Frequency of SMN1 exon 7 deletion in patients with spinal muscular atrophy in Kashmir

Meta Gene 15 (2018) 27–30 Contents lists available at ScienceDirect Meta Gene journal homepage: www.elsevier.com/locate/mgene Frequency of SMN1 exon 7 deletion in patients with spinal muscular atrophy in Kashmir MARK Shafia Syed, Mahrukh H. Zargar⁎, Arshad Pandith, Nabeela Khan, Rehana Ahmad, Qurteeba Mahajan, Wardha Qazi Advanced Centre for Human Genetics, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, J & K 190011, India A R T I C L E I N F O A B S T R A C T Keywords: Kashmir Spinal muscular atrophy Survival motor neuron Deletion Objective: Spinal muscular atrophy (SMA) is an autosomal recessive neuronal disorder resulting from degeneration of spinal motor neurons. Survival motor neuron (SMN) gene is the disease determining gene of SMA. In the present study molecular analysis was performed in 85 SMA patients as compared to 100 healthy controls from Kashmiri population. Method: The deletion in telomeric exon 7 of SMN1 gene was analysed in 85 SMA cases and 100 healthy controls by allele specific PCR followed by agarose gel electrophoresis. Result: The frequency of telomeric exon 7 deletion was 42.35% (36 of 85) in total SMA patients; 32 out of 61 SMA type I cases, 2 out of 13 type II SMA cases, 1 out of 6 type III cases and 1 out of 5 type IV SMA cases. Moreover the consanguinity was found in 57.64% of SMA patients. In healthy controls deletion was not found in any samples. Conclusion: There is a significant association of SMN1 homozygous exon 7 deletion and the occurance of SMA as compared to healthy controls. Although the frequency of SMN1 homozygous deletion in Kashmiri population is lower than the rest of the countries but we conclude the presence of exon 7 deletion in clinically suspected SMA patient should be treated as confirmation of diagnosis and therefore this test can be used as one of the useful tool for SMA diagnosis. 1. Introduction Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disorder and the second most common fatal autosomal recessive disorder after cystic fibrosis. With a worldwide incidence of 1/6000 to 1/10000 births and the carrier frequency of 1/ 50, it is the leading genetic cause of infant death globally (Roberts et al., 1970; Pearn, 1978; Czeizel and Hamula, 1989). Clinically, SMA is characterised by the progressive loss of alpha motor neurons in spinal cord anterior horn cells, leading to progressive proximal and symmetrical limb and trunk muscle weakness along with muscular atrophy. The routine activities such as walking, sitting up, crawling and controlling head movement are compromised. In more severe cases the muscles involved in breathing and swallowing are also involved. Poor muscle tone in SMA patients has been also associated with contractures and broken bones in children (Dubowitz, 1978). Depending upon the age of onset of symptoms, motor development milestones, severity of muscle weakness SMA has been categorised into four clinical subtypes by International SMA Consortium classification. ⁎ The most common and severe type is Type I SMA (Werdnig-Hoffmann I disease). This type has onset within first 6 months of life and the patients are not able to sit. Patients usually do not live past two years of age with severe respiratory distress being the most common cause of death. The type II SMA (also known as Dubowitz disease), is an intermediate form and symptoms usually has an onset from 6 months upto 2 yrs. of life. In this form of SMA children can sit up but still develop progressive muscle weakness and can never stand or walk on their own. The type III SMA (also called Kugelberg-Welander disease) has a chronic evolution and the symptoms appear after 2 yrs. of life. These patients are able to stand and walk unaided at least for some time in infancy but many later loose this ability. The type IV SMA (adult form) has an onset after 30 yrs. of life. The symptoms are mildest of all the forms and it mainly affects the proximal muscles of the extremities. The symptoms start gradually in adulthood with normal longevity (Zerrres and Davies, 1998). SMA has been frequently associated with a gene known as survival motor neuron 1 (SMN1) gene which is responsible for the production of a SMN protein. The SMN protein is essential for the maintenance of Corresponding author at: Advanced Centre for Human Genetics, Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir 190011, India. E-mail address: mhameedz@gmail.com (M.H. Zargar). http://dx.doi.org/10.1016/j.mgene.2017.10.005 Received 14 August 2017; Received in revised form 18 October 2017; Accepted 25 October 2017 2214-5400/ © 2017 Published by Elsevier B.V. Meta Gene 15 (2018) 27–30 S. Syed et al. Table 1 Frequency of SMN1 homozygous exon 7 deletion in different populations. Country No of Patients SMN1 EXON 7 deletion [Reference] Korea; 2001 Vietnam; 2003 South Africa(Johannesburg); 2007 Egypt; 2001 Russia; 2001 Brasilia; 1999 Algerian; 2009 India; 2005 Saudi Arabia; 1997 Morocco; 2003 Turkey; 2000 Germany; 1995 Spain; 1995 Holland; 1995 Japan; 2002 Tunis; 2006 Iran; 2004 UK; 1995 Kuwait; 2001 France; 1995 Taiwan; 1995 37 17 92 33 57 87 92 45 16 54 60 195 54 103 32 60 22 140 46 229 42 32% 41% 51% 55% 65% 69% 75% 76% 82% 83% 85% 90% 91% 93% 94% 95% 95% 97% 97% 98% 100% Cho et al. (2001) Duc Bach et al. (2003) Labrum et al. (2007) Shawky et al. (2001) Glotov et al. (2001) Kim et al. (1999) Sifi et al. (2013) Kesari et al. (2005) Al Rajeh et al. (1998) Bouhouche et al. (2003) Savas et al. (2000) Wirth et al. (1995) Bussaglia et al. (1995) Cobben et al. (1995) Akutsu et al. (2002) M'rad et al. (2006) Shafeghati et al. (2004) Rodrigues et al. (1995) Haider et al. (2001) Lefebvre et al. (1995) Tsai et al. (2001) both the inpatient and outpatient services from the Department of Neurology and Department of Paediatrics, Sher-i-Kashmir Institute of Medical Sciences Hospital and GB Panth Paediatrics Hospital in Kashmir. Data from all SMA patients was obtained from clinical examinations of patients and/or interviews with guardians. The study was approved by the ethics committee of the institute and all the patients/ guardians (in case of minors) and controls gave informed consent to participate in the study. specialised nerve cells called motor neurons. The SMN gene has been mapped to chromsomome 5q13.2. It contains 9 exons and encodes 294 aa protein. There are two highly identical copies of this gene: telomeric SMN1 and centromeric SMN2. Both share 99.8% sequence homology with only five nucleotide differences, one in intron 6, one in exon 7, two in intron 7, and one in exon 8 (Melki et al., 1990). These subtle differences do not alter the amino acids sequence, but it has an effect on mRNA splicing. The SMN1 transcript includes the exon 7 and produces a full-length and stable protein while SMN2 excludes the exon 7 and results in loss of a chunk of protein (Lorson and Androphy, 2000). The majority of SMN protein is produced by SMN1 gene and a small amount produced by the SMN2 gene. SMA phenotype is a result of SMN1 gene dysfunction. The SMN2 gene copy number affects the disease severity only because of the insufficient amount of protein produced by SMN2 gene as it excludes the exon 7. Several studies have shown a vast majority of SMA patients having the homozygous deletion in exon 7 in SMN1 gene but with varied frequency in different populations (Table 1) (Cho et al., 2001; Duc Bach et al., 2003; Labrum et al., 2007; Shawky et al., 2001; Glotov et al., 2001; Kim et al., 1999; Sifi et al., 2013; Kesari et al., 2005; Al Rajeh et al., 1998; Bouhouche et al., 2003; Savas et al., 2000; Wirth et al., 1995; Bussaglia et al., 1995; Cobben et al., 1995; Akutsu et al., 2002; M'rad et al., 2006; Shafeghati et al., 2004; Rodrigues et al., 1995; Haider et al., 2001; Lefebvre et al., 1995; Tsai et al., 2001). The SMN1 exon 7 deletion has been reported to be more prevalent among Spain, Holland, Japan, Tunis, Iran, UK, Kuwait, France and Taiwan with frequencies of > 90%, although lower frequencies have been found in Korea, Vietnam, Egypt and Johannesburg. Keeping in view the varied results of SMN1 exon 7 deletion in SMA patients across different populations, the present study has been undertaken to study the frequency of SMN1 exon 7 deletion in Kashmiri SMA cases. 2.2. Sample collection and DNA extraction About 2–3 mL of peripheral blood was collected from SMA patients and healthy controls in tubes containing ethylenediamine tetraacetic acid (EDTA) and DNA was isolated using a Zymogen (Irvine, CA, USA) DNA extraction kit. The quality of the DNA was checked by agarose gel electrophoresis. The extracted DNA was stored at − 20 °C for further use. 2.3. Mutation detection Allele specific PCR was used to detect exon 7 deletion in SMN1 gene using the primers described previously (Feldkötter et al., 2002). The primers specifically amplify exon7 of SMN1 gene and not SMN2 gene. To monitor the efficiency of PCR reaction, an internal control for the PCR was used. A control primers pair AAT1/2 was used to amplify a fragment of exon V of α1-antitrypsin gene (Newton et al., 1989). The primer sequences are given in Table 2. The PCR reaction was set in a final volume of 25 μl mixture containing 1× PCR buffer (Biotools), 0.2 mM dNTP mixture (biotools), 150 ng each primer (Sigma), 1 U Taq DNA polymerase (Biotools 5 U/μl), and 200 ng genomic DNA (0.2 μg/ μl). Amplification was done at 94 for 7 min, 30 cycles of 94 °C for 30 s, 58.5 °C for 30 s min, 72 °C for 30 s min followed by extension at 72 °C for 7 min. The PCR product was directly visualised under UV light after electrophoresis using 2–3% agarose gel (Genie, Bangalore, India). The PCR product of SMN1 was visualised at the corresponding size of 307 bp, while that of internal control AAT1/2 at 220 bp (Fig. 1). 2. Materials and methods 2.1. Study population A total of 85 SMA patients and 100 healthy controls from the Kashmiri population were included in the present study. The patient diagnosis was made in accordance with the guidelines set by the International SMA Consortium. The clinical classification of patients into four groups was done based on criteria given by International SMA Consortium (Zerrres and Davies, 1998). The patients were selected from 3. Result A total of 85 SMA patients and 100 healthy controls were studied for SMN1 exon 7 deletion from Kashmiri population. Of the 85 SMA patients, 61 cases were classified as type I SMA, 13 as type II SMA, 6 as 28 Meta Gene 15 (2018) 27–30 S. Syed et al. Table 2 Primers used for the PCR amplification. Gene SMN1 telSMNex7forw telSMNint7rev α1 antitypsin AATI AAT2 M 1 2 3 4 Primer sequence Amplicon size 5′-TTTATTTTCCTTACAGGGTTTC-3′ 5′-GTGAAAGTATGTTTCTTCCACGTA-3’ 307 bp 5’-TGTCCACGTGAGCCTTGCTCGAGGCCTGG-3′ 5′-GAGACTTGGTATTTTGTTCAATCATTAAG-3’ 220 bp 5 (Table 3).The mutation was present in 32 out of 61 type I SMA cases, 2 out of 13 type II SMA cases, 1 out of 6 type III SMA cases and 1 out of 5 type IV SMA cases (Table 4). The overall frequency of consanguineous marriages was 57.64%; 55.29% for 1st degree consanguineous marriages, 2.35% for 2nd degree consanguineous marriages and 42.35% for non-consanguineous marriages (Table 5). 4. Discussion SMA is a degenerative neuronal disease which has been associated with SMN gene. SMN gene has two nearly identical forms in humans, SMN1 and SMN2. The main difference between SMN1 and SMN2 gene is found in exon 7, which is critical for fully functional protein production. A single nucleotide change in the exon 7 (C in SMN1 and T in SMN2) is important for SMN RNA splicing. The resulting SMN1 mRNA includes exon 7 and produces a full length protein whereas the SMN2 mRNA excludes exon 7 and produces truncated protein (Melki et al., 1990; Lorson and Androphy, 2000). In SMA patients, a defect in SMN1 gene makes it unable to code for the SMN protein resulting in death of motor neuron cells. The defect is either a deletion occurring at exon 7 or due to other rare mutations. The homozygous exon 7 deletion in the SMN1 gene has been found in the majority of SMA patients in many studies (Table 1). In this study we analysed the homozygous SMN1 exon 7 deletion in 85 SMA affected patients as compared to 100 healthy controls from Kashmiri population. We found the deletions involving exon 7 were present in 42.35% of SMA patients and absent in all healthy controls (p < 0.0001). There was a higher frequency of homozygous deletion of SMN1 exon 7 in SMA type I cases (52.45%) than in type II, III & IV cases (15.38%, 16.66% and 20% respectively). In our study a lower frequency of SMN1 exon 7 deletion was observed than most of the earlier studies. Previously a lower frequency of exon 7 deletion was also observed among SMA population in Korea, Vietnam, Egypt and Johhanesburg (Cho et al., 2001; Duc Bach et al., 2003; Labrum et al., 2007; Shawky et al., 2001). In Vietnamese population the homozygous SMN1 deletion was found in 7 of 17 (41.17%) SMA patients (Duc Bach et al., 2003). In South African Black population SMN1 exon deletion was found in 51% (42 of 92) patients with SMA (Labrum et al., 2007). In Egyptian population, Shawky et al., has reported SMN1 exon 7 deletion to be present only 55% of cases studied (Shawky et al., 2001). Comparatively the SMN1 homozygous exon 7 deletion was higher in most of the studies conducted in other populations across the globe with frequencies > 90% (Table 1). This variability in the frequency of SMN1 exon 7 deletion in different populations could be explained by the fact that ethnic and geographic variations play an important role in the etiology of genetic diseases. In addition to exon 7 deletion the role of other mutations present in SMN1 gene in the pathogenesis of SMA cannot be excluded. Earlier some SMA cases have been found to be caused by other compound mutations, where one allele of SMN1 gene has exon 7 deletion and other allele has a subtle mutation. The studies have shown that the patients with these subtle mutations have significantly reduced SMN transcript levels which were comparable to those seen in patients with a homozygous deletion of SMN1 (Qu et al., 2012; Tiziano et al., 2010), thereby resulting in SMA. Fig. 1. Representative gel picture showing SMN1 amplicon at 307 bp and AAT1/2 amplicon at 220 bp. Lane M contains 100 bp DNA molecular weight marker. Lane 1,3 & 4 represents samples with non-deletion- having two bands at 220 bp + 307 bp. Lane 2 & 5 show samples with deletion having band only at 220 bp. Table 3 The frequency of homozygous SMN1 exon 7 deletion among SMA patients and healthy controls. Group No SMN exon 7 deletion P value Healthy Control SMA Patients 100 85 0 36 < 0.0001 Table 4 Frequency of homozygous SMN1 exon 7 deletion among Kashmiri SMA patients. Type I Type II Type III Type IV Total No of patients No of patients with SMN1 exon 7 deletion 61 13 6 5 85 32(52.45%) 2 (15.38%) 1(16.66%) 1(20%) 36(42.35%) Table 5 Frequency of Consanguinity in SMA patients. Degree of interbreeding No of patients Ist Degree 2nd Degree Total Consanguinity Unrelated 47 (55.29%) 2 (2.35%) 49(57.64%) 36 (42.35%) type III SMA and 5 as type IV SMA according to their medical history. In healthy controls the homozygous exon 7 deletion was not found in any of the samples. While in SMA cases the deletion was found in 42.35% of total SMA patients. A significant association was found between the homozygous exon 7 deletion and the occurance of SMA (p ≤ 0.0001) 29 Meta Gene 15 (2018) 27–30 S. Syed et al. Duc Bach, N., Hamim Sadewa, A., Takeshima, Y., et al., 2003. Deletion of the SMN1 andNAIP genes in Vietnamese patients with spinal muscular atrophy. Kobe J. Med. Sci. 49 (3–4), 55–58. Feldkötter, M., Schwarzer, V., Wirth, R., Wienker, T.F., Wirth, B., 2002. Quantitative analyses of SMN1 and SMN2 based on realtime light-cycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am. J. Hum. Genet. 70, 358–368. Glotov, A.S., Kiselev, A.V., Ivaschenko, T.E., Baranov, V.S., 2001. Analysis of deletions in SMN1, SMN2, and NAIP genes in spinal muscular atrophy patients from the northwestern region of Russia. Russ. J. 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Zerrres, K., Davies, K.E., 1998. 59th ENMC International Workshop: Spinal Muscular Atrophies: Recent Progress and Revised Diagnostic Criteria (17–19 April). Soestduinen, The Netherlands. In our study we found the rate of consanguinity was 57.64% in overall SMA cases. Most of the marriages in our region (Kashmir) are of consanguineous nature because of the cultural, social and religious background which has made Kashmiri population ethnically conserved through generations. As consanguinity is an important factor that contributes to the increased incidence of autosomal recessive disorder, the current data highlights the need for lowering the consanguineous marriages and the value of genetic counselling in such marriages to lower the prevalence of SMA in our Kashmiri population. Although the frequency of exon 7 deletion was lower in our SMA population than other populations, we suggest this test should be recommended to all suspected SMA patients before considering other invasive procedures like muscle biopsy and also to verify whether a patient has SMA or a different disorder that has phenotypical similarities. We conclude that the presence of a homozygous deletion in SMN exon 7 in a patient with clinically presumed SMA should be treated as confirmation of diagnosis, however a strongly suspected patient should be screened for other mutation in SMN1 gene as well. Currently as SMA has no treatment available, with the availability of genetic testing for SMA, an early diagnosis and appropriate genetic counselling to such families can be provided to decrease the risk of disease in future pregnancies. Acknowledgements This research was financially supported by the Sher-I-Kashmir Institute of Medical Sciences Hospital, Kashmir. Conflict of interest None declared. 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