(SMN) and Neuronal Apoptosis Inhibitory Protein ... - Springer Link

667_671_Mittal_JON_1714 02.06.2005 12:11 Uhr Seite 667

J Neurol (2005) 252 : 667–671 DOI 10.1007/s00415-005-0714-2

Akanchha Kesari Usha Kant Misra Jayantee Kalita Vijay Nath Mishra Sunil Pradhan Siddramappa Jagdish Patil Shubha Rajender Phadke Balraj Mittal

Received: 2 August 2004 Received in revised form: 14 October 2004 Accepted: 26 October 2004 Published online: 18 March 2005

A. Kesari · S. J. Patil · S. R. Phadke · Prof. Dr. B. Mittal () Dept. of Genetics Sanjay Gandhi Postgraduate Institute of Medical Sciences Raebareli Road Lucknow – 226014, India Tel.: +91-522/2668-005-8 extn. 2322 (Off), 2329 (Lab) Fax: +91-522/2668-017 / 2668-973 / 2668078 E-Mail: [email protected] [email protected]

ORIGINAL COMMUNICATION

Study of Survival of Motor Neuron (SMN) and Neuronal Apoptosis Inhibitory Protein (NAIP) gene deletions in SMA patients

U. K. Misra · J. Kalita · V. N. Mishra · S. Pradhan Dept. of Neurology Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow, India

■ Abstract In view of the paucity of deletion studies of survival of motor neuron (SMN) and neuronal apoptosis inhibitor protein (NAIP) genes in Indian SMA patients, this study has been undertaken to determine the status of SMN1, SMN2 and NAIP gene deletions in Indian SMA patients. Clinically and neurophysiologically diagnosed SMA

Introduction

■ Key words SMN1 · SMN2 · NAIP · spinal muscular atrophy

the neuronal apoptosis inhibitor protein (NAIP) gene [4]. A highly homologous copy of the SMN1 gene, the survival of motor neuron gene 2 (SMN2) is also present in the centromeric-most repeat of the chromosome 5. Lefebvre et al. [3] showed that the SMN1 gene is deleted or interrupted in 98.6 % of SMA patients. However, SMN2 is not directly associated with disease because homozygous deletions of SMN2 gene are also observed in 2–5 % of normal individuals. The high SMN1 deletion frequency in childhood SMA is supported by many studies [5–8]. In addition to deletion, the presence of subtle mutation on one of the chromosomes in a number of non deletion SMA patients have been described [5, 6, 9, 10] making SMN1 the responsible gene for proximal SMA. Type IV patients show only 20–30 % homozygous deletion of SMN1 gene [11, 12]. NAIP, the other candidate gene, shows homology with a baculoviral inhibitor of apoptosis and inhibits apoptosis in mammalian cells [13]. Absence of homozygous deletions of NAIP was found in 45 % of SMA type I

JON 1714

The spinal muscular atrophies belong to a heterogeneous group of disorders, which are responsible for early childhood mortality. Proximal SMA is an autosomal recessive neuromuscular disorder, which leads to symmetrical muscle weakness and atrophy. It has an estimated incidence of 1 in 10,000 live births [1], and a carrier frequency of 1 in 40. Clinically, proximal SMA is further divided into four types based on the age of onset and severity of the clinical course. Childhood onset SMA can be classified into three types (Type I–III) [2]. Type IV SMA is an adult form with onset after 30 years of age with variable severity. By molecular genetic analysis, all three forms of childhood SMA were mapped to chromosome 5q11.2-q13.3. In early 1995, two candidate genes were identified in the telomeric part of critical region harbouring a large inverted repeat: the survival of motor neuron 1 (SMN1) gene [3] and the gene encoding

patients were included in the study. A gene deletion study was carried out in 45 proximal SMA patients and 50 controls of the same ethnic group. Both SMN1 and NAIP genes showed homozygous absence in 76 % and 31 % respectively in proximal SMA patients. It is proposed that the lower deletion frequency of SMN1 gene in Indian patients may be due to mutations present in other genes or population variation, which need further study.


668

patients and in 18 % of type II and III SMA patients [4]. Similar results have been reported by other groups as well. Deletions in NAIP appear to be associated with increased severity of the disease, although the association is not absolute [4, 14]. Within the last few years, there have been several reports on SMN and NAIP gene deletions in SMA patients of different ethnic origins: including French [3], Chinese [12], African [15], American [16] and many other groups. However, detailed information pertaining to the analysis of these genes in clinically diagnosed SMA patients from India is still lacking. Hence, the aim of our study was to evaluate the status of SMN and NAIP genes in Indian patients referred with the diagnosis of proximal SMA.

Molecular analysis of SMN gene by PCR-RFLP Polymerase chain reaction was used to amplify exons 7 and 8 of SMN gene with an initial denaturation at 96 °C for 7 min was followed by 35 cycles (94 °C, 62 °C and 72 °C for 1 min each). The primer sequences for exons 7 and 8 were taken from Chang et al. (1997) [12]. PCR products of exons 7 and 8 were digested with Dra I and Dde I, respectively. The digested products were analysed on 10 % Poly acrylamide gel electrophoresis (PAGE) and photographed by Alpha Imager™ 1220 Documentation and Analysis System (Alpha Innotech Corporation, USA). Molecular analysis of NAIP gene by PCR All individuals were also tested for exon 5 deletion of NAIP gene. PCR conditions and primers used to amplify the exon 5 were identical to those of Somerville et al. (1997) [14]. The PCR was started with initial denaturation at 96 °C for 7 min followed by amplification for 30 cycles (94 °C, 60 °C and 72 °C for 1.30 min each) followed by a final extension for 10 min at 72 °C. The PCR products were analysed on 2 % agarose gel containing ethidium bromide.

Materials and methods ■ Subjects

Results

Patients from January 2000–January 2004 recruited from Genetic and Neurology outpatients departments (OPD) of Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow, were included in the study. A detailed clinical evaluation was recorded including age, sex, age of onset, present status of functional ability, associated features like respiratory muscle weakness, detailed antenatal history, reduced fetal movements, feeding difficulties and developmental history. Family history and consanguinity was also recorded. The patients with clinical suspicion of neuromuscular disorders other than proximal SMA were excluded from this study. Clinical diagnosis of SMA was based on the criteria of Munsat and Davies (1992) [17]. All patients showed features consistent with anterior horn cell involvement identified by neurological examinations, and EMG study carried out at the centre. The clinical features like symmetrical muscle weakness of trunk and limbs, proximal muscles weakness more than distal, lower limbs involvement more than upper limbs, fasciculations of tongue and tremor of hands were seen in the patients. Muscle biopsies were carried out in selected cases to confirm the diagnosis. The patients (if alive) were followed for 1–2 years. Fifty healthy age and sex matched individuals without any history of neurological disease and normal on neurological examination were included as controls.

■ Clinical Features

Deletion Analysis of SMN and NAIP gene

■ Methods Five ml blood was collected in EDTA tubes from 45 patients and 50 controls. DNA was extracted from blood by the standard salting out method [18].

Table 1 Clinical features of SMA patients

Forty five patients with proximal SMA (29 males, 16 females) compiled with the International inclusion criteria were included in the study. Twelve had type I SMA, 7 type II, 23 type III and 3 type IV SMA. All the patients and controls were of Indian ethnic origin and 17 were Muslim. In 8 Muslim patients a history of consanguineous marriage was present. The age of onset for neuromuscular disorder was from infancy to 3 months of age in type I SMA, 6 to 18 months in type II patients, 2.5 to 30 years in type III and in type IV it ranges from 39 to 58 years. Delayed motor milestones and hypotonia were observed in all type I patients. All the patients in the study showed the neurogenic patterns on EMG and their CPK levels were in normal range (Table 1).

The PCR amplification of exon 7 product digested with Dra I gave three products (188,149 and 39 bp); the 39bp product runs out of the gel (Fig. 1). Exon 8 PCR product digested with Dde I gave three products after digestion

Type of SMA

Type I

Type II

Type III

Type IV

Age of Onset Sex ratio (M:F) Positive family history (n) Consanguinity (n) Ethnicity (Hindu: Muslim) EMG CPK* (IU)

Birth to 3 Months 7:5 8 3 9:3 Neurogenic 222 (81 to 584)

6 to 18 Months 4:3 2 0 5:2 Neurogenic 157 (63–241)

2.5 to 30 Years 17:6 8 5 16:7 Neurogenic 238 (91–882)

39 to 58 Years 1:2 0 0 3:0 Neurogenic 204 (178–231)

* Values are in Median (Range), Normal range of CPK (20–200 IU); n Numbers of patients


669

Fig. 1 Exon 7 PCR-RFLP. Polyacrylamide gel photograph showing the undigested and digested products of SMN exon 7 after Dra I digestion. Lane 1, 3 undigested product of patient; lane 2, digested product of patient with SMN1 deletion; lane 4, digested product of patient without SMN1 gene deletion; Lane 5, undigested product of control; Lane 6, digested product of control with no SMN1 deletion. Lane 7, 50 bp DNA ladder.

(187, 123 and 64bp) (Fig. 2). The absence of 188 bp and 187 bp bands showed homozygous deletion of exons 7 and 8 of SMN1 gene respectively (Figs. 1, 2). Out of 45 SMA patients, 34 (76 %) showed homozygous deletion of SMN1 gene. In type I SMA patients, exon 7 of SMN1 gene was deleted in 91 %. There was slightly higher frequency of homozygous absence of SMN1 in type III than in type II SMA patients i. e. 78 % vs 72 %. Absence of both exons 7 and 8 was observed in 31 patients but 3 (9 %) patients showed only deletion of exon 7, whereas exon 8 of SMN1 gene was present (Table 2). None of Type IV patients showed homozygous absence of SMN1 gene. None of patients and controls showed SMN2 gene deletion. Fourteen (31 %) patients showed deletion of NAIP gene (Fig. 3). In our study, we observed 58 % of deletion

Fig. 2 Exon 8 PCR-RFLP. Polyacrylamide gel photograph showing the undigested and digested products of SMN exon 8 after Dde I digestion. Lane 1, undigested PCR product of SMA patient; lane 2 digested product of patient with SMN1 gene deletion; Lane 3, undigested product of normal control; lane 4 digested product of control without SMN1 deletion. Lane 5, 50 bp DNA ladder

Table 2 Deletion analysis results of SMN1 and NAIP gene in SMA patients Proximal SMA (n = 45)

Number of patients

Status of SMN1 gene Deletion

Status of NAIP gene Deletion

SMA I SMA II SMA III SMA IV

12 7 23 3

11 (91%) 5 (72%)a 18 (78%)b 0

7 (58%) 1 (14%) 6 (26%) 0

a two patients had deletion of exon 7 only; b one patient had deletion of exon 7 only n Number of patients

Fig. 3 Agarose gel photograph showing the products of NAIP-PCR. Lanes 1, 3 are type I patients with NAIP deletion. Lanes 2, 4 are type III patients with no deletion of NAIP gene. Lane 5 is type II SMA patient with NAIP gene deletion. Lane 6 is control sample.

of NAIP gene in Type I patients, but Type II and III patients showed 14 % and 26 % deletion respectively. Type IV patients did not show homozygous absence of NAIP gene. NAIP deletion in type I, II and III patients is in concordance with the reported frequency from other populations.

Discussion This study examines the clinical and genetic basis of SMA in Indian patients. It has been the experience of our laboratory that the clinical symptoms of SMA are not sufficiently specific to make a reliable diagnosis. EMG provides good supportive evidence for the diagnosis but may be inconclusive in some cases. There is no single diagnostic test; hence the diagnosis has to be based on clinical symptoms. Detection of deletion in SMN1 confirms the diagnosis of SMA and also provides reason for prenatal diagnosis. Hence, DNA test for SMN1 deletion has great importance in diagnosis of SMA. In our study 76 % of patients with SMA were found to be homozygous for deletion of SMN1 exon 7 and/or exon 8 and 31 % were found to be homozygous for NAIP deletion. The majority of type I patients showed large deletions of SMN1 and NAIP, whereas most of type II and III show deletions of SMN1 only. All the NAIP deleted samples also showed deletion of SMN1 gene. In none of our patients was NAIP deletion without SMN1 gene deletion detected. Since NAIP deleted individuals were a subset of those who had the SMN1 gene deletion, so NAIP deletion does not appear to be an independent risk factor for SMA [19]. In our study, 91 % of clinically defined type I SMA patients lacked both copies of SMN1, compared with 78 % and 72 % of type III and type II patients. In an earlier study from India, Dua et al. [20] reported approximately 55 % of floppy infants with clinical features of SMA had homozygous deletion of exon 7 of SMN1 gene. There may be several reasons for the variation in the frequency of SMN1 gene deletion in different studies. First, SMA is a part of a wide spectrum of anterior horn cells disorders that affect infants and young children and clinical


670

symptoms are overlapping and may not be sufficiently specific to make a reliable diagnosis of SMA. Patients who are referred for molecular diagnosis generally have symptoms, like hypotonia, proximal muscle weakness, and loss of ambulation. These symptoms are not specific to 5q linked SMA and in infants there are additional complications. Muscle testing and EMG examination at times may be difficult in a neonate to perform. This may be one of the reasons for discrepancy in the deletion frequency in our patients group. The other reason for the difference in the frequency may be the population variation as in South African black patients 65 % [21] had deletion, Saudi Arabian patients had 72 % deletion [22], Polish had 63 % [23] and Vietnamese showed 41 % [24] deletion, while many other studies showed more than 90 % of deletion in SMN1 gene. We have observed that 2 of our type II patients and 1 of type III patient had deletion of exon 7 only but retaining exon 8 of SMN1 gene, this may be due to a gene conversion event at this locus. The gene conversion event is more common in type II and III patients, than in type 1 SMA patients [25, 26]. Large deletions in type 1 patients may result from unequal cross-over of repeat units at SMN locus whereas gene conversion in type II and III SMA may be facilitated by sequence homology between SMN1 and SMN2 genes Only 31 % of the patients with SMA were found to have deletion of NAIP gene. In type I SMA higher percentage (58 %) of NAIP deletion has been observed as compared with type II and III SMA (Table 2). NAIP gene deletions frequencies in our patients are in concordance with the reported frequency from other populations [7, 27]. From the data, it is clear that NAIP deleted individuals are a subset of those who also have the SMN1 gene

deletion. NAIP gene deletion was not present in all type I patients and it was not absent in all type II and type III patients, as 14 % and 26 % of type II and type III patients had deletion of NAIP gene. NAIP was not deleted in many of type I patient despite a severe course and early onset of disease. Our data thus substantiate the view that majority of type I SMA cases result from large deletions encompassing both SMN and NAIP gene but it does not support the role of NAIP in disease severity. Taylor et al. [28] have shown that small mutations within the SMN1 gene do not disrupt NAIP but may still give rise to severe phenotype. These results suggest that NAIP may not have a direct role in SMA severity but simply delimits the extent of deletion within the SMA region [29]. We agree with others studies [25] that NAIP may be coincidentally deleted with SMN gene. The primary defect in SMA lies in the anterior horn cells.A subset of SMA patients present at birth with contractures and muscle atrophy, suggesting a defect in the development of anterior horn cells or early degeneration. There exists a small but significant number of neuromuscular disorders that fulfil the criteria for the diagnosis of SMA but have added features leading to uncertainty as to whether they are the same or different diseases. By detecting a mutation of SMN1 gene on the long arm of chromosome 5, it became possible to confirm the diagnosis of proximal spinal muscular atrophy. Point mutations have been reported in 3–4 % of patients without homozygous deletion of SMN1 gene [2, 3]. A negative result for homozygous absence of SMN1 decreases, but cannot entirely exclude, the probability that the patient has 5q linked SMA. It is possible that some of our patients may have mutations, which are present in other genes yet to be identified.

References 1. Ogino S, Leonard DG, Rennert H, Ewens WJ, Wilson RB (2002) Genetic risk assessment in carrier testing for spinal muscular atrophy. Am J Med Genet 110:301–307 2. Biros I, Forrest S (1999) Spinal muscular atrophy: Untangling the knot. J Med Genet 36:1–8 3. Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benechou B, Cruaud D, Millasseau P, Zeviani M, Le Paslier D, Frezal F, Cohen D, Weissenbach J, Munnich A, Meiki J (1995) Identification and characterization of a spinal muscular atrophydetermining gene. Cell 80:155–165

4. Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z, Farahani R, Baird S, Besner-Johnston A, Lefebvre C, Kang X (1995) The gene for neuronal apoptosis inhibitor protein is partially deleted in individuals with spinal muscular atrophy. Cell 80:167–178 5. Bussaglia E, Clermont O, Tizzano E, Lefebvre S, Bürglen L, Cruaud C, Urtizberea JA, Colomer J, Munnich A, Baiget M, Melki J (1995) A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients. Nature Genet 11:335–337

6. Hahnen E, Forkert R, Marke C, Rudnik-Schoneborn S, Schonling J, Zerres K, Wirth B (1995) Molecular analysis of candidate genes on chromosome 5q13 in autosomal recessive spinal muscular atrophy: evidence of homozygous deletions of the SMN gene in unaffected individuals. Hum Mol Genet 4:1927–1933 7. Velasco E, Valero C, Valero A, Moreno F, Hernandez-Chico C (1996) Molecular analysis of the SMN and NAIP genes in Spanish muscular atrophy (SMA) families and correlation between number of copies of cBCD541 and SMA phenotype. Hum Mol Genet 5:257–263


671

8. Bouhouche A, Benomar A, Birouk N, Bouslam N, Ouazzani R, Yahyaoui M, Chkili T (2003) High incidence of SMN1 gene deletion in Moroccan adult-onset spinal muscular atrophy patients. J Neurol 250:1209–1213 9. Rodrigues NR, Owen N, Talbot K, Patel S, Muntoni F, Ignatius J, Dubowitz V, Davies KE (1996) Gene deletions in spinal muscular atrophy. J Med Genet 33:93–96 10. McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes A (1997) Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet 60:1411–1422 11. Brahe C, Servidei S, Zappata S, Ricci E, Tonali P, Neri G (1995) Genetic homogeneity between childhood-onset and adult-onset autosomal recessive spinal muscular atrophy. Lancet 346:741–742 12. Chang JG, Jong YJ, Lin SP, Soong BW, Tsai CH, Yang TY, Hang CP, Wang WS (1997) Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents. Hum Genet 100:557–581 13. Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R, McLean M, Ikeda JE, MacKenzie A, Korneluk RG (1996) Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature 379:349–353 14. Somerville MJ, Hunter AGW, Aubry HL, Korneluk RG, Mac Kenzie AE, Surh LC (1997) Clinical application of the molecular diagnosis of spinal muscular atrophy. Am J Med Genet 69: 159–165

15. Wilmshurst JM, Reynolds L, Van Toorn R, Leisegang F, Henderson HE (2002) Spinal muscular atrophy in black South Africans: concordance with the universal SMN1 genotype. Clin Genet 62:165–168 16. Ogino S, Leonard DG, Rennert H, Wilson RB (2002) Spinal muscular atrophy genetic testing experience at an academic medical centre. J Mol Diagn 4:53–58 17. Munsat TM, Davies KE (1992) Meeting report: International SMA consortium meeting. Neuromuscul Disord 2: 423–428 18. Olerup O, Zetterquist H (1992) HLADR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 39:225–235 19. Crawford TO, Pardo CA (1996) The neurobiology of childhood spinal muscular atrophy. Neurobiol Dis 3:91–110 20. Dua T, Das M, Kabra M, Bhatia M, Sarkar C, Arora S, Sharma MC, Kalra V (2001) Spectrum of floppy children in Indian scenario. Indian Pediat 38: 1236–1243 21. Stevens G, Yawitch T, Rodda J, Verhaart S, Krause A (1999) Different molecular basis for spinal muscular atrophy in South African black patients. Am J Med Genet 86:420–426 22. Al-Rajeh S, Majumdar R, Awada A, alJumah M (1999) Application of DNAbased tests for diagnosis of spinal muscular atrophy in Saudi Arabia. East Mediterr Health J 5:1225–1229

23. Hausmanowa-Petrusewicz I (2001) Phenotype and genotype correlation in childhood spinal muscular atrophy. Neurol Neurochir Pol 35(Suppl 3): 29–35 24. Bach ND, Sadewa AH, Takeshima Y, Khanh TV, Sutomo R, Dao NT, Hoan NT, Dung VC, Hong DD, Harada Y Harada Y, Nishio H, Matsuo M (2003) Deletion of the SMN1 and NAIP Genes in Vietnamese Patients with Spinal Muscular Atrophy. Kobe J Med Sci 49:55–58 25. Campbell L, Potter A, Ignatius J, Dubowitz V, Davies KE (1997) Genomic variation and gene conversion in spinal muscular atrophy: implications for disease process and clinical phenotype. Am J Hum Genet 61:40–50 26. Talbot K, Rodrigues NR, Ignatius J, Muntoni F, Davies KE (1997) Gene conversion at the SMN locus in autosomal recessive spinal muscular atrophy does not predict a mild phenotype. Neuromuscul Disorders 7: 198–201 27. Rodrigues NR, Owen N, Talbot K, Ignatius J, Dubowitz V, Davies KE (1995) Deletions in the survival motor neuron gene on 5q13 in autosomal recessive spinal muscular atrophy. Hum Mol Genet 4:631–634 28. Taylor JE, Thomas NH, Lewis CM, Abbs SJ, Rodrigues NR, Davies KE, Mathew CG (1998) Correlation of SMNt and SMNc gene copy number with age of onset and survival in spinal muscular atrophy. Eur J Hum Genet 6:467–474 29. Simard LR, Rochette C, Semionov A, Morgan K, Vanasse M (1997) SMN(T) and NAIP mutations in Canadian families with spinal muscular atrophy (SMA): genotype/phenotype correlations with disease severity. Am J Med Genet 72:51–58