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The 42st National Conference of Child and Adolescent Neurology and Psychiatry and Allied Professions with international participation


SPINAL MUSCULAR ATROPHY – MOLECULAR GENETICS AND PATHOGENIC MECHANISMS

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ABSTRACT:

Spinal muscular atrophies (SMA) are a heterogeneous group of genetic disorders characterized by progressive degeneration of neurons in the spinal cord causing progressive muscle deficit. In this article we detail the genetic and pathogenetic features in SMA caused caused by mutations in chromosomal region 5qll.2-13.3 (spinal muscular atrophies 5q). SMA gene was discovered through studies of linkage analysis as 5q-5qll.2-13.3 region, comprising four duplicated genes, (centromeric and telomeric cop-ies). Mutations in the gene SMN1 are responsible for the disease, while SMN2 and NAIP genes contribute to its phenotype. AMS pathogenesis is not fully understanding yet. It seems that SMN protein has two roles in the cells: biogenesis of small nuclear ribonucleoproteins (splicing preARN messenger) and translocation of Pactin in axonal growth cones. Although SMN is expressed in all tissues, it affecting almost exclusively spinal cord motor neurons from reasons unknown yet.

 


 

I. INTRODUCTION

Spinal muscular atrophies (SMA) are a hetero-geneous group of genetic disorders characterized by progressive degeneration of neurons in the spinal cord causing progressive muscle deficit.
In this article we detail the genetic and patho-genetic features of spinal muscular atrophies caused by mutations in chromosomal region 5qll.2-13.3 in (spinal muscular atrophies 5q), which are transmitted autosomal recessive.

1.1. History

Different phenotypically features and the complexity of genotypic changes makes difficult to iden-tify the gene responsible for AMS.

Candidate gene region for SMA was located in 1990 by linkage studies, on the long arm of chromo-some 5, between 5qll.2-13.3, comprising a range of about 500kb. (2,19)

At this region were identified genic and nongenice sequence (DNA markers), duplicated by a variable number of times and oriented head to head. (11). Un-like most organisms,in humans the region contains at least four genes present in centromeric and telomeric copies. In 1995, Lefebvre et al. reported that the presence of a mutation on both alleles of the telomeric gene causes spinal muscular atrophy. This gene was called “survival motor neuron” (SMN1)(15) (figure 1).

Subsequently, Coovert et al have shown that centromeric gene SMN2 gene has a similar sequence in 99% with SMN1 and also produces, SMN protein but spinal muscular atrophy is caused only by mutations in SMN1 (8).

 

II. GENES IN SMA

The main gene responsible for the disease is SMN1.

In addition, the region on chromosome 5 were is found this, contains another three genes also dupli-cated and inverted, with the same funcţional features as SMN1 gene: telomeric copy is funcţional and centromeric copy dysfunctional. Furthermore, structural and funcţional integrity of the 3 genes influences SMA phenotype.

The three genes are:

  • BIRC1 gene (Baculoviral IAP Repeat-Con-taining Protein 1), known previously as neuronal apoptosis inhibitory protein -NAIP; the protein en-coded by this gene inhibits the apoptosis;
  • GTF2H2 gene (General Transcription Factor IIH, Polypeptide 2), or p44 encodes a subunit of the basal transcripting factor TFIIH, involved in transcription and repair DNA mediated by transcription;
  • SERF1A gene (Small Edrk-Rich Factor IA), previously known as H4F5 (Spinal Muscular Atro-phy-Related Gene), a gene with unknown function. (26). (figure 1).These genes are modifiers genes in spinal muscular atrophy Studies have indicated the presence of dele-tions of these genes in many cases of SMA, suggest-ing correlation between phenotype severity and the presence of these variations.

II. 1. Survival Motor Neuron Gene (SMN)

Pathogenic gene for SMA-SMN1, is the telomeric copy of the duplication (formerly named SMN-tel). Centromeric copy of the duplicate segment is homologous to SMN1 and has been called SMN2 (SMNcen). Molecular studies indicate that this duplication appeared from about 3 million years because it is present in chimpanzees and humans, but not in less evolved primates.

Number of the copies varies: one SMN1 copy in each chromosome in 82-96% of normal subjects, two SMN1 copies in each chromosome in 4 -18 % of normal subjects.

The presence of at least one copy is indispensable for the survival of motor neurons.
Structure: SMN gene has 20 kb and contain 9 ex-ons (1,2a, 2b, 3-8) (21). The exon 8 is not translated. The gene encodes SMN protein with 294 amino-acids.(32)

SMN1 gene (telomeric SMN)
The structure of SMN1 is very similar to SMN2: exons 7 and 8 contain specific nucleotide sequences, the sequence encoding of SMN2 differ by SMN2 by one nucleotide (840 C> T) in exon 7. (figure nr. 2) (21)

 

Figure 1. SMA Region (32)

 

SMN2 gene (Centromeric SMN)
SMN2 gene is located centromeric to the SMN1 gene and is very similar structurallywith SMN1 gene. SMNlşi SMN1 gene. SMN1 and SMN2 genomic sequences differ by five nucleotides , but one differ-ence is functionally important: C -> T transition located at a splicing regulatory site (enhancer splicing exons) of the exon 7 SMN2. (17)

This change modifies incorporating of the corresponding sequences in exons 7 in mRNA, resulting in synthesis of a shorter protein with greatly reduced efficiency. Therefore, SMN2 gene can produce a flill length protein, similar to SMN1, only the amount is approximately 10% of its expression products.

The number of SMN2 copies are individual varia-tion, the frequency of different genotypes are shown in Table. 1(17)

 

Figure 2. Localization of the nucleotides by which SMN1 can be distinguished from SMN2. (31)

 

Patients with SMA have two or more copies, fewer SMN2 gene is correlated with disease severity.

II.2. Neuronal Apoptosis Inhibitory Protein (NAIP)

In a separate publication Lefebvre et al. (15) and Roy et al. (25) have identified a different gene on chromosome 5ql3.1 – neuronal apoptosis inhibitory protein (NAIP).

NAIP gene is located near the SMN gene and is also duplicated in the region 5ql3. In this region there is a variable number of truncated copies. NAIP can be detected by PCR amplification of exon 5, which is present only in full-length gene. Deletion of exon 5 was present in 45% of SMA patients with type I and 18% in patients with type II and type III.

Recent experiments showing that NAIP suppress-es apoptosis in mammalian cells supports the idea that the protein acts as a negative regulator of apoptosis in motor neurons and when is deficiency or absence, contributing to the SMS phenotype. (12,24)

II.3. Basal transcription factor (Btf2-p44)

P44 gene, a subunit of basal transcription factor TFIIH, was also characterized and localized in theSMA region. There have been studies that showed that GTF2H2 gene (p44) is duplicated in the 5ql3 region, telomeric copy (p44t) is located near the SMA criticai region, while the centromeric copy (p44c) is located in the centromeric region (Figure 1).

Transcription factor TFIIH is a multifuncţional protein complex involved in transcription, DNA re-pair mechanisms and probably also in other cellular processes.

Have reported Studies that compared clinical data from 94 unrelated patients with SMA, with their genotype (gene SMN, NAIP, p44) and showed that large deletion involving these loci are associated with the phenotype of severe SMA (SMA type I). Moreover, the activity of different complexes TFIIH were compared in patients who had both p44 gene to those with homozygous deletion of p44 gene. The resulting data suggest that not only major rearranging of p44 gene affects the disease. However, the close association between large deletion of this region and the most severe SMA phenotype may suggest that the severity of clinical symptoms may be related to changes in p44 gene, either alone or with other modifiers factors associated to mutations of SMN gene. (18,29).

 

III. MUTATIONS IN SMA

The presence of duplications causes genomic in-stability of SMA region and offers the potential for unequal exchange of genetic material that modifies the number of copies of various sequence markers.

This feature is reflected in inter-individuals differ-ences in the number, position and orientation of multi-ples genetic markers located in this criticai region SMA. The presence of two identical copies of a gene reduces the pressure of natural selection that should maintain a low number of mutations. Thus, although humans and chimpanzees have a variable number of copies of the SMN gene on each chromosome, only humans have different sequences in exons 7 and 8 causing degrada-tion of SMN in a partially functional gene.

 

Table I. Number of SMN2 copies

 

 

Mutations responsible for most cases of SMA ap-pearance can be:

  • Deletions of one or more exons of the SMN1 gene;
  • Small muations, ussually missense mutations

For the disease is necessary that both alleles present
mutations. In some cases may be associated a deletion on one allele with a small mutation on the other.

The mechanisms of deletions could be:

  • Deletion by unequal recombination during paternal meiosis, leading to loss of genetic material, including the SMN1 gene or a portion of this
  • Conversion the SMN1 gene în SMN2 gene-from unequal recombination can produce a unidirecţional transfer of informationbetweenneighboringgenes very similar, so one of the genes remains unchanged, and the other is modified, receiving a part of the sequences from the first one (11).The result is a chromosome with no SMN1 sequence but a corresponding increase in the number of SMN2 copies with one. (9)

Point mutations occur through errors of replica-tion of the polymerase.

III.1. SMN 1 Deletion and conversion mutations

Approximately 94% of patients with clinically typical AMS have absence of both copies of SMN1
exons 7. SMN1 loss can occur by deletion (typically a large deletion that includes the entire gene) or gene conversion to SMN2. (21)

 

Figure 3. Schematic representation of SMN , with the position of the markers and genes.(31)

 

There are studies using two polymorphic markers, initially with AG1-CA (C272) and later C212, located at the end 5 of the SMN gene. Genotype with SMN2 copy on each chromosome, with loss of NAIP occurs in chromosomes with SMA type I and involves a large deletion. In types II and III although lack SMN1, often have a chromosome with a copy of SMN2 and the other chromosome with two copies of SMN2. In the SMA types II and III, SMN1 gene is missing, but NAIP gene is present as the markers are present at the 5 ‘ end of the SMN1 gene. When SMN1 gene is not detected but markers shows that the locus is still present, another mechanism should be considered besides deletion, most likely mechanism is the conversion of SMN1 into SMN2, leading to increased of SMN2 copies. (figure nr. 3) (3,31)

Based on the finding that the SMN2 gene can generate, however, a small amount of funcţional protein, has been proposed the hypothesis that increased SMN2 copy number decreases the severity of the disease through the production increased of funcţional SMN protein levels.

III. 2. SMN 1 Small intragenic mutations

In a substantial number of patients with SMA who do not lack both copies of SMN1 were identified point mutations in the SMN1 gene. These mutations provide strong evidence that SMN1 gene is indeed the gene for SMA, while intragenic mutations in SMN1 are associated with SMA phenotype regard-less of NAIP or other genes status. (21)
Wirth et al reported that 18 of 501 patients with SMA (6 of 265 type I, 6 of 122 type II and 6 of 114 type III) with identifiable mutations in both 5 chromosomes have a point mutation and a mutation type deletion (30). Thus, the proportion of alleles with point mutations were highest în SMA type III and the lowest in type I.

Although the absence of both copies of SMN1 is a reliable and sensitive evaluation for most patients with SMA, about 5% have other mutations in the SMN1, which will not be detected by methods used for testing deletion [homozygous]. Due to the high frequency of deletion most of these patients will be heterozygous compounds with a deletion on one SMNlallele and occurrence of point mutations or other small mutation on the other allele.
Were described missense and frame shift mutations. Although mutations can occur throughout the
of frameshift mutations wich can be improved by increasing the number of SMN2 copies. (31)

 

Figure 4. Small mutations in SMN1 (23)

 

 

III. 3. De novo SMN1 mutations

In about 2% of cases of spinal muscular atrophy one parent is a carrier of a mutation in the gene SMNl.The second allele are a mutation de novo. De novo deletions occurs frequently during meiosis due to chromosomal recombination defects between sisters chromatids (21)

III. 4. SMN Polymorphisms

Most polymorphisms are present in the promoter region and intron 6 (20). Ogino et al. reported five subjects unrelated who had two alleles different in length due to a single inserts of thymidină in a polythymidină existing tract (8T) in SMN1 intron 6 (IVS6-24dupT or IVS6-24_IVS6-23insT). Two were symptomatic (one with Type II SMA and other type III) and the other three were asymptomatic. Impairment of non-coding regions, away from regulatory sites suggests, however, that these changes unlikely to have effects on protein function. The presence of symptomatic cases may suggest the association of other factors. (21). There are 31 polymorphisms reported to present: 12 to the promoter, one in intron 1, one in exon 2a, one in the intron exon 3:16 6 (20,21).

 

IV. SMN PROTEIN

IV. 1. SMN Protein is a very widespread protein with a molecular weight of 38 kilodaltons (kDa). It is present in both the cytoplasm and the nucleus of body cells. In nucleus is concentrated in the structures called gems that overlap or are very close to Cajal bodies. Cajal bodies contain high levels of factors involved in transcription and processing of various types of nuclear RNA.

Based on the finding that the SMN2 gene can generate, however, a small amount of funcţional protein, has been proposed the hypothesis that increased SMN2 copy number decreases the severity of the disease through the production increased of funcţional SMN protein levels.

The number of gems in tissues or cell lines in the patients with SMA correlates inversely with disease severity, patients with type I have with little or they are missing.

SMN is also present in granules of axons of neu-rons, which are rapidly transported bidirectionally. SMN is richly represented in the growth cones and probably in the postsinaptic membrane of the neuro-muscular junction.
SMN protein levels are regulated during develop-ment as follows: they are elevated during embryonic period and then decrease during the postnatal period. It is possible that the disease starts when SMN levels fall below a criticai threshold and the severity of disease depends, in part, of the timing of this decrease during development.

The SMN protein has more motifs identified so far: (figure nr. 5):

  • domain rich in lysine that is encoded by exon 2
  • Tudor domain encoded by exon 3
  • Domain with pliprolina encoded by exons 4 and 5
  • Domain rich in tyrozine -glicine (Y-G) encoded by exon 6IV.2 SMN protein functions

From the data currently reported in the literature it seems that SMN protein cell has two roles: snRNP Biogenesis (splicing preARN messenger) and trans-location of P actin in axonal growth cones. (13)

 

Figure 5. A diagram of SMN showing the exons and domains.(4)

 

a. SMA and splicing AMS
To understand the pathogenesis of spinal muscular atrophy we make a review of some theoretical data about the transformation of genetic informa-tion (DNA) into protein. Such genetic information is transcribed initially resulting premesenger RNA, then through complex process of maturation RNA premesager becomes messenger RNA (has only exons) and then through a process called translation decoding genetic information is carried in a specific sequence of amino acids (figure nr. 6).

 

 

Figure 6. Decoding of the genetic information (9 modifi ed)

 

The gene consists of exons (funcţional regions of gene), alternating with intron (non-codingsequences). In the process of maturation of premessenger RNA in messenger RNA intronii are cut and removed, then ex-onii be tied “together”, a process that is called splicing. SMN has an important role in splicing, which is why we detail the process a bit. Splicing depends on the existence of nucleotids sequences signal located at the border nucleotids exon-intron:

  • site 5’GT (GU în ARN)
  • site 3’AG
  • branch site – wich contains nucleotid A

The splicing mechanism is accomplish in three steps: (9):

1. The cut of exon – intron junction 5’GU
2. Attachement of terminal nucleotid G to the nucleotid A of the branch site and mahe a loop structure
3. The cut of the intron at the site 3’AG, removal of it and put togheter of exonic RNA

In general the splicing is accomplished by a mac-romolecular complex named In general matisarea este realizată de un complex macromolecular denumit spliceosome, wich recognize these sites.The splicing process of premessenger RNA is criticai for the expression of the gene and is transported by sliceosome în the nucleus.

The major compounds of the spliceosome are:

  • SMN complex
  • five small nuclear ribonucleoproteins (snRNP) rich în uridine (UI, U2, U5 si U4/U6 – UsnRNP). Each of UsnRNP (excepted U6) is composed by a small nuclear RNA molecule (snRNA), seven common proteins and more proteins specifics for each snRNA. (14)

SMN complex is composed by SMN protein (wich are oligomerisazed at the carboxi site) and a lot of adiţional proteins, called gemins (6, 7, 22) (figure nr. 8). It has been shown in several studies that Gemin 5 is the protein that bound RNA -snARN-binding-of the SMN complex as it binds directly and specifi-cally at the end 3 of snARN.

This, Gemini 5 serves to specify snARN among all cellular RNA for snRNP assembly function. (14).In the SMN complex besides the major compo-nents of SMN (gemins) that are stable associated, could be identified several proteins. Part of them are Sm proteins (B or B2, Dl, D2, D3, E, F and G) wich are arranged in a Sm core on a sequence rich in uridine of each snARN called “Sm site”. (33)

 

Figure 7. SMN complex (7)

 

 

SMN complex binds Sm proteins through some ar-eas rich in arginine and glycine (RG) found in three of them-SMB, SmDl, SmD3, and also trough additional interactions of gemins with Sm.The asociation with RG domains is strong increased by symmetry methylation of these fields, process developed by a complex called methylosome 20 S complex (containing JBP1 methyl-transferase (PRMT5), plCln and MEP50). (Figure 8)

SMN protein itself binds directly via Tudor do-main to these areas symmetrical dimethyl-arginine and methylation of these proteins increase binding Sm to protein SMN complex (Figure 8).

During the assembly of snRNP, initially to each snARN in nucleus, in the process of maturation, is added at the 5 ‘ end a 7 methyl guanosine molecule which form a specific structure called “cap”-m7g cap. Subsequently these snARN are exported from nucleus in cytoplasm, where the complex is associated with SMN. After assembly, 7-methyl guanosine cap of small nuclear RNA is hypermetilated in 2,2,7-tri-methyl guanosine cap.

Properly assembled snRNP is then imported into the nucleus, where specific protein-snRNP are associated to form fully funcţional snRNP.These splice RNA premessenger . Because splicing must be exe-cuted in an efficient and timely manner, the assembly of snRNP must take place efficiently and smoothly. SMN complex is essential here. SMN complex directly recognizes and binds both protein and RNA components of snRNP and facilitate their interaction, thus providing a strict specificity for their assembly process. (Figure Nr. 8).

b. Other functions of SMN protein
SMN may also have other functions in motor neurons. In the neurons, SMN proteins binds het-erogeneous nuclear ribonucleoproteins, that binds the 3′-region untranslated of the P actin messenger RNA. This interaction is required for efficient transport of P actin messenger RNA in growth cones. P actin messenger RNA and protein localized in the growth cone are known to be required for axonal growth, like the actin cytoskeleton as is the force required for growth cone mobility. (10)

 

V. PATHOGENIC MECHANISMS IN SMA

Although it is known that mutations in the gene SMN1 cause low levels of SMN protein in patients SMA, the exact sequence of events leading to selective death pathogenic of spinal motoneurons is not fully known.Two hypotheses have been proposed to explain the mechanism of SMA. The first suggests that in-terruption of assembly snRNPs affects the splicing of a selective group of genes that are important for motor neuron turnover. The second suggests that the SMN has a function in the axons that is interrupted in SMA. It is also possible that both hypotheses are somehow overlapped, thus lowering of snRNP assembly can influence splicing of significant genes for axons. Although there is evidence to support both hypotheses are not yet clear evidence wich of them is correct or if both apply. (4)

The difference between SMN1 and SMN2 (trans-lational silent of C-T transition located at exon 8) cause frequently skipping of exon 7 during splicing of transcripts of SMN2. (17). Therefore, although SMN produce full-length transcripts, most mature transcripts from SMN2 did not have exon 7. These transcripts encode a truncated protein (SMN A 7) wich is rapidly degraded and the signs of the disease are probably the result of deficiency of protein full-length SMN. (8,16,27,28).

 

Figure 8. SMN 1 and SMN2 transcripts (4)

 

Figure 9. Steps of assembly SnRNPs (1):

 

 

 

A fundamental step in molecular pathogenesis of SMA is altered splicing of the transcripts derived from SMN2 compared with transcripts derived from SMN1. SMN full-length transcripts are encoded by nine exons (1,2 a, 2b, 3-8) and exons 1-7 are translated into SMN protein. Have been proposed two models to explain the inhibitory effect of C-to T transition in including exon 7 in SMN2. (28).

According to the exons splicing enhancer model, exon 7 of SMN1 contains a heptamer sequence motif that recruits splicing factor SF2/ASF, which promotes inclusion of exon 7. When this motif is interrupted by the C – toT transition, which is present in SMN2 transcripts, the SF2/ASF factor is not recruited, splice site 3′ is not recognized and exon 7 is excluded.The exons splicing silencer model proposes that the C – to T transition creates an inhibitor element that interacts with Al nuclear ribonucleopro-tein , a splicing factor that suppress including exon 7. These models are not necessarily mutually exclusive and both mechanisms can occur simultaneously. The specific pattern of splicing SMN2 transcripts is likely to be specific to each type of cell, depending on the concentration of different splicing factors in different cell types. In motor neurons is unknown.

Loss of amino acids encoded by exon 7 causes the production of SMN protein with severely deficient oligomerization and stability low of SMN monomers and are rapidly degraded. Thus SMN 1 loss cause lowers levels of SMN in most tissues.

 

CONCLUSIONS

Although many discoveries have been made in genetics of SMA 5q, which allowed the identification of genes and mutations involved, pathogenic mechanisms are not fully known.

It is known that SMN protein plays an important role in splicing by assembly the small specific nuclear ribonucleoproteins. Small nuclear ribonu-cleoproteins assembly is clearly altered in SMA, but it is unknown if other specific reactions dependent on SMN are affected. Although S MN is expressed in all tissues, there are affected almost exclusively motor neurons in spinal cord, specificity which is not fully understand.

 

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Correspondence to:
Niculina Butoianu, Str. Panait Istrati nr. 79, sect 1, cod poştal 011546, Bucureşti