Elevated inflammatory responses and targeted therapeutic intervention in a preclinical mouse model of ataxia-telangiectasia lung disease.

Ataxia-telangiectasia (A-T) is an autosomal recessive, multisystem disorder characterized by cerebellar degeneration, cancer predisposition, and immune system defects. A major cause of mortality in A-T patients is severe pulmonary disease; however, the underlying causes of the lung complications are poorly understood, and there are currently no curative therapeutic interventions. In this study, we examined the lung phenotypes caused by ATM-deficient immune cells using a mouse model of A-T pulmonary disease. In response to acute lung injury, ATM-deficiency causes decreased survival, reduced blood oxygen saturation, elevated neutrophil recruitment, exaggerated and prolonged inflammatory responses and excessive lung injury compared to controls. We found that ATM null bone marrow adoptively transferred to WT recipients induces similar phenotypes that culminate in impaired lung function. Moreover, we demonstrated that activated ATM-deficient macrophages exhibit significantly elevated production of harmful reactive oxygen and nitrogen species and pro-inflammatory cytokines. These findings indicate that ATM-deficient immune cells play major roles in causing the lung pathologies in A-T. Based on these results, we examined the impact of inhibiting the aberrant inflammatory responses caused by ATM-deficiency with reparixin, a CXCR1/CXCR2 chemokine receptor antagonist. We demonstrated that reparixin treatment reduces neutrophil recruitment, edema and tissue damage in ATM mutant lungs. Thus, our findings indicate that targeted inhibition of CXCR1/CXCR2 attenuates pulmonary phenotypes caused by ATM-deficiency and suggest that this treatment approach represents a viable therapeutic strategy for A-T lung disease.

Bi-allelic mutations in HARS1 severely impair histidyl-tRNA synthetase expression and enzymatic activity causing a novel multisystem ataxic syndrome.

Mutations in histidyl-tRNA synthetase (HARS1), an enzyme that charges transfer RNA with the amino acid histidine in the cytoplasm, have only been associated to date with autosomal recessive Usher syndrome type III and autosomal dominant Charcot-Marie-Tooth disease type 2W. Using massive parallel sequencing, we identified bi-allelic HARS1 variants in a child (c.616G>T, p.Asp206Tyr and c.730delG, p.Val244Cysfs*6) and in two sisters (c.1393A>C, p.Ile465Leu and c.910_912dupTTG, p.Leu305dup), all characterized by a multisystem ataxic syndrome. All mutations are rare, segregate with the disease, and are predicted to have a significant effect on protein function. Functional studies helped to substantiate their disease-related roles. Indeed, yeast complementation assays showing that one out of two mutations in each patient is loss-of-function, and the reduction of messenger RNA and protein levels and enzymatic activity in patient's skin-derived fibroblasts, together support the pathogenicity of the identified HARS1 variants in the patient phenotypes. Thus, our efforts expand the allelic and clinical spectrum of HARS1-related disease.

GARS-related disease in infantile spinal muscular atrophy: Implications for diagnosis and treatment.

The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.

Ubiquitously Expressed Proteins and Restricted Phenotypes: Exploring Cell-Specific Sensitivities to Impaired tRNA Charging.

Aminoacyl-tRNA synthetases (ARS) are ubiquitously expressed, essential enzymes that charge tRNA with cognate amino acids. Variants in genes encoding ARS enzymes lead to myriad human inherited diseases. First, missense alleles cause dominant peripheral neuropathy. Second, missense, nonsense, and frameshift alleles cause recessive multisystem disorders that differentially affect tissues depending on which ARS is mutated. A preponderance of evidence has shown that both phenotypic classes are associated with loss-of-function alleles, suggesting that tRNA charging plays a central role in disease pathogenesis. However, it is currently unclear how perturbation in the function of these ubiquitously expressed enzymes leads to tissue-specific or tissue-predominant phenotypes. Here, we review our current understanding of ARS-associated disease phenotypes and discuss potential explanations for the observed tissue specificity.

Alanyl-tRNA Synthetase 2 (AARS2)-Related Ataxia Without Leukoencephalopathy.

Mutations in the mitochondrial alanyl-tRNA synthetase gene, AARS2, have been reported to cause leukoencephalopathy associated with early ovarian failure, a clinical presentation described as "ovarioleukodystrophy." We present a sibling pair: one with cerebellar ataxia and one with vision loss and cognitive impairment in addition to ataxia. Neither shows evidence of leukoencephalopathy on MRI imaging. Exome sequencing revealed that both siblings are compound heterozygous for AARS2 variants (p.Phe131del and p.Ile328Met). Yeast complementation assays indicate that p.Phe131del AARS2 dramatically impairs gene function and that p.Ile328Met AARS2 is a hypomorphic allele. This work expands the phenotypic spectrum of AARS2-associated disease to include ataxia without leukoencephalopathy.

Cysteinyl-tRNA Synthetase Mutations Cause a Multi-System, Recessive Disease That Includes Microcephaly, Developmental Delay, and Brittle Hair and Nails.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.

Genomic variation within alpha satellite DNA influences centromere location on human chromosomes with metastable epialleles.

Alpha satellite is a tandemly organized type of repetitive DNA that comprises 5% of the genome and is found at all human centromeres. A defined number of 171-bp monomers are organized into chromosome-specific higher-order repeats (HORs) that are reiterated thousands of times. At least half of all human chromosomes have two or more distinct HOR alpha satellite arrays within their centromere regions. We previously showed that the two alpha satellite arrays of Homo sapiens Chromosome 17 (HSA17), D17Z1 and D17Z1-B, behave as centromeric epialleles, that is, the centromere, defined by chromatin containing the centromeric histone variant CENPA and recruitment of other centromere proteins, can form at either D17Z1 or D17Z1-B. Some individuals in the human population are functional heterozygotes in that D17Z1 is the active centromere on one homolog and D17Z1-B is active on the other. In this study, we aimed to understand the molecular basis for how centromere location is determined on HSA17. Specifically, we focused on D17Z1 genomic variation as a driver of epiallele formation. We found that D17Z1 arrays that are predominantly composed of HOR size and sequence variants were functionally less competent. They either recruited decreased amounts of the centromere-specific histone variant CENPA and the HSA17 was mitotically unstable, or alternatively, the centromere was assembled at D17Z1-B and the HSA17 was stable. Our study demonstrates that genomic variation within highly repetitive, noncoding DNA of human centromere regions has a pronounced impact on genome stability and basic chromosomal function.

Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.