of SMA revealed 28 patients with homozygous Exon 7 was amplified with intron 6 primer R111 (2 µM) and deletions in SMN1 or/and NAIP genes [Table 2]. Out of these 28 mismatch primer X7-Dra (2 µM) in 35 cycles of 1 min at 94oC, 1 SMA patients showing deletions, the maximum number (54%) min at 55oC and 1 min at 72oC.[6,11] PCR products (25 µl) along were of Type II followed by Type I (39%) and only 7% were Type with 1 x enzyme buffer SM (25 µl) were subsequently digested to III. The classification into different types was based on the ISMAC. completeness with restriction enzyme Dra I (2 units) (Sigma) SMA I: Eleven SMA I patients showed homozygous deletions [Table 1]. for SMN1 gene and/or NAIP gene [Table 2]. Their ages ranged By using a mismatch primer (X7-Dra) on the 3’ end of exon 7, between 1.5 months to 2 years. Ten patients were between 1.5 a G to A change is introduced at positive 875 of the cDNA. This months to 10 months; only one patient was age two years, who change creates a Dra I site (TTTAAA). The third T nucleotide died shortly. All these patients were unable to sit or hold head and of the sequence is present only in the SMN 2 gene and is a C in the were floppy infants. EMG was possible only in four of the 11 SMN 1 gene, thus the enzyme cuts only the SMN 2 amplified patients and showed AHC (anterior horn cell disease). PCR product.[6,11] Hence, after digestion two bands are seen, one The deletion analysis for SMN1 and NAIP genes of these at 188 bp (indicating SMN 1) and the other at 176 bp patients showed 73% to have homozygous deletions of both SMN1 (representing SMN 2) as gene amplified products. gene (exons 7 and 8) and NAIP gene (exon 5) (Figure 1 lane 4c, For exon 8, DNA was amplified with primers 541C960 and 7c and 10c), while 27% showed deletions of exons 7 and 8 of only 541C1120 (2 µM each), with the same PCR conditions, except the SMN1 gene. None of the patients of SMA type I revealed a for an annealing temperature of 59oC instead of 55oC.[6,11] PCR deletion of only exon 7 of the SMN gene. It was observed in these products (25 µl) were subsequently digested with restriction 11 patients that a severe phenotype is manifested mainly when enzyme Dde I (2 units). deletions occur in both exon 7 and exon 8 of SMN and/or NAIP gene [Table 2]. Table 1: Classification of spinal muscular atrophies.[13] SMA II: Fifteen patients showing homozygous deletions were Designation Symptom Course Survival classified as SMA II, which is the largest group in our study onset (54%).