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B.1.12 Identifique a los miembros de la alta dirección que no sean a su vez consejeros ejecutivos, e indique la remuneración total devengada a su favor
10) Ejercer aquellas otras competencias asignadas a dicha Comisión en el Reglamento del Consejo
Next-generation sequencing studies have disproportionally examined the genetics Western and Asian populations over African and South American individuals. This is especially the case for the genetics of rare diseases like ALS which affects people worldwide. I was given the opportunity to sequence a small cohort of Argentinian ALS patients to search for potential disease-causing variation. Previous work into the genetic makeup of Argentina revealed a heterogeneous population with a composition of 65% European, 31% Indigenous American and 4% African (Avena et al., 2012). Another paper has analysed C9orf72 in a small Argentinian ALS cohort to discover expansions in a single (1/47) sporadic subject and a single (1/3) familial case (Itzcovich et al., 2016). The aim of the present study was to explore the genetics of 26 ALS patients from Argentina.
7.2. MATERIALS AND METHODS
A total of 26 ALS patients from Buenos Aires, one of which had ALS-FTD, were analysed. Firstly for C9orf72 repeat expansions and then for 24 relevant genes using the panel described in Chapter 6. Subjects included in the study consisted of 8 familial ALS, 19 sporadic ALS and 1 sporadic ALS-FTD. The average age of onset was 61 (range 45-83) and the ethnicity was self-reported as Latin except for two of Caucasian heritage. However, the clinician who examined these patients believes all but one patient to be of European heritage. Additionally
ATXN2 screening was performed on all subjects to detect repeat expansions. My colleague,
Lucia Schottlaender, performed the analysis of C9orf72 and ATXN1 using methods previously described (Koutsis et al., 2012; Schottlaender et al., 2015).
7.3. RESULTS
Two patients harboured the C9orf72 repeat expansion (8%). In the remaining 24 subjects, 122 variants were uncovered by NGS, 75 of which were common SNPs (present in >1% of the population), 14 were synonymous, 34 were intronic and 9 were rare, coding mutations. Three
135 patients had an ATXN2 intermediate repeat. Of the 24 patients fully sequenced, 8 had potentially damaging modifications in SOD1, TARDBP, FUS, UBQLN2, VCP, CHMP2B, SETX and ATXN2 which may have caused their disease (Table 18-19).
Subje
ct
Gene Variant Disease Escorial El Site of onset
Age of On set Ev olutio n (months ) ATXN2 scr een Ge nde r Ethnic ity
1 SOD1 G86S fALS (probable) Possible Limbs 50 17 Normal range F Latin
2 FUS S135N sALS Possible Limbs 74 24 Normal range M Latin
3 SETX D1077N sALS-FTD Probable Bulbar + FTD 72 30 Normal range F Caucasian
4 VCP R155H fALS (probable) Probable Limbs 45 48 repeats 22/27 M Latin
5 TARDBP N378D fALS (probable) Definite Limbs 52 96 22/27
repeats F Latin 6 FUS D490N fALS (probable) Definite Bulbar 48 7 repeats 22/27 F Latin
UBQLN2 P497S
7 SOD1 D84G fALS (probable) Possible Limbs 47 24 Normal range M Latin
8 CHMP2B R32Q fALS (probable) Probable Limbs 83 24 Normal range M Caucasian
9 SOD1 E22G sALS Probable Limbs 48 36 Normal range M Latin
10 SOD1 E22G sALS Possible Limbs 67 15 Normal range M Latin
11 SOD1 E22G sALS Definite Bulbar/ upper
limbs 56 48 Normal
range F Latin
12 C9orf72 Expansion sALS Probable Limbs 65 20 Normal range M Latin
13 C9orf72 Expansion sALS Definite Bulbar 67 24 Normal range F Latin
T a b l e 1 8 . L i s t o f A r g e n t i n i a n p a t i e n t s w i t h r a r e m u t a t i o n s .
Subje
ct
Gene Variant change Nuc.
Ex on Chr Position Gen o typ e dbSNP in silico pre d ic tion Our verd ict
1 SOD1 G86S G256A 4 21 33039587 het - D D
2 SOD1 E22G A65G 1 21 33032147 het U D
3 FUS S135N G404A 5 16 31195598 het rs61732970 T T
4 SETX D1077N G3229A 10 9 135203756 het rs145097270 U T
5 VCP R155H G464A 5 9 35065360 het rs121909329 U D
6 TARDBP N378D A1132G 6 1 11082598 het - U D
7 FUS D490N G1468A 14 16 31202358 het - U D
UBQLN2 P497S C1489T 1 X 56591795 hom - U U
8 SOD1 D84G A251G 4 21 33039582 het - D D
9 CHMP2B R32Q G95A 2 3 87289909 het - U U
T a b l e 1 9 . D e t a i l s o n r a r e m u t a t i o n s u n c o v e r e d ; D = d a m a g i n g ; U = u n k n o w n ; T = t o l e r a t e d ; N u c = n u c l e o t i d e .
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7.4. DISCUSSION
A selection of 26 ALS patients from Argentina were scrutinised in 26 genes of interest for any disease-causing mutations. 13 of these patients have one or more likely damaging variants which may have contributed to their disease. Two of these included the SOD1 mutations D84G and G86S which are both in exon 4. The latter of these has been observed previously as an aggressive ALS phenotype in a Japanese kindred (Takazawa et al., 2010). This matches the clinical progression of our patient who was diagnosed within a month of onset and lived for only 17 months.
Another patient presented with the known alteration N378D in TARDBP (Tsai et al., 2011; Ticozzi et al., 2011). This patient also had an intermediate CAG repeat expansion in ATXN2 which is known to increase a patient’s risk of ALS (Elden et al., 2010). However, at 27 repeats, this is at the cusp of what is considered a risk with many papers declaring a higher number in order to get significant results (van Damme et al., 2011; Conforti et al., 2012). For this reason it has been suggested that the lower limit may diverge in different populations (Lee et al., 2011). A FUS S135N variant in a sporadic sample was established which has both been observed in ALS (Rademakers et al., 2010) and in one control subject (Huey et al., 2012) indicating it is likely to be benign. Additionally, another patient presented with a novel FUS D490N variant which lies in the RGG-rich domain between the disease-causing variants R487C and R495X (van Blitterswijk et al., 2012b). This patient additionally has an ATXN2 intermediate expansion and the homozygous P497S variant in UBQLN2. This variant is located on the same codon as P497H which was shown to impair the protein degradation pathway in cell cultures (Deng et al., 2011).
VCP revealed only one known mutation which was found in an individual with a family history
of ALS: R155H which has been reported previously in both ALS and IBMPFD as one of the most common VCP variants (Johnson et al., 2010; González-Pérez et al., 2012; Cady et al., 2015; IBMPFD: Watts et al., 2004; Viassolo et al., 2008; Jacquin et al., 2013). Examination of
CHMP2B revealed the novel variant R32Q which coincided with an intermediate ATXN2
repeat expansion. The subject with FTD and Caucasian ethnicity harboured a D1077N variant in SETX however this variant did not segregate in a family with ALS (Arning et al., 2012). Lastly there were 34 intronic variants of unknown significance.
137 Excluding the two SNPs which were questioned in previous publications, the incidence of potentially damaging variants in our ALS cohort for each gene stands at 8% in C9orf72, 19% in SOD1, 12% in ATXN2 and 4% in TARDBP, FUS, UBQLN2 and VCP. This amounts to 34% of patients able to be genetically explained, which includes 62% of familial cases and 28% of sporadic subjects. This is higher than might be expected which could be due to the low subject count.
Additionally, the frequency of multiple mutations occurring in the same patient stands at 12%. This was entirely from the familial subjects. Most publications examining the prevalence of
ATXN2 repeat expansions excluded patients where a mutation was already discovered in
another gene. In the present report, all three of the subjects with an intermediate expansion had a mutation in a known ALS gene, therefore, these would have all been excluded in other studies.
As the number of patients in this cohort are relatively few, a larger replication study needs to be completed. NGS has shown that the genetics of ALS within South American countries are potentially similar to that of European decent with SOD1 being the most common cause of ALS. Given that there is a chance most of these patients are in fact of European decent, this finding is not surprising. We will be performing further analysis on these patients to determine their genetic makeup.
This work is currently in preparation for submission:
Morgan S, Schottlaender L, Hardy J, Holden H, Pittman A. Comprehensive investigation of causal genes in an Argentinian cohort with amyotrophic lateral sclerosis. 2016.
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