Neuromuscular

Original research

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Identification of a pathogenic mutation in ARPP21 in
patients with amyotrophic lateral sclerosis
Oriol Dols-­Icardo ‍ ‍,1,2 Álvaro Carbayo ‍ ‍,3,4,5 Ivonne Jericó,6,7
Olga Blasco-­Martínez,8 Esther Álvarez-­Sánchez,1,2 Maria Angeles López Pérez,8
Sara Bernal,4,9 Benjamín Rodríguez-­Santiago,4,9,10 Ivon Cusco,9 Janina Turon-­Sans,3,4,5

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Manuel Cabezas-­Torres,3,4 Marta Caballero-Ávila ‍ ‍,3,4,5 Ana Vesperinas,3,4,5
Laura Llansó,3,4,5 Inmaculada Pagola-­Lorz,6,7 Laura Torné,6,7 Natalia Valle-­Tamayo,1,2
Laia Muñoz,1,2 Sara Rubio-­Guerra ‍ ‍,1,2 Ignacio Illán-­Gala ‍ ‍,1,2
Elena Cortés-­Vicente ‍ ‍,3,4,5 Ellen Gelpi,11 Ricard Rojas-­García ‍ ‍3,4,5

► Additional supplemental ABSTRACT

material is published online Background and objective Between 5% and 10% WHAT IS ALREADY KNOWN ON THIS TOPIC
only. To view, please visit the ⇒ Between 5% and 10% of amyotrophic lateral
journal online (https://d​ oi.​org/​ of amyotrophic lateral sclerosis (ALS) cases have a
10.​1136/​jnnp-​2024-​333834). family history of the disease, 30% of which do not sclerosis (ALS) cases have a family history of
have an identifiable underlying genetic cause after a the disease. 30% of familial ALS cases do not
For numbered affiliations see comprehensive study of the known ALS-­related genes. have an identifiable underlying genetic cause
end of article. after a comprehensive study of the known ALS-­
Based on a significantly increased incidence of ALS in
a small geographical region from Spain, the aim of this related genes. Incidence rates of ALS are fairly
Correspondence to homogeneous across European and American
Dr Ricard Rojas-­García; ​rrojas@​ work was to identify novel ALS-­related genes in ALS
santpau.​cat and Dr Oriol Dols-­ cases with negative genetic testing. populations.
Icardo; o​ dols@​santpau.c​ at Methods We detected an increased incidence of both WHAT THIS STUDY ADDS
sporadic and, especially, familial ALS cases in a small
OD-­I and ÁC contributed ⇒ This work supports the causative role of
region from Spain compared with available demographic
equally. cyclic AMP regulated phosphoprotein 21
and epidemiological data. We performed whole genome
(ARPP21) in ALS and adds to the extensive
Received 18 March 2024 sequencing in a group of 12 patients with ALS (5 of them
list of RNA-­binding proteins involved in ALS
Accepted 17 June 2024 familial) from this unique area. We expanded the study
Published Online First 2 July pathophysiology. Further studies are required
to include affected family members and additional cases
2024 to elucidate the contribution and frequency of
from a wider surrounding region.
this variant in ARPP21 to the genetic basis of
Results We identified a shared missense mutation
the disease. Peaks of incidence of the disease in
(c.1586C>T; p.Pro529Leu) in the cyclic AMP regulated
small geographical areas may help identify the
phosphoprotein 21 (ARPP21) gene that encodes an
underlying genetic causes of ALS.
RNA-­binding protein, in a total of 10 patients with ALS
from 7 unrelated families. No mutations were found in HOW THIS STUDY MIGHT AFFECT RESEARCH,
other ALS-­causing genes. PRACTICE OR POLICY
Conclusions While previous studies have dismissed a ⇒ ARPP21 should be reclassified as an ALS-­
causal role of ARPP21 in ALS, our results strongly support causing gene, and included in genetic
ARPP21 as a novel ALS-­causing gene. screenings along with other known genes.

INTRODUCTION ALS-­causing mutations have an impact on genes

Amyotrophic lateral sclerosis (ALS) is a devastating encoding RNA-­binding proteins, such as transactive
motor neuron disease, characterised by muscle response DNA-­binding protein 43 (TDP-­43), fused
weakness and atrophy secondary to upper and in sarcoma (FUS) and T cell-­restricted intracellular
lower motor neuron degeneration, leading to respi- antigen-­1 (TIA1), among others, highlighting the
ratory insufficiency and death within a mean of 3–5
central role of RNA metabolism in the disease.7
years.1 Over the last decades, an evident overlap of
© Author(s) (or their However, despite the rapidly growing number
clinical, genetic and neuropathological features has
employer(s)) 2025. Re-­use of ALS-­ related genes, genetic diagnosis remains
permitted under CC BY-­NC. No been established between ALS and frontotemporal
dementia (FTD).2–5 elusive in approximately 30% of patients with
commercial re-­use. See rights
and permissions. Published by Although most cases are considered sporadic fALS.8 9 The disease has a complex genetic archi-
BMJ Group. (sALS), approximately 5–10% of ALS cases have a tecture, and oligogenic and polygenic models of
previous family history of ALS and are classified as inheritance have gained relevance.10 In this era of
To cite: Dols-­Icardo O,
Carbayo Á, Jericó I, et al. J familial ALS (fALS).6 To date, over 40 genes have protein-­targeted therapies and genetically oriented
Neurol Neurosurg Psychiatry been related to ALS, most showing a Mendelian treatments, discovery and detection of new genetic
2025;96:132–139. autosomal dominant inheritance pattern. Several variants and implicated genes is paramount.10
132 Dols-­Icardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834
 Neuromuscular
especially when several cases aggregate in a family, might suggest
the disorder has an underlying genetic substrate.13

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Here we report the identification of a mutation in the cyclic
AMP-­regulated phosphoprotein 21 (ARPP21) gene, supporting
its role as a novel ALS-­causing gene and expanding the reper-
toire of RNA-­binding proteins associated with ALS. We describe
the clinical phenotype of 10 patients from 7 unrelated families
carrying the same mutation in ARPP21.

MATERIALS AND METHODS

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Case selection and study design
Since 1998 our register has prospectively included cases of ALS
seen at the Motor Neuron Clinic at the Neuromuscular Diseases
Unit of Hospital Sant Pau (Barcelona), a national referral
centre for neuromuscular diseases. Between 2009 and 2022 we
observed an unusual number of cases of ALS (especially familial
cases) at our outpatient clinic, from a small region in the prov-
ince of La Rioja in Spain. Figure 1 illustrates the study design.
For epidemiological justification (figure 1 – box 1) we reviewed
our database and contacted neurology departments from local
centres in the region. Eligible population: We selected new cases
with diagnosis of ALS from the region, visited between January
2009 and December 2022.
Study population: For genetic analysis (figure 1 – box 2) we
selected cases from the study area (see figure 2) with available
DNA samples, performing whole genome sequencing (WGS).
Patients fulfilled ALS-­El Escorial revised criteria for probable,
probable laboratory-­supported, or definite ALS.14 Both sporadic
and fALS cases were included. fALS was defined as having
a family member with a diagnosis of ALS in first or second-­
degree relatives, or having a first-­degree family member with
a confirmed FTD in the formal neuropsychological examina-
tion, according to current proposed criteria.6 All patients went
through a targeted ALS-­gene panel prior to WGS (online supple-
Figure 1 Flowchart indicating the steps followed in the study and the mental table 1 in supplementary materials). Patients with a
number of patients at each phase. ALS, amyotrophic lateral sclerosis; previously identified ALS-­causing mutation in this custom panel
ARPP21, cyclic AMP regulated phosphoprotein 21; fALS, familial ALS; MND, were excluded from this part of the study (WGS analysis), as
motor neuron disease; WGS, whole genome sequencing. they already had a known underlying genetic substrate for the
disease.
After identifying the candidate variant, we expanded the anal-
Prospective, population-­based studies show that incidence ysis with the aim of increasing the series of mutation carriers.
rates of ALS are relatively uniform across European and Amer- We performed Sanger sequencing in other cases of sALS and
ican populations, ranging between two and three cases per year fALS from the surrounding geographical areas and also in their
per 100 000 inhabitants.11 12 Peaks of incidence in small regions, affected family members (figure 1 – box 3). In cases carrying

Figure 2 (A) Map of Spain and its regions. (B) Map of La Rioja province and its regions. Regions belonging to the south-­eastern area of La Rioja, where
an increased frequency of amyotrophic lateral sclerosis cases was detected, are shaded in green. White dots mark the location on the map of the six towns
of origin of the seven families.
Dols-I­ cardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834 133
 Neuromuscular
the candidate variant, mutations in other known ALS-­FTD genes FS>60,000, FS>200,000, ReadPosRankSum<−8.0, ReadPos-
were ruled out through exome sequencing (see online supple- RankSum>20.0, MQRankSum<−12.5, QD<2.0, MQ<40.0).

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mental table 1 in supplementary materials). We also retained variants where at least one sample contained
the variant with a Depth ≥10.
Demography and epidemiology Functional annotations were added using SnpEff V.5.0 with
The expected number of cases in the area during the study period the hg38.22 Variants were annotated with SnpSift V.5.023 using
was calculated using incidence rates from previous studies and population frequencies, conservation scores and deleteriousness
demographic data of those in the catchment area over 18 years. predictions from dbNSFP V.4.1a.24 We also used other sources of
Demographic information on the geographical region was annotations, such as gnomAD (V.3.1.2), CADD (V.1.6), InterVar
obtained from the official demographic records between 2009 and Clinvar (V.20200602).25–27 In order to check the frequency
and 2022 in the Spanish National Institute of Statistics (INE— of the ARPP21 variant in the Spanish population, we used the

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Instituto Nacional de Estadística).15 16 Collaborative Spanish Variant Server which comprises exomes
The minimum incidence was calculated using the numbers of and genomes from 2105 unrelated Spanish individuals.28
new cases identified from the area and the population at risk Somalier software29 was used with WGS and whole exome
in the study period. We compared the observed cases with the sequencing (WES) data to assess relatedness among all probands
expected number of cases in the area, to obtain a ratio. included in the study (coefficient of relationship as a measure of
the degree of consanguinity). Briefly, the software considers for
Clinical features each comparison: (1) the number of sites where one sample is
We recorded demographic and clinical variables. Demographic hom-­ref and another is hom-­alt; (2) the number of sites where
data included sex, date and place of birth and place of origin the samples have the same genotype; (3) the number of sites
of the family. Clinical variables included age at disease onset, where both samples are heterozygotes; and (4) the number of
disease duration, region of onset of motor symptoms (spinal or sites where both samples are homozygous alternate.
bulbar). The clinical phenotype was categorised depending on WGS data was used to identify shared genotypes that might
the presence of upper (primary lateral sclerosis), lower motor suggest a common haplotype in the four mutation carriers with
neuron signs (progressive muscular atrophy) or both (ALS). available WGS data. A merged VCF file of ARPP21 gene ±1
The presence of cognitive and/or behavioural impairment and megabase combining variants from the four patients was obtained
whether or not they met clinical criteria for FTD and its variants, to explore shared blocks of genotypes. LDhap web-­based tool
were also noted.17–19 (https://ldlink.nih.gov/?tab=ldhap) was used to obtain specific
population haplotype frequencies of all haplotypes observed for
a list of 30 query genetic variants (the maximum accepted as
Family history input) flanking the shared ARPP21 candidate variant and part
We recorded previous cases of ALS in the family as part of the
of the shared haplotype (23 of them were available in LDhap).
routine clinical evaluation. As possible indicators of an under-
We selected the Iberian population in Spain (IBS) population to
lying FTD we also recorded previous history of FTD, early-­onset
obtain allelic frequencies, in addition to the other populations in
cognitive impairment (defined as younger than 65 years of age at
Europe and all other super populations available (African (AFR),
symptom onset), cognitive impairment with marked behavioural
American (AMR), East Asian (EAS) and South Asian (SAS)).
impairment but no definite diagnosis, and diagnosis of other
The presence of the disease-­causing mutation in ARPP21 was
neurodegenerative diseases.
validated using Sanger sequencing in all mutation carriers using
the following forward (5’ ​GACA​GTGG​TGTG​CATC​TTGTG 3’)
Genetic analyses and reverse (3’ ​GAGACATTCGCACATACCCC 5’) primers.
Library preparation and sequencing Once the ARPP21 variant was confirmed, other ALS and FTD
WGS was performed at the National Centre for Genomic Anal- causing genes were screened using WES and mutations in 58
ysis (Centro Nacional de Análisis Genómico—CNAG-­ CRG, ALS-­FTD-­related genes were ruled out (see online supplemental
Barcelona, Spain). Paired-­end multiplex libraries were prepared table 1 in supplementary materials for the complete list of
following the manufacturer’s instructions. Libraries were loaded genes). WES was used to study gene coding exons and flanking
to Illumina flow cells for cluster generation prior to producing regions (±50 base pairs). Briefly, DNA libraries were prepared
150 base read pairs on a NovaSeq 6000 instrument following using kapa reagents (Roche) and sequenced in NextSeq 500
the Illumina protocol. Image analysis, base calling and quality or NextSeq 1000 instruments (Illumina) to obtain 2×150 base
scoring of the run were processed using the manufacturer’s soft- pair reads. Fastq files were mapped to the human genome using
ware Real Time Analysis and followed by generation of FASTQ BWA,30 variants were called through GATK variant calling,21
sequence files. and annotated using ANNOVAR.31 The C9orf72 hexanucleotide
repeat expansion was discarded by means of rpPCR (repeat-­
Bioinformatic and genetic analyses primed Polymerase Chain Reaction), as previously described.32
Reads were mapped to human build GRCh38 with BWA-­MEM
V.0.7.17.20 Alignment files containing only properly paired,
Ethics
unique mapping reads without duplicates were processed using
The study was conducted in accordance with the Declaration of
Picard V.2.20 (http://broadinstitute.github.io/picard/) to add
Helsinki. All participants gave their written informed consent to
read groups and to remove duplicates. The Genome Analysis
participate in the study.
Tool Kit (GATK V.4.1.9.0)21 was used for local realignment
and base quality score recalibration. Joint variant calling was
done using HaplotypeCaller, CombineGVCFs and Geno- RESULTS
typeGVCFs from GATK following the GATK best practices Epidemiology
pipeline. Variants were hard filtered using GATK Variant- The south-­
eastern region of the province of La Rioja is a
Filtration (BaseQRankSum>4.0 || BaseQRankSum<−4.0, 1219.42 km2 area in north-­
eastern Spain (figure 2). Between
134 Dols-­Icardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834
 Neuromuscular

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Figure 3 Family trees of the seven families in which the p.Pro529Leu mutation in ARPP21 was detected. Patients with ALS are shaded in black. Cases
presenting an early onset dementia or dementia with prominent behavioural features are marked with a red square at the top right corner. The proband
in each family is marked with an arrow and a ‘P’. Cases that harbour the p.Pro529Leu mutation in ARPP21 are outlined in red and marked with an
asterisk. The healthy individual (Family 4— II.3) with negative genetic testing is framed in a green box. No DNA was available from the other patients
with ALS or patients with early-­onset cognitive-­behavioural impairment in the pedigrees. ALS, amyotrophic lateral sclerosis; ARPP21, cyclic AMP regulated
phosphoprotein 21; FTD, frontotemporal dementia.

2009 and 2022, it had a mean population of 43 433, of whom 12 of the 15 patients were eligible for further genomic analysis.
31 324 were over 18 years of age. The population density was One patient was excluded from this part of the study because an
35.62 persons/km2. Figure 1 illustrates the study design and alternative genetic cause was found (non-­sense pathogenic muta-
flowchart of patients studied in each phase. tion in NEK1—c.3107C>G; p.Ser1036X—NM_001199397.3).
Considering a mean ALS incidence of 1.4–2.47 cases/100 000 No DNA was available for analysis in the remaining two patients.
person/year,11 12 we calculated an expected number of cases of
0.44–0.77 cases per year in this area, equivalent to 5–10 patients
during the study period (2009–2022). In reference to fALS, Genetics
assuming a frequency of 5–10% of fALS, the expected number Detection of the ARPP21 mutation
of cases in the area would be 0.02–0.08 cases/year or one new WGS was performed in 12 unrelated patients (5 fALS and 7
case every 12.5–50 years. sALS). We found a shared heterozygous missense mutation in
Between 2009 and 2022, we visited 15 patients from the ARPP21 (c.1586C>T; p.Pro529Leu (NM_001385486.1 or
study area who met the diagnostic criteria for ALS. 7 of the 15 ENST00000417925.5)) in four unrelated patients with ALS
(46.6%) had a family history of ALS and were considered as (three fALS) from four different families (figure 3—Families
fALS. In fALS cases, known ALS-­causative mutations were ruled 1–4) (table 1). The high frequency of this almost unique variant,
out through exome sequencing analysis or a custom gene panel. together with its predicted deleteriousness (CADD_phred=26.7;
The comparison between the observed frequency and the Polyphen HDIV and HVAR=Deleterious and Mutation Tast-
expected frequency resulted in an increased frequency of ALS er=Deleterious) and high GERP (Genomic Evolutionary Rate
cases in the area, with a ratio of 1.5–2.6 for total ALS cases and Profiling) score (6.07), which predicted a huge evolutionary
7–24.5 for fALS cases. constraint, led us to prioritise this variant in ARPP21 as a

Table 1 Clinical features of patients with ALS carrying the ARPP21 mutation
Family Patient Region of onset Motor phenotype Cognitive symptoms Disease duration Study
1 Fam 1 II.1* Spinal ALS No 13 WGS
2 Fam 2 III.3 Spinal ALS No – WES
3 Fam 2 III.1* Spinal ALS No 16 WGS
4 Fam 3 II.2* Spinal ALS No – WGS
5 Fam 4 II.1* Bulbar ALS-F­ TD bvFTD 31 WGS
6 Fam 5 III.11* Bulbar ALS No 16 WES
7 Fam 5 IV.1 Spinal ALS No 15 WES
8 Fam 6 III.3* Spinal ALS No – WES
9 Fam 7 III.2 Spinal ALS No 17 WES
10 Fam 7 III.3* Spinal ALS No 4 WES
Probands of each family are marked with an asterisk (*). ‘Disease duration’ is expressed in months (M).
ALS, amyotrophic lateral sclerosis; ARPP21, cyclic AMP regulated phosphoprotein 21; bvFTD, behavioural variant of FTD; FTD, frontotemporal dementia; MND, motor neuron
disease; WES, whole exome sequencing; WGS, whole genome sequencing.

Dols-I­ cardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834 135

 Neuromuscular
potential causative mutation. The coefficients of the relationship Regarding family history, only one patient did not have a
obtained using Somalier suggest that the probands with available family history of ALS, but had a younger sibling with an early

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WGS and WES data are unrelated (data not shown). This variant onset dementia (Family 4 in figure 3). Moreover, two other fami-
is absent in the population database gnomAD (V.3.1.2) and lies (Families 5 and 7 in figure 3) had a family history of early-­
carried by 4 out of 1 384 130 chromosomes in the latest updated onset dementia.
version of gnomAD (V.4.0.0), where the mutation is annotated
as p.Pro563Leu (NM_001385562.1 or ENST00000684406.1). Pedigrees and case descriptions
We did not find any mutation carrier among the 2105 unre- Table 1 summarises the main clinical features of the series.
lated Spanish individuals included in the Collaborative Spanish Figure 3 shows the family trees.
Variant Server.28
Our analysis disclosed a common haplotype shared by the four

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Family 1
mutation carriers with available WGS data, which included 442 Proband (II.1) presented with progressive paraparesis in their
SNPs (Single Nucleotide Polymorphisms), encompassing 256 Kb late 40s, with diffuse upper motor neuron signs. Family history
(chr3:35605944–35862077). This haplotype was also found in was notable for a younger sibling (II.4) who had died from ALS
WGS data from two of the eight non-­mutation carriers included in their 30s, and was hence diagnosed with fALS, with initial
in this study. We further assessed its frequency in the general negative genetic testing, dying of respiratory insufficiency within
population using LDhap. The frequency of the haplotype in the 13 months from symptom onset. No clinical information was
IBS was 7.48%, the same as in the Toscani in Italy. Interestingly, available concerning the proband’s parents.
this haplotype was observed at a higher frequency in the British
in England and Scotland (GBR) population (11.5%) or at a lower
Family 2
frequency in the Finnish in Finland population (5.6%). The
Proband (III.1) was a patient in their late 30s with a family
haplotype was almost absent in AFR or EAS populations (<1%),
history of ALS in the grandfather on the father’s side (I.1) and
and at low frequencies in the ad mixed AMR population (2.7%).
in three of his aunts (II.2; II.6; II.8). The patient presented with
Notably, 9% of the SAS population carried this haplotype.
spinal onset ALS and died within 16 months from symptom
onset. Conventional genetic testing was negative but the hetero-
zygous variant in ARPP21 was identified. A cousin (III.3) was
Expanded study in the region
diagnosed with ALS and carries the ARPP21 variant.
We subsequently broadened the analysis to affected family
members of mutation carriers (hence new fALS cases) and other
new patients with sALS and fALS from a wider surrounding Family 3
geographical area. Proband (II.2) is an adult patient with no relevant medical history,
Using Sanger sequencing we analysed 22 new patients with but a family history of a brother (II.4) who died of spinal onset
ALS, 8 of whom were fALS. We found the candidate variant in ALS. There were no medical records available for their parents.
6 novel individuals: 1 affected relative in Family 2, and 5 more The patient was diagnosed with ALS in their late 60s following
cases from 4 unrelated families, resulting in a final count of 10 progressive distal upper and later lower limb weakness. Despite
ARPP21 mutation carriers from 7 unrelated families (see pedi- the slow progression of the disease, neurophysiological testing
grees in figure 3). We were able to study only one unaffected confirmed a motor neuron disease. At the time of publication of
family member (Family 4—II.3), who did not harbour the candi- the present work, the patient was alive.
date variant. We did not find any patient with ALS among the
families who did not carry the candidate variant in ARPP21. Family 4
Other ALS-­FTD-­related mutations were ruled out through Proband (II.1) was a patient in their 60s who presented with a
WES, including the p.Arg92Cys variant in GLT8D1 (see online sporadic, bulbar onset ALS, with a disease duration of 3 years.
supplemental table 1). No other rare variants were found in the The patient showed profuse behavioural and cognitive impair-
GLT8D1 gene in ARPP21 mutation carriers. In the proband of ment, suggestive of an ALS-­FTD, but no formal neuropsycholog-
Family 5 (III.11), we detected a heterozygous variant in SQSTM1 ical testing was available.
(c.1175C>T; p.Pro392Leu), for which there is no consensus A younger brother (II.2) had been diagnosed with an early-­
regarding its pathogenicity for ALS-­FTD. The rare variant in onset dementia before 65 years of age, but no formal cognitive-­
SQSTM1 was not carried by his affected family member (Family behavioural evaluation was performed. Both their parents had
5—IV.1) who also carried the mutation in ARPP21 described in died from unknown causes before 60 years of age. No further
this study, thus demonstrating that did not segregate with disease detailed information was available on their family. The patient’s
and making any potential association with ALS unlikely. younger sibling (II.3), older than 70 years of age, who showed
no signs or symptoms suggestive of motor neuron disease, was
genetically tested and did not harbour the ARPP21 mutation.
Clinical features and family history
Table 1 shows the main clinical features of the series. Five patients Family 5
were men (50%). The mean age at onset was 59.5 years (SD Proband (III.11) was an elderly patient with an unremarkable
11.37) and the mean disease duration was 16 months (SD 7.96). personal medical history. This patient presented with a bulbar
At the date of publication, three patients (30%) were alive. The onset motor neuron disease with progressive limb weakness and
region of onset was bulbar in two patients (20%) and spinal in respiratory failure, leading to death within 16 months.
the others (n=8; 80%). The clinical phenotype was classic ALS There was a profuse history of ALS in the family (see family
(ie, presence of upper and lower motor neuron signs) in all 10 tree: II.3, II.8, III.4, III.7, III.10, IV.1, IV.6). Furthermore, an
patients (100%), with no cases of progressive muscular atrophy uncle of the proband (II.6), whose son and granddaughter died
or primary lateral sclerosis in the series. Cognitive-­behavioural of ALS and was hence an obligate carrier, was diagnosed with
impairment was present in one patient (10%). unspecified dementia and died before 50 years of age. After the
136 Dols-­Icardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834
 Neuromuscular
ARPP21 mutation was detected in the proband, the only affected the shorter disease duration might be independently attributed
family member (IV.1) with available DNA was retrospectively to the p.Pro529Leu mutation in ARPP21.

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confirmed to harbour the same mutation. Interestingly, the four mutation carriers identified through our
WGS approach shared a common haplotype. This haplotype was
Family 6 present in two of the eight non-­mutation carriers in our study
Proband (III.3) was a patient in their late 60s, with a family with available WGS data, and it is also found in other worldwide
history of two cases of ALS (III.2, II.2). The proband developed populations. In this context, beyond the Spanish population, its
progressive paraparesis evolving to upper limb weakness and high frequency in the GBR and SAS populations, together with
dysphagia, and neurophysiological examinations compatible the fact that this haplotype is found at very low frequencies in
with ALS. At the time of submission of this work, the patient AMR, AFR or EAS populations, suggests that people carrying
this mutation might be restricted to specific ancestries. In this

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was alive.
sense, the ARPP21 mutation, although not definitely linked
to ALS, has previously been detected in patients of European
Family 7 ancestry, primarily from UK,34 which is part of the GBR popula-
Proband (III.3) was an adult in their 60s who presented with a tion. Indeed, the haplotype is almost null in the EAS population,
rapidly progressive tetraparesis, followed by respiratory insuf- which might explain why this mutation has not been found in
ficiency and death within the first 4 months of disease onset. large cohort studies from China35 and Australia34 36 that have
The patient’s brother (III.2) died of ALS 10 years prior to the previously described other rare variants in ARPP21 or GLT8D1,
proband’s diagnosis, and was retrospectively confirmed to carry but were unable to confirm their pathogenic role. Large ALS
the ARPP21 mutation. A cousin (III.5) was also diagnosed with cohorts have been previously used to identify novel disease-­
early-­onset dementia before 60 years of age. Their father died of causing variants, such as those included in project MiNE which
pancreatic cancer in his early 70s. comprises samples from diverse countries across the world (from
Spain, but also Brazil, the USA, Australia and Russia, among
DISCUSSION others). As demonstrated by the differences in the presence of
Following an increased frequency of cases in the small region the ARPP21 risk haplotype around the world, using such diverse
of the south-­eastern La Rioja province in Spain, we identified populations may have precluded the identification of extremely
the p.Pro529Leu mutation in ARPP21 in 10 patients from 7 rare disease-­causing variants using rare genetic burden associ-
unrelated families, showing a presumably autosomal dominant ation analyses. It is important to note that our approach was
pattern. Familial cases (ie, fALS) accounted for almost 50% of based on the identification of a higher than expected burden of
the total cases from the area, none of whom harboured any other ALS cases in a remote region. For this reason, we implemented
known ALS-­causing mutations. a unique strategy that diverged from previously used methods.
From a clinical point of view, patients carrying the p.Pro529Leu To date, the most recent reviews on the genetics of ALS do
variant presented a shorter survival (mean 16 months) than not consider ARPP21 to be an ALS-­causing gene,8 nor was it
that described in the literature for ALS,12 with no differential included in a recent study comprising a set of 90 ALS-­associated
clinical features. Although no systematic cognitive assessment genes selected from multiple databases.37 In addition, an OMIM
was performed, one patient showed prominent cognitive and number has not yet been assigned to the ARPP21 gene, and in the
behavioural impairment. Furthermore, three relatives (one of ALS Online Database (ALSoD; https://alsod.ac.uk)38 the gene is
them an obligate carrier— Family 5 II.6) from different fami- still categorised as ‘tenuous evidence’, meaning that it has been
lies had been diagnosed with early-­onset dementia with marked associated with ALS in small studies that have not stood up to
behavioural symptoms, but no apparent motor neuron disease. replication. Our study demonstrates that ARPP21, or at least the
It cannot be ruled out that the ARPP21 mutation could also be p.Pro529Leu mutation in this gene, is consistently associated
related to FTD, and clinical presentation at any point might fall with ALS and should be considered in future genetic studies.
within the ALS-­FTD spectrum (ie, ALS, FTD or ALS-­FTD), as The ARPP21 protein is an RNA-­ binding protein widely
has been observed in other ALS-­FTD-­related genes.33 Unfor- expressed in the human brain39 and based on the results of our
tunately, no formal cognitive testing, neuroimaging or DNA study, adds to the long list of RNA-­binding proteins implicated in
samples were available from these patients. Further studies will ALS, such as TDP-­43, TIA1 or FUS. It is involved in RNA metab-
be required to confirm this hypothesis. olism, an event known to be deregulated and central to ALS
In 2019, Cooper-­Knock et al34 described the segregation of pathophysiology.40 Importantly, a common set of ARPP21 target
a mutation (p.Arg92Cys) in the glycosyltransferase 8 domain-­ RNAs with related functions have been identified through iCLIP.
containing protein 1 (GLT8D1) gene and the mutation described These RNAs have a prominent role in messenger RNA splicing
in the present study (p.Pro529Leu mutation in ARPP21) in an and processing as previously demonstrated by gene ontology
autosomal dominant ALS pedigree from the UK. Additional enrichment analyses.39 In addition, in cells expressing ARPP21
screening of both genes in 103 ALS cases disclosed 4 new treated with stress inductors (arsenite, clotrimazole or heat
GLT8D1 mutation carriers, 3 of which also harboured the shock), the recruitment of ARPP21 to stress granules is induced.
p.Pro529Leu mutation in ARPP21. However, after a rare variant Importantly, the C-­terminal sequence of the protein (where the
burden analysis in the Project MinE data set, the authors priori- p.Pro529Leu mutation is located) is necessary and sufficient to
tised mutations in the glycosyltransferase domain of GLT8D1 as induce its recruitment to these membraneless dynamic struc-
causative, suggesting only a secondary and putative synergistic tures. Although its domains have not been fully characterised,
effect for the mutation in ARPP21 in double-­mutation carriers. the mutation described in our study (p.Pro529Leu) is located in
This conclusion was based on an observed shorter disease dura- the C-­terminal sequence and in close proximity to an intrinsi-
tion in patients carrying both mutations (<16 months),34 similar cally disordered region with compositional bias to Proline resi-
to that of patients in our series who carry the p.Pro529Leu dues (low-­complexity regions) of ARPP21. We speculate that
variant in isolation. Therefore, we show that the mutation this mutation might result in an increased propensity of ARPP21
described in our study is sufficient to cause ALS, and suggest that to interact with and localise to stress granules, a mechanism
Dols-I­ cardo O, et al. J Neurol Neurosurg Psychiatry 2025;96:132–139. doi:10.1136/jnnp-2024-333834 137
 Neuromuscular
previously described in ALS-­related mutations in other RNA-­ Funding This study was funded by the Instituto de Salud Carlos III (Ministerio
binding proteins such as FUS,41 TIA142 or TDP-­43, and directly de Asuntos Económicos y Transformación Digital, Gobierno de España) through

J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2024-333834 on 2 July 2024. Downloaded from http://jnnp.bmj.com/ on April 9, 2025 by guest.
the projects PI19/01543, PI23/00845 to RR-­G, and PI18/00326, PI21/01395 to
related to the pathophysiology of ALS43 and other neurodegen- OD-­I, cofunded by the European Regional Development Fund/European Social
erative diseases.44 Thus, beyond the implication of ARPP21 in Fund (ERDF/ESF), ’A way to make Europe’/’Investing in your future’. OD-­I receives
RNA metabolism and processing, we suggest that this mutation funding from the Fundación Española para el Fomento de la Investigación de la
could alter the dynamics of stress granules by increasing their Esclerosis Lateral Amiotrófica (FUNDELA - ’Por un mundo sin ELA’), Fundación HNA
aberrant formation and thus impacting on ALS pathogenesis. (’Premio Investigación científica de salud’) and the Alzheimer’s Association (AARF-­
22-­924456). II-­G is a senior Atlantic Fellow for Equity in Brain Health at the Global
Our study has some limitations. First, although our results Brain Health Institute (GBHI), and receives funding from the GBHI, the Alzheimer’s
suggest the causal role of the mutation described herein, DNA Society (GBHI ALZ UK-­21-­720973) and the Alzheimer’s Association (AACSF-­
was available and genetic testing was consented to only in a few 21-­850193). II-­G was also supported by the Juan Rodés Contract (JR20/0018) and
family members, thus precluding a complete segregation study. PI21/00791 from Instituto de Salud Carlos III (Ministerio de Asuntos Económicos y

Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies.
Transformación Digital, Gobierno de España).
Given the variability in the age of onset of the disease, segrega-
tion analysis, especially involving some of the younger asymp- Map disclaimer The inclusion of any map (including the depiction of any
boundaries therein), or of any geographical or locational reference, does not imply
tomatic relatives, should be interpreted cautiously. Nevertheless, the expression of any opinion whatsoever on the part of BMJ concerning the legal
we strongly believe that the presence of the same variant in 10 status of any country, territory, jurisdiction or area or of its authorities. Any such
patients with ALS (9 fALS) from 7 unrelated families in a small expression remains solely that of the relevant source and is not endorsed by BMJ.
geographical area with a high incidence of ALS is a strong argu- Maps are provided without any warranty of any kind, either express or implied.
ment in favour of its pathogenicity. Further studies are required Competing interests None declared.
both in our country and worldwide to elucidate the contribu- Patient consent for publication Consent obtained directly from patient(s).
tion and frequency of this and other variants in ARPP21 to the Ethics approval This study involves human participants and was approved by
genetic basis of the disease, as well as functional validation of the Hospital de la Santa Creu i Sant Pau Ethics Comittee (Study ID: 20/016 HSCSP).
mutation pathogenicity. Second, clinical information on patients’ Participants gave informed consent to participate in the study before taking part.
families was collected retrospectively and was often incomplete, Provenance and peer review Not commissioned; externally peer reviewed.
especially regarding cognitive and behavioural symptoms, for Data availability statement Data are available upon reasonable request.
which specific standardised cognitive-­ behavioural tests could
Supplemental material This content has been supplied by the author(s). It
have provided a wider phenotypical spectrum of the variant. has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have
In conclusion, our results support the causative role of a been peer-­reviewed. Any opinions or recommendations discussed are solely those
mutation (p.Pro529Leu) in ARPP21 in ALS. This work provides of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
robust epidemiological and genetic arguments in favour of muta- responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
tion pathogenicity. We also discuss disease-­causing mechanisms
of the translations (including but not limited to local regulations, clinical guidelines,
associated with the mutation, supporting the role of ARPP21 as terminology, drug names and drug dosages), and is not responsible for any error
an independent, novel ALS-­causing gene, adding to the complex and/or omissions arising from translation and adaptation or otherwise.
genetic architecture of the disease and expanding the landscape Open access This is an open access article distributed in accordance with the
of RNA-­binding proteins altered in ALS. Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-­commercially,
Author affiliations and license their derivative works on different terms, provided the original work is
1
Sant Pau Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant properly cited, appropriate credit is given, any changes made indicated, and the use
Pau, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Barcelona, Spain is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
2
Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas
(CIBERNED), Madrid, Spain ORCID iDs
3
Motor Neuron Disease Clinic, Neuromuscular Diseases Unit, Department of Oriol Dols-­Icardo http://orcid.org/0000-0003-2656-8748
Neurology, Hospital de la Santa Creu i Sant Pau, Institut d’Investigació Biomèdica Álvaro Carbayo http://orcid.org/0000-0001-9282-8603
Sant Pau (IIB Sant Pau), Barcelona, Spain Marta Caballero-Ávila http://orcid.org/0000-0001-9850-8504
4
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Sara Rubio-­Guerra http://orcid.org/0000-0001-7652-8029
Madrid, Spain Ignacio Illán-­Gala http://orcid.org/0000-0002-5418-2052
5
Department of Medicine, Universitat Autonoma de Barcelona, Barcelona, Spain Elena Cortés-­Vicente http://orcid.org/0000-0002-1428-1072
6
Neuromuscular and Motor Neuron Diseases Research Group, Department of Ricard Rojas-­García http://orcid.org/0000-0003-1411-5573
Neurology, Hospital Universitario de Navarra, Pamplona, Spain
7
Health Research Institute of Navarra (IdisNa), Pamplona, Spain
8
Department of Neurology, Hospital San Pedro, Logroño, La Rioja, Spain REFERENCES
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