Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease.

The failure of DNA ligases to complete their catalytic reactions generates cytotoxic adenylated DNA strand breaks. The APTX RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. Here, we report X-ray structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes POLß and FEN1. Our results uncover a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. Further, based on comprehensive biochemical, X-ray and solution NMR results, we define a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and a dominant AOA1 variant unexpectedly allosterically modulates APTX active site conformations.

Germ cell arrest associated with aSETX mutation in ataxia oculomotor apraxia type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive neurodegenerative disorder characterized by cerebellar atrophy, peripheral neuropathy and oculomotor apraxia. It is caused by mutations in the SETX gene that encodes senataxin, a ubiquitously expressed protein that mediates processes, including transcription, transcription termination, DNA repair, RNA processing, DNA-RNA hybrid (R-loop) elimination and telomere stability. In mice, senataxin is essential for male germ cell development and fertility through its role in meiotic recombination and sex chromosome inactivation. AOA2 is associated with hypogonadism in women, but there are no reports of hypogonadism or infertility in men. We describe the first case of human male infertility caused by germ cell arrest in a man with AOA2. Our patient has a homozygous mutation in the SETX gene (NC_000009.11:g.135158775dup), which results in a frameshift and premature protein termination (NM_015046.6:c.6422dup, p.[Ser2142Glufs*23]). In accordance with the murine phenotype, testis histology revealed disrupted seminiferous tubules with spermatogonia and primary spermatocytes, but absent spermatids. Collectively, these data support an essential role of senataxin in human spermatogenesis, and provide a compelling case that men with AOA2 should be counselled at diagnosis about the possibility of infertility.

Ribonucleotide triggered DNA damage and RNA-DNA damage responses.

Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

Unique Ataxia-Oculomotor Apraxia 2 (AOA2) in Israel with Novel Variants, Atypical Late Presentation, and Possible Identification of a Poison Exon.

AOA2 is a rare progressive adolescent-onset disease characterised by cerebellar vermis atrophy, peripheral neuropathy and elevated serum alpha-fetoprotein (AFP) caused by pathogenic bi-allelic variants in SETX, encoding senataxin, involved in DNA repair and RNA maturation. Sanger sequencing of genomic DNA, co-segregation and oxidative stress functional studies were performed in Family 1. Trio whole-exome sequencing (WES), followed by SETX RNA and qRT-PCR analysis, were performed in Family 2. Sanger sequencing in Family 1 revealed two novel in-frame SETX deletion and duplication variants in trans (c.7009_7011del; p.Val2337del and c.7369_7371dup; p.His2457dup). Patients had increased induced chromosomal aberrations at baseline and following exposure to higher mitomycin-C concentration and increased sensitivity to oxidative stress at the lower mitomycin-C concentration in cell viability test. Trio WES in Family 2 revealed two novel SETX variants in trans, a nonsense variant (c.568C > T; p.Gln190*), and a deep intronic variant (c.5549-107A > G). Intronic variant analysis and SETX mRNA expression revealed activation of a cryptic exon introducing a premature stop codon (p.Met1850Lysfs*18) and resulting in aberrant splicing, as shown by qRT-PCR analysis, thus leading to higher levels of cryptic exon activation. Along with a second deleterious allele, this variant leads to low levels of SETX mRNA and disease manifestations. Our report expands the phenotypic spectrum of AOA2. Results provide initial support for the hypomorphic nature of the novel in-frame deletion and duplication variants in Family 1. Deep-intronic variant analysis of Family 2 variants potentially reveals a previously undescribed poison exon in the SETX gene, which may contribute to tailored therapy development.

Ataxia Oculomotor Apraxia Type 1 in the Siblings of a Family: A Novel Mutation.

Although AOA1 (ataxia oculomotor apraxia1) is one of the most common causes of autosomal recessive cerebellar ataxias in Japanese population, it is reported from all over the world. The clinical manifestations are similar to ataxia telangiectasia in which non-neurological manifestations are absent and include almost 10% of autosomal recessive cerebellar ataxias. Dysarthria and gait disorder are the most two common and typical manifestations. Oculomotor apraxia is usually seen a few years after the manifestations start. APTX gene on 9p13.3 chromosome is expressed in the cells of all human body tissues and different mutations had been discovered. Here we report two siblings (a girl and a boy) of consanguineous parents visited at Mofid Pediatrics Hospital in 2015, with history of gait ataxia, titubation, tremor, and oculomotor apraxia around five yr old and after that. The brother showed symptoms of disease earlier and more severe than his sister did. After ruling out the common etiologies of progressive ataxia, we did genetic study for AOA1 that showed a homozygous frameshift mutation as c.418_418 del was found. This mutation was not reported before so this was a new mutation in APTX gene.

Novel mutations in ataxia telangiectasia and AOA2 associated with prolonged survival.

Ataxia telangiectasia (AT) and ataxia oculomotor apraxia type 2 (AOA2) are autosomal recessive ataxias caused by mutations in genes involved in maintaining DNA integrity. Lifespan in AT is greatly shortened (20s-30s) due to increased susceptibility to malignancies (leukemia/lymphoma). Lifespan in AOA2 is uncertain. We describe a woman with variant AT with two novel mutations in ATM (IVS14+2T>G and 5825C>T, p.A1942V) who died at age 48 with pancreatic adenocarcinoma. Her mutations are associated with an unusually long life for AT and with a cancer rarely associated with that disease. We also describe two siblings with AOA2, heterozygous for two novel mutations in senataxin (3 bp deletion c.343-345 and 1398T>G, p.I466M) who have survived into their 70s, allowing us to characterize the longitudinal course of AOA2. In contrast to AT, we show that persons with AOA2 can experience a prolonged lifespan with considerable motor disability.

Senataxin protects the genome: Implications for neurodegeneration and other abnormalities.

Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive disorder characterized by cerebellar atrophy, peripheral neuropathy, loss of Purkinje cells and elevated α-fetoprotein. AOA2 is caused by mutations in the SETX gene that codes for the high molecular weight protein senataxin. Mutations in this gene also cause dominant neurodegenerative disorders. Similar to that observed for other autosomal recessive ataxias, this protein protects the integrity of the genome against oxidative and other forms of DNA damage to reduce the risk of neurodegeneration. Senataxin functions in transcription termination and RNA splicing and it has been shown to resolve RNA/DNA hybrids (R-loops) that arise at transcription pause sites or when transcription is blocked. Recent data suggest that this protein functions at the interface between transcription and DNA replication to minimise the risk of collision and maintain genome stability. Our recent data using SETX gene-disrupted mice revealed that male mice were defective in spermatogenesis and were infertile. DNA double strand-breaks persisted throughout meiosis and crossing-over failed in SETX mutant mice. These changes can be explained by the accumulation of R-loops, which interfere with Holiday junctions and crossing-over. We also showed that senataxin was localized to the XY body in pachytene cells and was involved in transcriptional silencing of these chromosomes. While the defect in meiotic recombination was striking in these animals, there was no evidence of neurodegeneration as observed in AOA2 patients. We discuss here potentially different roles for senataxin in proliferating and post-mitotic cells.

A novel mutation in the aprataxin (APTX) gene in an Iranian individual suffering early-onset ataxia with oculomotor apraxia type 1(AOA1) disease.

BACKGROUND: Ataxia with oculomotor apraxia type 1 (AOA1) shows early onset with autosomal recessive inheritance and is caused by a mutation in the aprataxin (APTX) gene encoding for the APTX protein. METHODS: In this study, a 7-year-old girl born of a first-cousin consanguineous marriage was described with early-onset progressive ataxia and AOA, with increased cholesterol concentration and decreased albumin concentration in serum. PCR and direct DNA sequencing was performed after DNA extraction. RESULTS: Sequencing analysis revealed a novel homozygous deletion in c.643 and A>T single nucleotide polymorphism in c.641 in exon 6 of the APTX gene [ENST00000379825]. CONCLUSION: It seems that this region of exon 6 is probably a hot spot; however, no deletions have been reported in exon 6 yet.

Cognitive functions in ataxia with oculomotor apraxia type 2.

BACKGROUND: Ataxia with oculomotor apraxia type 2 (AOA2) is characterized by cerebellar atrophy, peripheral neuropathy, oculomotor apraxia, and elevated serum alpha-fetoprotein (AFP) levels. The disease is caused by a recessive mutation in the senataxin gene. Since it is a very rare cerebellar disorder, no detailed examination of cognitive functions in AOA2 has been published to date. The aim of the present study was to investigate the neuropsychological profile of a 54-year-old patient with AOA2. METHODS: A broad range of neuropsychological examination protocol was administered including the following domains: short-term, working- and episodic-memories, executive functions, implicit sequence learning, and the temporal parameters of speech. RESULTS: The performance on the Listening Span, Letter Fluency, Serial Reaction Time Task, and pause ratio in speech was 2 or more standard deviations (SD) lower compared to controls, and 1 SD lower on Backward Digit Span, Semantic Fluency, articulation rate, and speech tempo. CONCLUSION: These findings indicate that the pathogenesis of the cerebrocerebellar circuit in AOA2 is responsible for the weaker coordination of complex cognitive functions such as working memory, executive functions, speech, and sequence learning.