Case Report published: 08 November 2019 doi: 10.3389/fgene.2019.01109 Haplotype Analysis of the First A4V-SOD1 Spanish Family: Two Separate Founders or a Single Common Founder? Cecilia Garcia 1,2,3†, Jose Manuel Vidal-Taboada 1,2,3†, Enrique Syriani 2,3, Maria Salvado 1,2,3,4, Miguel Morales 2,3 and Josep Gamez 1,2,3,4* 1ALS Unit, Neurology Department, Vall d’Hebron University Hospital, Barcelona, Spain, 2 Vall d’Hebron Research Institute (VHIR), Barcelona, Spain, 3 European Reference Network on Rare Neuromuscular Diseases (ERN EURO-NMD), Barcelona, Spain, 4 Department of Medicine, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Despite the genetic heterogeneity reported in familial amyotrophic lateral sclerosis (ALS) (fALS), Cu/Zn superoxide-dismutase (SOD1) gene mutations are the second most common cause of the disease, accounting for around 20% of all families (ALS1) and isolated sporadic cases (sALS). At least 186 different mutations in the SOD1 gene have been reported to date. The possibility of a single founder and separate founders have Edited by: Anne Marja Remes, been investigated for D90A (p.D91A) and A4V (p.A5V), the most common mutations University of Oulu, Finland worldwide. High-throughput single nucleotide polymorphism genotyping studies have Reviewed by: suggested two founders for A4V (one for the Amerindian population and another for the Fan Jin, Zhejiang University, China European population) although the possibility that the two populations are descended Adriano Chio, from a single ancient founder cannot be ruled out. We used 15 genetic variants spanning University of Turin, Italy the human chromosome 21 from the SOD1 gene to the SCAF4 gene, comparing them *Correspondence: with the population reference panels, to demonstrate that the first A4V Spanish pedigree Josep Gamez josepgamez.bcn@gmail.com shared the genetic background reported in the European population. † These authors have contributed Keywords: SOD1, A4V, p.A5V, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, ALS1, equally to this work founder effect Specialty section: This article was submitted to INTRODUCTION Genetic Disorders, a section of the journal About 10% of amyotrophic lateral sclerosis (ALS) cases are familial (fALS), and genetics is the Frontiers in Genetics discipline that has made the greatest contribution to understanding the complexity of the disease’s Received: 04 March 2019 pathogenesis, with a major impact on clinical practice, especially in the field of genetic counseling. Accepted: 16 October 2019 Since the discovery of mutations in the SOD1 gene linked to ALS in 1993, an increasing number of Published: 08 November 2019 genes have been reported as associated with the disease, justifying the term “genetic heterogeneity Citation: of ALS”. At least 31 major genes and 2 different genetic loci with dominant, recessive, and X-linked Garcia C, Vidal-Taboada JM, patterns of inheritance have been identified for fALS, and an increasing number of susceptibility Syriani E, Salvado M, Morales M and or modifying gene loci have been suggested for fALS and several sporadic ALS (sALS) cases. The Gamez J (2019) Haplotype Analysis ALSoD database currently lists genetic variants in 126 genes as associated to ALS. Gene–gene and of the First A4V-SOD1 Spanish Family: Two Separate Founders or a gene–environment interactions have also been suggested as playing a major role in the disease’s Single Common Founder?. appearance and phenotype (Andersen and Al-Chalabi, 2011; van Blitterswijk et al., 2012; Abel et al., Front. Genet. 10:1109. 2013; Al-Chalabi et al., 2013; Leblond et al., 2014; Renton et al., 2014; Jones et al., 2015; Al-Chalabi doi: 10.3389/fgene.2019.01109 et al., 2017; Brown and Al-Chalabi, 2017; Hardiman et al., 2017; van Es et al., 2017; Chia et al., 2018). Frontiers in Genetics | www.frontiersin.org 1 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family Mutations in the gene encoding Cu/Zn superoxide dismutase obtaining informed consent. There were no skipped generations. (SOD1) are the second most common cause of fALS cases The affected individuals were clinically characterized according worldwide, accounting for approximately 20% of all families. to gender, age at onset, initial topography, signs of dementia, and ALS1 is the designation for fALS linked to the SOD1 gene (MIM survival time (Supplementary Table S1). 105400). At least 186 different mutations in the SOD1 gene have The proband, a 53-year-old male (II:1), noticed weakness in his been described, of which over 90% are missense, confirming its left leg beginning 7 months previously, which he attributed to a allelic heterogeneity (ALSoD1). The SOD1 mutations reported traumatism he had experienced when working in construction. in ALS1 pedigrees are primarily associated with a dominant An initial electromyographic study showed denervation in all four inheritance pattern and high penetrance, despite occasionally extremities. Transcranial magnetic stimulation and brain and spinal being found in apparently sporadic or recessive cases. Clinical MRI were normal. Cerebrospinal fluid, hemogram, biochemical heterogeneity, including gender predominance, age at symptom screening, and serological tests for neurotropic infectious diseases onset, site of onset, penetrance, and progression of the disease, has all presented normal values, except for creatine kinase, which also been reported in many ALS1 families. The type of mutation was 735 U/l (normal values < 135). The patient subsequently and the resulting phenotype are strongly correlated in some cases experienced pain, with frequent falls in the months after his first (Andersen et al., 2003a; Andersen, 2003b; Andersen, 2006). examination, when muscle strength in his upper limbs and right However, information about the clinical–genetic correlations for lower limb was completely normal. His Amyotrophic Lateral most of these mutations is scarce. As a result, clinical and genetic Sclerosis Functional Rating (ALSFRS-R) score was 44. In the data from families with ALS1 from around the world are being next visit, 3 months later, we observed weakness and amyotrophy compiled in the constantly updated ALS database (ALSoD1). This in the left upper limb, particularly in the proximal muscles. We database provides clinicians with information on research into observed generalized fasciculations, which were more marked the genetic characterization of ALS1 and other forms of familial in the muscles on the left side of the body, where it was difficult ALS, as well as new candidate genes. to elicit deep tendon reflexes. Eight months after the initial visit, Epidemiological studies report that D90A is the most the patient was admitted to the emergency ward complaining of common mutation worldwide, although the most frequent dyspnea and orthopnea, and non-invasive mechanical ventilation mutation in North America is A4V (formally designated p.A5V, was fitted. The patient was offered a gastrostomy, but declined. The rs121912442), which accounts for 50% of North American ALS1 patient died 19 months after symptom onset. families (ALSoD1, Watanabe et al., 2000; Andersen, 2003b; Two years later, the proband’s 47-year-old brother (II:5) Andersen, 2006). The most striking features of the p.A5V came to our clinic due to presenting clumsiness and difficulties mutation are rapid progression, with a mean survival time of climbing stairs, running and walking. He had experienced less than 2 years from clinical onset, predominant lower motor recurring episodes of cramp in both calves in the 2 months before neuron symptoms, and its rarity in the European population. his visit. We observed weakness and amyotrophy in the proximal The possibility of two founder haplotypes—one Native American muscles of the right lower limb and a minimal loss of muscle and another European—has recently been suggested (Rosen, mass in the first interosseous of the right hand. Deep tendon 2004; Broom et al., 2008; Armon, 2009; Saeed et al., 2009; Tang reflexes were normal, except for the patellar and Achilles reflexes. et al., 2018). The p.A5V-SOD1 mutation has been described Fasciculation was observed in the musculature of the four in a very small number of families in Europe, and never in the extremities. His ALSFRS-R rating at this point was 39. Like his Spanish population. brother, neurophysiological studies showed signs of denervation Here we report the clinical characterization and high- in three regions and an absence of upper motor neuron signs. throughput single nucleotide polymorphism (SNP) genotyping The patient declined any follow-up, life support, ventilation or of the first p.A5V (A4V) Spanish ALS kindred with high nutritional measures. He died 17 months after clinical onset. penetrance, predominant lower motor neuron involvement, fast In their family, their two sisters requested genetic screening for progression, short survival times, and no cognitive impairment. familial forms of ALS, as they remembered that their father had died at 57 years of age due to respiratory insufficiency after 2 years of weakness in the upper limbs. To the best of our knowledge, this MATERIAL AND METHODS pedigree contains no other cases of neurodegenerative disorders, including frontotemporal dementia and Parkinson’s disease. Subjects This study was carried out following the protocol approved by the Hospital Universitari Vall d’Hebron Institutional Review Board Genetic Studies with written informed consent from all subjects in accordance Selection of Single Nucleotide Polymorphisms for with the Declaration of Helsinki. Analysis of the SOD1 Haplotype Our pedigree originated in north-western Spain. The simplified In order to saturate the SOD1 region with genetic markers, we pedigree of the family is shown in Figure 1. We examined two performed a scientific literature search of haplotypes previously ALS patients and two healthy individuals in the pedigree after described in the SOD1 gene. We consulted various databases (1K Genomes, dbSNP, and Ensembl Web sites) for SNPs in the SOD1 1http://alsod.iop.kcl.ac.uk/mutations/mutationsFoundGeneOnly.aspx?gene_ region of the IBerian populations in Spain (IBS) population, and id=SOD1. Web site access on Oct 8th 2019. analyzed 14 SNPs and 1 repeat. Thirteen SNPs were selected from Frontiers in Genetics | www.frontiersin.org 2 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family FIGURE 1 | Pedigree of the family studied. The proband is indicated by an arrow (II:1). The haplotypes of the progenitors are inferred from the haplotypes of the offspring. Highlighted in red the genetic variation causing A5V mutation, in bold the single nucleotide polymorphisms that determine the differences between haplotypes of the IBS population. Haplotype (variation order): rs4817415, rs2070422, rs1008270, rs9974610, rs2173962, rs202445, rs121912442, rs4816405, Rs2070424, rs1041740, CA_repeat, rs2833475, rs16988427, rs2833481, rs2070423, rs2833483. the results of 79 A4V families (Broom et al., 2008) (rs4817415, The entire coding regions of SOD1, FUS, TARDBP, and rs2070422, rs1008270, rs9974610, rs2173962, rs4816405, PFN1 genes and the C9orf72 expansion were screened using rs2070424, rs1041740, rs2833475, rs16988427, rs2833481, Sanger sequencing. rs2070423, rs2833483); and 1 additional SNP (rs202445) and a CA repeat from a study of 54 patients (Saeed et al., 2009) and Analysis of Data genetic data from an isolated Chinese case (Tang et al., 2018). LDhap software (Machiela and Chanock, 2015) was used with The locations of all markers and their previous designations are a 1K Genomes dataset to identify inferred haplotype blocks in shown in Supplementary Table S2. the Iberian (IBS) population. Linkage disequilibrium between SNPs was calculated using LDMatrix software (LDLink). The Genotyping of Normal and Mutant p.A5V SOD1 p.A5V haplotypes detected in our Spanish patients were Alleles in SOD1 compared with the haplotypes associated in American, Swedish, The SNP rs121912442 (SOD1 p.A5V) region was amplified by and Chinese populations (Broom et al., 2008; Tang et al., PCR and the allele detected by Sanger sequencing. DreamTaq 2018). The genetic data for the comparisons with the African Master Mix was used under standard conditions according to the (ACB, ASW, ESN, GWC, LWK, MSL, YRI), Asian (CHB, CHS, manufacturer’s instructions. Primer sequences and PCR cycling CDX, JPT, KHV), European (CEU, FIN, GBR, IBS, TSI), and conditions are shown in Supplementary Table S3. PCR products Mixed American (CLM, MXL, PEL, PUR) populations were were purified with ExoSAP-IT™ (Thermo Fisher Scientific Inc.) obtained from all the available subjects (n = 2,312 individuals prior to automated Sanger sequencing using a BigDye® v3.1 from 19 populations, including 102 IBS subjects) of the 1K terminator Cycle Sequencing Kit in an ABI3730XL® (Thermo Genomes Project (phase 3, version 5) (The 1000 Genomes Fisher Scientific Inc., USA). DNA sequences were analyzed using Project Consortium, 2015). Haplotype assembly was carried the Finch TV 1.5.0 software. out by manual phasing of the alleles from the different variants analyzed. Genotyping of Single Nucleotide Polymorphisms and Microsatellite PCR Amplification of CA-repeat region was performed, and the RESULTS products were analyzed by Sanger sequencing. The results were validated by Fragment Size Analysis using an ABI3730XL® system Clinical Characteristics of the p.A5V-SOD1 and GeneMapper 4.0 Software (Applied Biosystems). Most SNPs Pedigree were genotyped by PCR amplification and Sanger sequencing The clinical phenotype in the affected members in our family—a as indicated above. In other cases, SNPs were detected by allele- mean age of onset of 51.7 years, spinal onset, predominant lower specific PCR using standard conditions and an internal PCR motor neuron signs, and a survival time of 20 months—is consistent control. Their primers and annealing temperature are shown with the phenotype in the American, Italian, and Swedish families. in Supplementary Table S3. PCR primers were designed using No cognitive impairment in either brother was detected in the Primer3 version 4.0. (Untergasser et al., 2012). neuropsychological test (Supplementary Table S1). Frontiers in Genetics | www.frontiersin.org 3 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family Genetic Results Haplotype Analysis Mutation analysis of the SOD1 gene by direct PCR sequencing We analyzed the 15 genetic variants spanning the human revealed a C-to-T transition at nucleotide position 14 (c.14C > T, chromosome 21 from the SOD1 gene to the SCAF4 gene Supplementary Figure S1) leading to a p.A5V (rs121912442, (Supplementary Table S2). The genetic markers around the A4V in the old nomenclature) sequence change at protein level in SOD1 gene were genotyped to infer the SOD1 haplotypes of the the two affected ALS patients: the proband (individual II:1) and ALS patients and the family members. Based on the genotypes of his brother (individual II:5) (Table 1). This exon 1 mutation was the four siblings, it was possible to identify four different inferred not found in the two healthy members of the family (individuals haplotypes present in the family and propose the haplotypes of II:3 and II:4) who requested details of their genetic situation the affected father and the mother (Figure 1). (Supplementary Figure S1). Fourteen of the 16 SNPs analyzed in the SOD1 gene No mutations were detected in the FUS, TARDBP, and PFN1 were detected in subjects of the IBS population from the 1K genes, and no C9orf72 expansions were identified in the two Genomes Project. Eight phased haplotypes of the SOD1 region ALS patients. were inferred in the IBS population with different frequencies TABLE 1 | SOD1 p.A5V mutation founder haplotype in IBS population compared to other populations. RS number SOD1 A5V founder haplotype Patients genotype USA* SWE* CHN** IBS II:1 II:5 rs4817415 C C − C C/C C/A rs2070422 T C C C C/C C/C rs1008270 A A − A A/A A/A rs9974610 A A − A A/A A/A rs2173962 T T − T T/T T/T rs202445 - - − T T/T T/T rs121912442 T T T T T/C T/C rs4816405 G C C/G C C/C C/C rs2070424 G A A A A/A A/A rs1041740 C T C/T T T/T T/T rs2833475 G A A A A/A A/A rs16988427 C T T/C T T/T T/T rs2833481 C T T T T/T T/T rs2070423 T C C/T C C/C C/C rs2833483 C T T/C T T/T T/T *Data from Broom et al., 2008. **Data from Tang et al., 2018. (-) Not available. FIGURE 2 | Haplotypes inferred in the IBS population (1K Genomes Project). The IBS haplotype_ID codification is based on the frequency of the haplotype in the IBS population, and in the case of the most frequent (IBS haplotype_ID = 1) the presence of the SOD1 p.A5V protein mutation (1V). (*) The haplotype 1V and allele rs121912442T are only present in the Spanish ALS patients with the SOD1 p.A5V mutation. Frontiers in Genetics | www.frontiersin.org 4 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family (Figure 2). The haplotypes were named (1 to 8) according to DISCUSSION their frequency in the population, ranging from higher to lower frequency (Figure 2). The mutation p.A5V-SOD1 detected in Redefining the p.A5V-SOD1 Phenotype. the ALS patients is only compatible with haplotype 1 (haplo1V), Reasons for the Disease’s Aggressive which is the most common in the IBS population (haplo1A) Course (Figure 2 and Table 1). According to the nomenclature proposed, Over 186 SOD1 mutations have been reported in amyotrophic the family has the IBS haplotypes 1A, 1V, 4, and 1A* (Figure 1). lateral sclerosis (ALSoD). Previous epidemiological studies The linkage disequilibrium of these 14 SNPs was calculated suggest that p.D91V is the most common worldwide, followed for the IBS population (Supplementary Figure S2). A by p.I113T and p.A5V. The latter is the most commonly conserved haplotype block, in high linkage disequilibrium, reported in the United States, accounting for 41% of the was detected between the start of intron 1 and the end of SOD1, mutations identified in that population. It is one of the most and including the SCAF4 gene. The SOD1 exon 1, including the aggressive mutations, as the mean survival time is under 2 c.14C > T (p.A5V) mutation, is located outside this conserved years. The mutation has rarely been described among European haplotype in the IBS population (Supplementary Figure S2). populations. This is the first time that the p.A5V-SOD1 has Using data for all the populations deposited in the 1K Genomes been reported in the Spanish population. Project, the analysis of linkage disequilibrium showed the With the rare exception of a 73-year-old male presenting same results for the conservation of this SOD1 haplotype block facial diplegia and unilateral vocal cord paralysis as the initial (data not shown). symptoms of ALS (Salameh et al., 2009), the phenotype associated The founder p.A5V-SOD1 haplotype in our patients is the with p.A5V in the American population is characterized by rapid same as the one found in the Swedish population, and differs progression and short survival. Patients present predominantly from the founder haplotype observed in patients from North lower motor neuron signs, with limb weakness in 51% of patients, America (Table 1). and bulbar symptoms in less than 11% of cases (Cudkowicz We determined all the inferred SOD1 haplotypes for these et al., 1997; Juneja et al., 1997; Andersen, 2003b; Andersen, 2006; 14 SNPs in the IBS, European, Asian, African, and Mixed Broom et al., 2008; Saeed et al., 2009). In a recent review of 57 American populations, using all the available subjects from p.A5V-SOD1 cases, the mean survival time was 1.4 years, with the 1K Genomes Project as a control group (Figure 3). A clinical onset at a mean age of 50.0 years, and a male-female total of 21 phased inferred haplotypes were detected in all the ratio of 1.3:1. Other characteristics associated with this SOD1 populations. The European haplotype (p.A5V-EUR) was the mutation include fast progression, predominantly spinal onset, most common in all the populations. The American haplotype high penetrance, involvement of primarily lower motor neuron (p.A5V-USA) was the second most common worldwide. signs, and an absence of dementia (Bali et al., 2017). However, both haplotypes appear with similar frequencies in However, the cases reported in European families differ the Asian population (0.386 for p.A5V-EUR, 0.385 for p.A5V- slightly from those in America. For example, Andersen reported USA) (Figure 3). the clinical characteristics of a Swedish family (Andersen et al., FIGURE 3 | Inferred haplotypes around the SOD1 gene detected in all populations from the 1K Genomes Project. (A) 21 inferred haplotypes. (B) Haplotype frequencies and counts are shown for each subpopulations: African (ACB, ASW, ESN, GWC, LWK, MSL, YRI); European (CEU, FIN, GBR, IBS, TSI); East Asian (CHB, CHS, CDX, JPT, KHV); South Asian (GIH, PJL, BEB, STU, ITU); Mixed American (CLM, MXL, PEL, PUR); all (African + European + East Asian + South Asian + Mixed Americans). EUR, haplotype base for p.A5V-EU. USA, haplotype base for p.A5V-USA. Frontiers in Genetics | www.frontiersin.org 5 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family 1997; Andersen, 2007). One case had leg onset, another had hand for p.A5V patients worldwide, rather than one founder, as onset, another had shoulder onset, and a further case had bulbar previously assumed (Broom et al., 2008; Saeed et al., 2009). Some onset. These individuals were aged between 56 and 68 years old, authors suggest that the European founder effect is older than the and survival ranged from 8 months to 2 years. In the members Amerindian, hypothesizing that the American families were in of the two Italian families reported in 2001, information was fact descendants of the European founder. Other authors argue available for two probands. One had shoulder onset at 55 years that the European origin is different from the American one old, and died 8 months later. The other proband presented hand (Rosen, 2004; Armon, 2009). onset at 57 years old. No information about this individual’s After analyzing the SOD1 haplotypes in different populations survival time is available, although the course was rapidly (European, Asian, African, and all the populations analyzed in progressive (Gellera et al., 2001). Our family’s phenotype—a the 1K Genomes Project), we found that the SOD1 haplotype in mean age of onset of 51.7 years, spinal onset, predominant lower which the p.A5V mutation is located in European fALS patients motor neuron signs, no cognitive alteration, and a survival (p.A5V-EU) is the most common in the population worldwide, time of 20 months—is consistent with the phenotype in these and the haplotype for American p.A5V-SOD1 patients (p.A5V- European families. Interestingly, none of the women inherited USA) is the second most common. These variants appear to be the mutation. Whether this was due to chance or a protective linked to the most common haplotypes, and not linked to rare factor is unknown. haplotypes. The haplotype p.A5V-USA is the most common The reasons why p.A5V-SOD1 predicts fast progression and (0.385) in the East Asian population and the p.A5V-EU haplotype short survival are unknown. In vitro studies have pointed to this is the second most common (0.306), with little difference between mutation leading to a decline in dimerization capacity, a loss their allelic frequencies. However, in the European population, of metalation (a 30-fold decrease in zinc-binding affinity), and the haplotype p.A5V-EU is the most common haplotype (0.298), aberrant oligomerization, leading to misfolding and aggregation far below the frequency of the haplotype p.A5V-USA (0.062). in the form of insoluble toxic inclusions within motor system The haplotypes p.A5V-EU (0.037) and p.A5V-USA (0.045) are cells, a key pathological hallmark of ALS. There is some rare in the African populations (as shown in Figure 3). variability in the propensity of SOD1 mutants to aggregate, which Based on these data, we propose two possible hypotheses for could be related to the duration of the disease. High-aggregation the origin of this mutation in two different haplotypes: propensities have been described for p.A5V, which could be the factor responsible for the disease’s short duration (Prudencio A Single Origin for All p.A5V-SOD1 et al., 2009; Zhao et al., 2014; Farrawell et al., 2018; Maurel et al., Families Worldwide 2018; Srinivasan and Rajasekaran, 2018). Information from previous linkage analyses suggests an Another alternative and complementary hypothesis to origin for p.A5V around 13,000 years ago, probably in Asia. explain the aggressive nature of p.A5V is its position in the The p.A5V mutation appears in the p.A5V-EU haplotype and tertiary structure of the protein. Interestingly, of the 30 mutations would have disseminated in Europe and Asia. A homologous reported in exon 1, most of those in residue A5 (A4 in the previous recombination could have occurred in an ALS American family, nomenclature) predispose to a rapid progression and survival of leading to the linkage of the p.A5V mutation to the p.A5V-USA under 24 months (Syriani et al., 2009). According to the tertiary haplotype. A founder effect in North America could explain structure of SOD1, residue A5 is located in the first β-strand that the p.A5V mutation associated to the p.A5V-USA haplotype in forms part of the dimer interface. A5 mutations affect SOD1 most (82%) cases in the American families (Rosen et al., 1994; dimerization and/or destabilize the Greek key β-barrel because Rosen, 2004; Broom et al., 2008; Armon, 2009; Saeed et al., A5 packs into the SOD1 monomer’s hydrophobic core (Getzoff 2009) and to the p.A5V-EU in only 18% of the other fALS cases. et al., 1990; Deng et al., 1993; Cardoso et al., 2002; Galaleldeen Only the p.A5V mutation linked to the haplotype p.A5V-EU et al., 2009; Schmidlin et al., 2009; Schmidlin et al., 2013; is detected in Asia and Europe. Interestingly, linkage analysis Kumar et al., 2018). of the haplotype of SOD1 SNPs, a highly conserved haplotypic block (with a strong correlation) is observed after exon 1, but does not contain exon 1. Founder Effect Two earlier studies investigated a possible founder effect in Several Origins (One for Asians, One for Americans, the cases of North American and European origin. Following and Perhaps One or More for Europeans) these haplotype studies, there is evidence to suggest at least Previous studies investigating the haplotypic region around two different origins: one European (probably a Scandinavian SOD1 have suggested different mutational events to explain founder effect) and one Amerindian (Native Americans). The the mutation in USA and European ALS patients (Rosen, 2004; latter presents in 82% of North American families with the Broom et al., 2008, Eisen et al., 2008; Saeed et al., 2009). It is p.A5V-SOD1 mutation. In European populations, p.A5V-SOD1 possible that the mutation in A5 was generated in the haplotypes has been reported in Scandinavia (three cases) and Italy (six p.A5V-USA and p.A5V-EU independently, since the two cases). An isolated case has recently been reported in China, haplotypes are the most frequent in the population worldwide, which shares most of the European haplotype. These findings and the locus at codon 5 of SOD1 would be prone to mutations could be interpreted in terms of the existence of various founders (a hotspot) since four additional mutations (p.A5T, p.A5S, p.A5F, Frontiers in Genetics | www.frontiersin.org 6 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family and p.A5P) have been described in this codon (Syriani et al., site of onset, and survival were similar to those reported 2009; Pratt, 2014). mainly in North American kindreds, in a few European From the molecular point of view, the most parsimonious families and in one Asian individual. SNP and haplotype hypothesis would be a single origin for the A5V mutation. analyses identify 21 haplotypes worldwide for the SOD1 Our hypothesis of a common founder for p.A5V is supported genomic region. Our family shares the haplotype reported in by research investigating who first colonized America and the founder European effect rather than the more frequent when they did so. These genetic studies showed that the first Amerindian haplotype. inhabitants of the Americas came from a single Siberian population, who used the Bering Land Bridge to migrate from Beringia to the Americas sometime after 16,500 years ago DATA AVAILABILITY STATEMENT (Bonatto and Salzano, 1997; Goebel et al., 2008). We propose a Eurasian origin for the A5V mutation in the haplotype Publicly available datasets were analyzed in this study. This data p.A5V-EU. A Eurasian individual could have introduced the can be found here: http://www.internationalgenome.org/data. A5V mutation of the haplotype p.A5V-EU in America, and the mutation could have recombined in their descendants to obtain the p.A5V-USA haplotype linked to the p.A5V mutation. ETHICS STATEMENT A genetic drift in America could therefore explain the founder effect of p.A5V in the haplotype p.A5V-USA being the most This study was carried out in accordance with the frequent ALS1 mutation in USA. A similar genetic phenomenon recommendations of the Hospital Universitari Vall d’Hebron was described for the D90A mutation. Initial studies proposed Institutional Review Board (VdHIRB) with written informed two different founder effects (one for heterozygous cases and one consent from all subjects. All subjects gave written informed for homozygous cases) (Al-Chalabi et al., 1998). After increasing consent in accordance with the Declaration of Helsinki. The the sample set and the markers, they concluded that there was protocol was approved by the VdHIRB. a single founder for all cases. These studies for this mutation showed that the D90A mutation arose in Eurasia approximately 20,000 years ago (Parton et al., 2002). AUTHOR CONTRIBUTIONS Asian or Amerindian origins have been proposed for the A5V mutation based on their high frequency in USA and the JG and JV-T conceived the study. JG and MS collected the haplotype p.A5V-USA being the most frequent haplotype in clinical information. CG, JV-T, ES, MM, and JG analyzed the Asian populations (Rosen 2004; Broom et al., 2008; Saeed et al., data. JV-T and CG conducted the bioinformatics analysis. CG, 2009). The hypothesis of these two origins (Amerindian and JV-T, and JG drafted the manuscript. CG and JV-T contributed Asian) is contradicted by the failure to detect the A5V mutation equally to this work. All the authors approved the final version in the p.A5V-USA haplotype in ALS cases from Africa, Europe, of manuscript. Asia, and among Native Americans. Current data, including our results, indicate that the A5V mutation cases have been associated to the p.A5V-EU haplotype in different populations FUNDING from Europe, USA, and Asia. This study has been supported by Instituto de Salud Carlos The SOD1 haplotypes inferred from the IBS population III (grant numbers PIS-FEDER PI16/01673 and PI19/00593). enabled us to identify the haplotypes of the four members of JG and JV-T are the recipients of grant 2017SGR00939 from a Spanish ALS family of Caucasian origin with two affected Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) of brothers with the p.A5V-SOD1 mutation. The SOD1 haplotype the Generalitat de Catalunya. associated to the A5V mutation in our Spanish cases is the most frequent in the IBS and European populations. To our knowledge, our Spanish family is unrelated to the other p.A5V reported cases from Sweden (three subjects from one family) or ACKNOWLEDGMENTS Italy (six subjects from two families). A more detailed haplotype The authors are indebted to the patients and their relatives for analysis of the European fALS cases with this mutation will be their cooperation. We thank the multidisciplinary ALS Unit necessary to investigate whether there is a common origin for Team from Vall d’Hebron University Hospital. the p.A5V-SOD1 mutation in the European ALS cases, or if it is a consequence of different mutational events. This research could be carried out through an international collaborative consortium SUPPLEMENTARY MATERIAL to enroll and analyze these ALS families, as in the study suggested by an Italian group (Gellera et al., 2001). The Supplementary Material for this article can be found online at: In conclusion, this is the first report on the p.A5V-SOD1 https://www.frontiersin.org/articles/10.3389/fgene.2019.01109/ mutation in the Spanish population. The age at onset, full#supplementary-material Frontiers in Genetics | www.frontiersin.org 7 November 2019 | Volume 10 | Article 1109 Garcia et al. First A4V SOD1 Spanish Family REFERENCES Canada: clinical features, neurophysiology and ethical issues in management. Amyotroph. 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The p.E22G mutation in the Cu/Zn absence of any commercial or financial relationships that could be construed as a superoxide-dismutase gene predicts a long survival time: clinical and genetic potential conflict of interest. characterization of a seven-generation ALS1 Spanish pedigree. J. Neurol. Sci. 285, 46–53. doi: 10.1016/j.jns.2009.05.011 Copyright © 2019 Garcia, Vidal-Taboada, Syriani, Salvado, Morales and Gamez. Tang, L., Ma, Y., Liu, X., Chen, L., and Fan, D. (2018). Identification of an A4V This is an open-access article distributed under the terms of the Creative Commons SOD1 mutation in a Chinese patient with amyotrophic lateral sclerosis without Attribution License (CC BY). The use, distribution or reproduction in other forums the A4V founder effect common in North America. Amyotroph. Lateral Scler. is permitted, provided the original author(s) and the copyright owner(s) are credited Frontotemporal Degener. 19, 466–468. doi: 10.1080/21678421.2018.1451895 and that the original publication in this journal is cited, in accordance with accepted The 1000 Genomes Project Consortium (2015). A global reference for human academic practice. No use, distribution or reproduction is permitted which does not genetic variation. Nature 526, 68–74. doi: 10.1038/nature15393 comply with these terms. Frontiers in Genetics | www.frontiersin.org 9 November 2019 | Volume 10 | Article 1109