Study of Genes of Spinal Muscular Atrophy in Children

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1 Chin J Contemp Pediatr Vol. 3 No. 1 Feb. 2001, (, ) [ ] (SMA) (SMN) (NAIP), SMA PCR PCR - 15 SMA ( 4, 3, 8 ) 20 SMA 30 SMN 7,8 NAIP 5,6 7 SMA 6 SMN 7 8,1 7 8 ;8 SMA 1 7 8,7 SMN ;15 SMA NAIP 5 / 6 SMA SMN 7,8,, SMA SMN, SMN 7,8,NAIP SMA, [ ] ; (SMN) ; (NAIP) [ ] R744 [ ] A [ ] (2001) Study of Genes of Spinal Muscular Atrophy in Children ZHAN G Li2Fang, YAN G Xiao2Su, XIAO Bo Depart ment of Neurology, Xiangya Hospital, Cent ral South U niversity, Changsha , China Abstract : Objective To study the survival motor neuron ( SMN) gene exon 7 and 8 and neuronal apoptosis inhibitory protein ( NAIP) gene exon 5 and 6 in Chinese patients with Type SMA, and to confirm the relationship between the deletions of SMN and NAIP genes with SMA phenotype. Methods PCR and PCR - enzyme methods were used to detect the deletions of NAIP gene exon 5 and 6 and SMN gene exon 7 and 8 in 15 SMA ( ) patients, 20 healthy relatives and 30 normal controls. Results Deletions of exon 7 and 8 of the telomeric SMN gene were 4/ 4, 2/ 3, 1/ 8 in Type,, SMA patients, respectively. One patient with Type lacked exon 7 but retained exon 8. No deletion was found in the controls (0/ 50). No deletion of exon 5 and 6 of the NAIP gene was detected in the patients, healthy relatives and controls. Conclusions Deletions of SMN gene exon 7 and 8 examined by PCR - enzyme digestion could be recommended as an accurate gene diagnostic method for SMA with Type and. However, the method was not as useful in Type as in and for the diagnosis of SMA. lower in Chinese SMA patients. The frequency of NAIP deletion was Key words : Spinal muscular atrophy ; Survival motor neuron gene ; Neuronal apoptosis inhibitory protein ; Child ( spinal muscular at ro2, 4 SMA phy, SMA) 5q 13. 1, (Survival moter neuron, SMN) 5q13 ( neuronal apoptosis inhibito SMA 5q ry protein, NAIP ) P44 [ ] ; [ ] [ ] ( :9621) [ ] ( ),, 6

2 1,: B TF2p44 RNA Exon7F5 A GACTA TCAACTTAA TTTCTGA TCA3 ; H4F5 [1 ], SMN Exon7R5 CCTTCCTTCTTTTTGA TTTTGTTT3 ; SMA Exon 8 R 5 CTACAACACCCTTCTCACA G 3 ;, SMNt, Exon 8 F5 GTAA TAACCAAA TGCAA TGTGAA3 ; SMNc, 5 ( Exon 5 R 5 CTCTCA GCCTGCTCTTCA GA T 3 ; %) SMA SMNt ( Exon 5 F 5 AAA GCCTCTGACGA GA GGA TC 3 ; 7 8 7), Exon 6 F 5 TGCCACTGCCA GGCAA TCTAA 3 ; SMA ; Exon 6 R 5 CA TTTGGCA TGTTCCTTCCAA 3 ; SMN NAIP ( 5 6) SMA PCR - Exon 1 3 R 5 A TGCTTGGA TCTCTA GAA TGG3 PCR 15 SMA PCR : SMN 7,8 SMN,NAIP, SMA 95 5 min,95 45 s,55 45 s, SMA 5 h NAIP 5, SMA 20 ; 4 min,94 30 s,55 60 s,72 1 min, 30, 72 7 min,5 30 SMA 435 bp,6 120 bp, ( ENMC) bp PCR [2 SMA ],,, 4, 6 puc19dna/ Msp I ; 3, 6 ; 8, ml ( ), 2. 1 [35 ] Exon 1 3 F5 CCA GCTCCTA GA GAAA GAA GGA 3 ; min, 35, min,pcr 190 bp SMN 7,8 : 7,8 PCR 15 l, DraI 7 U,DdeI 3. 6 U 30 l, 37 2 DNA PCR, SMA 1 1 SMA Table 1 Clinical data of Type SMA patients EM G ( + ) SMN,NAIP SMN 7 PCR,, bp 68 bp 2, SMA 190 bp, 2 NAIP 5 13 PCR 164 bp,24 bp 3 (24 bp, ), 435 bp 243 bp 2, bp 1, 1 ; SMN SMA 190 bp,120 bp,68 bp 3 13 PCR 120 bp 243 bp 2 8 PCR,, bp 1 7

3 3 (13, ) 3,4 SMA SMN,NAIP 2 2 SMA SMN,NAIP Table 2 Results of the molecular analysis of SMN and NAIP genes in Type SMA patients ( %) SMN Exon 7 SMN Exon 8 NAIP Exon 5 NAIP Exon (100) 4 (100) 0 (0) 0 (0) 3 3 (100) 2 (66. 7) 0 (0) 0 (0) 8 1 (12. 5) 1 (12. 5) 0 (0) 0 (0) 3 NAIP 5 PCR 1-5 : - SMA ;6-7 ; ;8-9 : ;M : :pucdna/ Msp Figure 3 Amplified PCR products of exon 5 of NAIP gene. Lane 1-5 : Type - of SMA patients ; Lane 6-7 : relatives of SMA patients ; Lane 8-9 : normal controls ; M : molecular weight markers : pucdna/ Msp 1 SMN :, SMA ;3-4 : SMA ;5-6 : SMA ;7 : ; M :pucdna/ Msp Figure 1 Restriction enzyme mapping of amplified PCR products of exon7 of SMN gene. Lane l - 2 : Type of SMA patients ; Lane 3-4 : Type ; Lane 5-6 : relatives of SMA patients ; Lane 7 : normal controls ; M : molecular weight markers : pucdna/ Msp 4 NAIP 6 PCR 1-5 : - SMA ;6-7 ; ;8 : ;M : :pucdna/ Msp Figure 4 Amplified PCR products of exon 6 of NAIP gene. Lane 1-5 : Type - of SMA patients ; Lane 6-7 : relatives of SMA patients ; Lane 8 : normal controls ; M : molecular weight markers : pucdna/ Msp NAIP 5,6 2 SMN 8 PCR 1-2 :, SMA ;3 : SMA ;4 : ;5 : PCR ;M : :pucdna/ Msp Figure 2 Restriction enzyme mapping of amplified PCR products of exon 8 of SMN gene. Lane l - 2 : Type of SMA patients ; Lane 3 : Type ; Lane 4 : normal controls ; Lane 5 : undigested PCR products ; M : molecular weight markers : pucdna/ Msp 7 SMA,6 SMN 7 8,1 7 8 SMA 1 SMN 7 8, SMA 1 SMN 15 SMA 3 SMA 3 : Werdnig - Hoffmann, 6,, 2 8, 618,

4 1,:, 2 ; Banyer [10 ] 6 Kugelberg - Welander,217, (6 p 21. 3) 1 5 q 13. 1, SMA, 9,, 5 q SMA DNA 1995 Necker Lefebvre [3 ] SMN SMA, SMA, %(213/ 229) SMA 7 8 SMA, 5. 7 %(13/ 229) 7, SMA 8, 1. 3 % (3/ 229) SMN ;,NAIP ( 5, SMA NAIP (45 %) SMN,7 8, (18 %) 90 %98 %, 7 8 SMA NAIP 5,6, 3 SMA SMN, SMN (5/ 101) [11 ], 55 SMA [12 (7 ) SMA SMN NAIP 5 ] % (7/ 7), %(6/ 7),1 7 NAIP SMA, 8, 8 NAIP SMN [11 SMA,1 16 ], 7 8,7 ( 2 5 ) 7 / 8 7,8, ;, SMA SMN, SMA SMN SMN 7 8,, Fujii [6 ] 3 NAIP SMA, SMA 2 (66 %,2/ 3) SMN 7 / 8 [ ] SMA,SMN,PCR,,, SMA, PCR SMN SMA SMN : SMN 7,8, 6,7 [7 ] SMA 5 q, SMN 7 [8 ] 8,, Stevens [9 ] 29 SMA SMN,35 % SMN, 6,,,6 p q (246 ) SMA (127 ) 1 / 6) SMA,, SMA SMN [1 ] Scharf J M, Endrizzi M G, Wetter A, et al. Identification of a candidate modifying gene for spinal muscular atrophy by compara2 tive genomics [J ]. Nat Genet, 1998, 20 (1) : [2 ] Munsat TL, Davies KE. International SMA consortium meeting [J ]. Neuromuscular Disord, 1992, 2 (5/ 6) : [ 3 ] Lefebvre S, B rglen L, Reboullet S, et al. Identification and 9 characterization of a spinal muscular atrophy determining gene [J ]. Cell, 1995, 80 (1) : [ 4 ] Roy N, Mahadevan MS, Mclean M, et al. The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy [J ]. Cell, 1995, 80 (1) : [ 5 ] Van der Steege G, Grootscholten PM, Van der Vlies P, et al. PCR - based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy [ J ]. Lencet, 1995, 345 (8955) : [ 6 ] Fujii T, Miyajima T, Ito M, et al. Utility and intricacy of molec2 ular diagnosis of spinal muscular atrophy [J ]. No To Hattatsu, 1999, 31 (6) : [ 7 ] Hahanen E, Schonling J, Rudnik2Schoneborn S, et al. Missense

5 3 mutation in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy [J ]. Hum Mol Genet, 1997, 6 (5) : [ 8 ] Wirth B, Herz M, Wetter A, et al. Quantitative analysis of sur2 vival motor neuron copies : identification of subtle SMN1 muta2 tions in patients with spinal muscular atrophy. genotype - pheno2 type correlation and implications for genetic counseling [J ]. Am J Hum Genet, 1999, 64 (5) : [ 9 ] Stevens G, Yawitch T, Rodda J, et al. Different molecular basis for spinal muscular atrophy in South African black patients [J ]. Am J Med Genet, 1999, 86 (5) : [10 ] Banyer JL, Goldwurm S, Cullen L, et al. The spinal muscular atrophy gene region at 5q has a paralogous chromosomal re2 gion at 6p [J ]. Mamm Genome, 1998, 9 (3) : [11 ] Chang J G, Jong YJ, Liu SP, et al. Molecular analysis of sur2 vival motor neuron ( SMN ) and neuronal apoptosis inhibitory protein ( NAIP) genes of spinal muscular atrophy patients and their parents [J ]. Hum Genet, 1997, 100 (5-6) : [12 ],,,1 SMN NAIP [J ]1 ( ),1999, 21 ( ) : ( : ) 1, (, ) [ ] R [ ] E,, : 52 / min,,,, 5,, 160 / min,, 7 cm, 6 cm, 2. 5 cm :WBC / L,N 0. 55,L 0. 38,Hb 91 g/ L, / L :, I,aVL,V 5,V 6 Q,ST - T :, :??,, 3 1, 38 39,, 10 %20 % 1 : WBC 23, / L, / L 30 mm/ h,,,,,,, 7. 5 mm,,, 6 mm, 1 1,,, Q,ST - T 1 RVOT : ;RCA : ;LCA : ; RA : ;AO : ;LA : ( : ) [ ] ; [ ] [ ] ( ),,, 10