Missense mutations in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy (SMA)
Missense mutations in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy (SMA)Eric Hahnen, Jutta Schönling, Sabine Rudnik-Schöneborn, Heidrun Raschke, Klaus Zerres and Brunhilde Wirth*
Institute of Human Genetics, Wilhelmstrasse 31, D-53111 Bonn, Germany
Received January 23, 1997;Revised and Accepted February 21, 1997
Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor neuron gene (SMN) is a strong candidate for SMA and present in two highly homologous copies (telSMN and cenSMN) within the SMA region (5q11.2-q13.3). More than 90% of SMA patients show homozygous deletions of at least exon 7 of telSMN, whereas absence of cenSMN seems to have no clinical consequences. In 23 non-deleted SMA patients, we searched for intragenic mutations of the SMN genes in exons 1-7 and the promotor region by single strand conformation analysis. We identified two different missense mutations, S262I and T274I, in exon 6 of telSMN in three independent SMA families, providing further evidence for the telSMN gene as a SMA determining gene. Both mutations, as well as two previously described mutations (Y272C and G279V) are located within a highly conserved interval from codon 258 to codon 279 which seems to be an important functional domain of the telSMN protein. Recently, this region has been shown to contain a tyrosine/glycine-rich motif, which is also present in various RNA binding proteins, suggesting a potential role of SMN in RNA metabolism. Missense mutations might be useful for in vivo and transgenic experiments and further investigations on understanding the function of the telSMN protein.
The proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder affecting ~1:10 000 live births. The disease is characterised by degeneration of [alpha]-motor neurons in the spinal cord, which results in weakness and atrophy of voluntary muscles. Due to the extremely variable clinical picture of SMA, affected individuals have been classified into three groups considering the age of onset and achieved motor milestones (1 ,2 ). Two candidate genes for proximal SMA, the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP), both localised within a duplicated and inverted region on 5q11.2-q13.3, have been shown to be frequently deleted in SMA patients (3 ,4 ).
The NAIP gene, which is present in only a single functional copy and several truncated pseudogenes, is homozygously deleted in ~50% of patients with severe SMA and <20% of patients with milder manifestations, but also in ~2% of carriers and controls (4 ,9 ,10 ).
The SMN gene is present in two highly homologous and functional copies (telSMN and cenSMN). Both copies encompass ~28 kb on genomic level and are composed of nine exons (1-2a, 2b-8), encoding identical amino acid sequences (3 ,5 ). Only five base pair differences at the 3' end of the genes (from intron 6 to exon 8) and a frequent polymorphism in exon 2a allow to distinguish the SMN counterparts by SSCA and restriction site assays (3 ,5 -7 ). Independent of the clinical severity, >90% of SMA patients carry homozygous deletions of exon 7 or exons 7 and 8 of telSMN (3 ,8 -10 ). In patients showing homozygous deletions of telSMN exon 7 but not exon 8, the remaining exon 8 has been shown to be part of a hybrid SMN gene, giving evidence for unequal rearrangements or gene conversion events between both SMN copies as molecular mechanisms responsible for the disease (11 ,12 ). Absence of cenSMN is found in ~5% of unaffected individuals and seems to have no apparent phenotypic effect (3 ).
Interestingly, homozygous deletions of telSMN have also been found in healthy siblings and parents of SMA patients-a finding that may question the essential role of telSMN for the development of SMA (9 ,10 ,13 ). However, so far seven different mutations within telSMN have been identified in non-deleted SMA patients, giving evidence for a causal involvement of telSMN in the pathogenesis of SMA. Lefebvre et al. (3 ) detected two small deletions in the splice sites of introns 6 and 7, presumably resulting in aberrant splicing of the telSMN transcripts. Two further microdeletions in exon 3 and a 11 bp duplication in exon 6 have been described, causing frameshifts and premature stop codons, respectively (14 -16 ). Up to now, only two missense mutations (Y272C, G279V, localized in exons 6 and 7) have been identified (3 ,22 ).
Previous deletion analysis of telSMN in 191 SMA patients mainly of German origin has shown homozygous absence of at least exon 7 in 172 patients (90%; 9 ). The remaining patients show distinct clinical signs for autosomal recessive SMA, but were neither homozygously deleted for telSMN nor the NAIP gene. In order to identify intragenic mutations of telSMN in 23 non-deleted SMA patients (four type I, two type II and 17 type III SMA), we performed single strand conformation analysis (SSCA) of exons 1-7 as well as the promotor region.
In two independent German SMA families (SI and PE), the patients SI-1378 (type II SMA), PE-2549 and PE-2593 (both of type III SMA) display identical band shifts in exon 6 (Fig. 1 ), caused by an ACT -> ATT transversion in codon 274 as shown by direct sequencing of exon 6 PCR products. In order to assign this mutation, resulting in an amino acid exchange from threonine to isoleucine (T274I), to the centromeric or the telomeric copy of the SMN gene, we cloned RT-PCR products of the whole SMN transcripts derived from patients SI-1378 and PE-2549. By sequencing of clones containing the centromeric or the telomeric exons 7 and 8 we could show that the T274I missense mutation belongs in both cases to telSMN and is not found in clones containing cenSMN transcripts (Fig. 2 ). As reported before, part of the transcripts derived from cenSMN undergo alternative splicing resulting in a transcript lacking exon 7 (3 ; Fig. 2 ).
Due to the high deletion frequency of telSMN in SMA patients, intragenic mutations of telSMN seem to be very rare. Including the T274I and S262I missense mutations presented in this study, nine different mutations within telSMN have been reported so far, giving further evidence for telSMN as a major SMA causing gene: Bussaglia et al. (14 ) and Brahe et al. (16 ) identified different microdeletions in exon 3 (codons 133-134 and 147-148), resulting in frameshifts and premature stop codons, respectively; Parsons et al. (15 ) identified an 11 bp duplication in exon 6 (nucleotides 801-811), also leading to a frameshift and a premature stop codon, and Lefebvre et al. (3 ) reported two different microdeletions in the splice sites of introns 6 and 7, most probably causing aberrant splicing of telSMN transcripts. While all these mutations affect the entire structure of the telSMN gene product and most likely result in a loss of function of the telSMN protein, only two missense mutations, Y274C and G279V, localised in exon 6 and 7 have been described up to now (3 ,22 ). Interestingly, all mutations concerning the coding region of telSMN were located in exons 3, 6 and 7, which indicates that these exons represent hotspots for mutation events.
In only three out of 23 independent SMA patients, we identified two different intragenic mutations of telSMN. In patients SI-1378 (type II SMA), PE-2593 and PE-2549 (both of type III SMA), the latter of which are haploidentical siblings, we found T274I missense mutations which seem to have a common founder since they reveal identical haplotypes associated with this mutation. Additionally, we identified a loss of inheritance of paternal C212 and Ag1-CA marker alleles in family SI, giving evidence for a compound heterozygous state of the patient SI-1378: the paternal SMA chromosome carries a large scale deletion, the maternal SMA chromosome a T274I mutation of telSMN, indicating that this patient does not have an intact telSMN. The second missense mutation (S262I) was identified in a type III SMA patient of Australian origin.
Interestingly, all four missense mutations identified up to now (S262I, Y272C, T274I and G279V) are localised in a small interval from codon 262 to 279. The region of codon 258 through 277 has been shown to be 100% conserved between human, mouse and rat (C. J. DiDonato and L. Simard, in preparation) and therefore, can be considered to be an important functional domain of the telSMN protein. In cases of S262I and T274I mutations, hydrophobic isoleucine is substituted for hydrophilic serine and threonine, respectively, which obviously impairs telSMN function. Recently, Talbot et al. (22 ) identified a Tyr/Gly-rich dodecapeptide motif (YxxG)3 spanning codons 268-279 of the SMN gene. This motif is present and highly conserved in several RNA binding proteins found in different species (Caenorhabditis elegans, Artemia, Drosophila and Schizosaccharomyces pombe), emphasising a potential role of the SMN protein in RNA metabolism. All missense mutations described up to now are located within this motif (Y272C, T274I, G279V) or within the highly conserved adjacent region (S262I) underlining the functional importance of this domain.
Missense mutations of telSMN might be useful for further investigations on understanding the cellular function of the telSMN protein. If these mutations do not cause a complete loss of function of the telSMN protein, they might be used for in vivo and transgenic experiments, if a knock-out of the SMN mouse homologue, which seems to be present in only a single copy in the mouse genome (19 ,20 ) results in a lethal phenotype.
In the cases of the remaining 20 patients (four of type I, one of type II and 15 of type III SMA) the molecular cause for the disease is still unknown. Maybe these patients carry mutations of telSMN, which have not been detected by SSCA, or other genes are responsible for the SMA phenotype. In order to further clarify the molecular basis of the disease in these patients, sequencing of the telSMN gene on cDNA level and carrier analysis of these families as recently reported by McAndrew et al. (20 ) have to be performed.
The patients fulfilled the diagnostic criteria for proximal SMA defined by the International SMA Consortium (1 ). Clinical details are as follows. Patient SI-1378 (SMA II). First symptoms at the age of 5 months with delayed motor development. Never walked unaided, sat weakly. Diagnosis established by clinical picture and muscle biopsy in the first year of life. Progressive course with scoliosis and contractures from age 2 years, frequent episodes of respiratory infections from 8 years of age. Current age 11.5 years, temporary assisted ventilation required.Patient PE-2549 (SMA III). First walking difficulties with frequent falling at 3.5 years. Neurological examination at 4 years (normal CK, neurogenic EMG, normal conduction velocity). Progressive weakness in climbing stairs and rising from floor, using an electric wheelchair from 16 years. Last information at 20 years.Patient PE-2593 (SMA III). Onset at 2 years with a similar picture to his brother (PE-2549) (waddling gait, difficulties in climbing stairs and rising). Diagnosis established at 2 years, 8 months on the basis of EMG and muscle biopsy, CK-activity was normal. More severe course in comparison to his brother, chairbound at 5 years, marked weakness in arm and shoulder girdle muscles from 15 years. Last seen at 18 years of age.Patient DI-935 (SMA III). Onset at 17-18 years with difficulties in walking and climbing stairs. Diagnosis of SMA made at the age of 21 years by elevated CK levels, neurogenic findings in EMG and muscle biopsy. Slow progression, still able to walk at last information at 27 years.
The patients and their relatives have been analysed with polymorphic multicopy markers (Ag1-CA and C212) and tested for homozygous deletions in the SMN and NAIP genes as described in detail (9 ,17 ). DNA was isolated from peripheral venous blood samples by the salting out method (21 ).
Exon 6 and flanking intronic sequences were amplified in 25 [mu]l containing 80 ng of genomic DNA, 10 pmol of each primer (E6FI, 5'-ccagactttactttttgtttactg and E6R, 5'-cataactacaaaaaaattgtcagg), 120 [mu]M dNTPs, 1* Cetus PCR-buffer and 1 U Taq polymerase (Gibco-BRL). Cycling conditions include a 7 min initial denaturation at 94oC followed by 30 cycles of 45 s at 94oC, 45 s at 56oC, 45 s at 72oC and a final extension of 7 min at 72oC in a Perkin-Elmer Cetus Cycler 9600. Non-radioactive SSCA and silver staining was performed as described in detail (9 ), except that the gels were run at 70 V.
RNA was isolated from Epstein-Barr virus transformed leucocytes using the TRIzol kit (Life Technologies). First strand cDNA synthesis was initiated with oligo-dT primers and 4 [mu]g total RNA, using M-MLV reverse transcriptase (Gibco-BRL) according to the supplier's protocol. The single stranded SMN cDNAs were amplified with primer E1B (5'-atgaattcgtttgctatggcgatgagcagcg) localised within exon 1 and E8B (5'-tgtctagaactgcctcaccaccgtgctgg) localised within exon 8 using the Boehringer Expand High Fidelity PCR System. Reactions were carried out in 50 [mu]l volumes, including 5 pmol of each primer, 200 [mu]M dNTPs, 1* Expand HF buffer with 15 mM MgCl2 and 2.6 U enzyme mix. Cycling conditions include a 2 min initial denaturation at 94oC followed by 30 cycles of 15 s at 94oC, 30 s at 67oC and 1 min at 72oC, whereby the elongation time was extended for 20 s in each subsequent cycle beginning at cycle 11, and a final extension of 7 min at 72oC in a Perkin-Elmer Cetus Cycler 9600.
The genomic 6 kb fragments containing exons 6 and 7 of the SMN genes were amplified with the primers E6FII (5'-tgatgatgctgatgctttggga), localised in exon 6 and 541C770 (3 ) using the Boehringer Expand High Fidelity PCR System. Reactions were carried out as described above, except that 250 ng genomic DNA and 10 pmol of each primer were used. Cycling conditions include a 2 min initial denaturation at 94oC followed by 30 cycles of 15 s at 94oC, 30 s at 60oC and 4 min at 68oC and a final extension of 7 min at 68oC, the elongation time increased by 20 s per cycle beginning at cycle 11.
PCR products were cloned into pUC18 vector using the SURE-Clone-kit (Pharmacia) according to the supplier's protocol. Sequencing was performed with USB sequencing kit (Amersham) using vector and internal primers. For direct sequencing of exon 6 PCR products a biotinylated reverse primer was used followed by single strand separation with magnetic beads (Dynal).
We are grateful to the SMA families and the clinicians for their cooperation. We kindly acknowledge the support of Prof. G. Morgan from the Sydney Children's Hospital. We thank S. Raeder for excellent technical assistance and Th. Schmidt, S. Weiland and S. Uhlhaas for reading the manuscript. This research was funded by the Deutsche Forschungsgemeinschaft, E.H. is supported by the Herber-Reek Foundation. Furthermore, we thank ENMC and MDA.
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20 McAndrew, P.E., Parsons, D.W., Mendell, J.R., Burghes, A.H.M. and Prior, T.W. (1996) Gene-dosage of cen and tel SMN genes for assessment of sequence conversion SMA status, and carrier detection. Am. J. Hum. Genet., 59, 1568.
21 Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 3, 1215.
22 Talbot, K., Ponting, C. P., Theodosiou, A. M., Rodriques N. R., Surtees, R., Mountford, R. and Davies K. E. (1997) Missense mutation clustering in the survival motor neurone gene: a role for a conserved tyrosine and glycine rich region of the protein in RNA metabolism? Hum. Mol. Genet., 6, 497-500.
*To whom correspondence should be addressed. Tel: +49 228 287 2344; Fax: +49 228 287 2380; Email: bwirth@uni-bonn.de
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