Exome sequencing reveals pathogenic mutations in the LARS2 and HSD17B4 genes associated with Perrault syndrome and D-bifunctional protein deficiency in Moroccan families.

BACKGROUND: Syndromic hearing loss (SHL) is characterized by hearing impairment accompanied by other clinical manifestations, reaching over 400 syndromes. Early and accurate diagnosis is essential to understand the progression of hearing loss and associated systemic complications. METHODS AND RESULTS: In this study, we investigated the genetic etiology of sensorineural hearing loss in three Moroccan patients using whole exome sequencing (WES). The results revealed in two families Perrault syndrome caused by LARS2, p. Asn153His; p. Thr629Met compound heterozygous variants in two siblings in one family; and p. Thr522Asn, a homozygous variant in two sisters in another. The patient in the third family was diagnosed with D-bifunctional protein deficiency (D-BPD), linked to compound heterozygous mutations p. Asn457Tyr and p. Val643Argfs*5 in HSD17B4. Molecular dynamic simulation results showed that Val643Argfs*5 does not prevent HSD17B4 protein from binding to the PEX5 receptor, but further studies are recommended to verify its effect on HSD17B4 protein functionality. CONCLUSION: These results highlight the effectiveness of WES in identifying pathogenic mutations involved in heterogeneous disorders and the usefulness of bioinformatics in predicting their effects on protein structure.

LARS2-Perrault syndrome: a new case report and literature review.

BACKGROUND: Perrault syndrome is a rare recessive and genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and gonadal dysgenesis in females. Mutations in seven different genes have been identified: HARS2, HSD17B4, CLLP, C10orf, ERAL1, TWNK and LARS2. To date, 19 variants have been reported in 18 individuals with LARS2-Perrault syndrome. CASE PRESENTATION: Here we describe the case of an 8-year-old girl with compound heterozygous missense mutations in the LARS2 gene. We identified two missense mutations [c.457A > C, p.(Asn153His) and c.1565C > A, p.(Thr522Asn)] and subsequent familial segregation showed that each parent had transmitted a mutation. CONCLUSIONS: These results have implications for genetic counseling and provide insight into the functional role of LARS2. This case highlights the importance of an early diagnosis. Systematic genetic screening of children with hearing loss allows the early identification of a Perrault syndrome in order to ensure specific endocrinological surveillance and management to prevent secondary complications. Clinical data are compared with the other cases reported in the literature.

Biallelic mutations in LARS2 can cause Perrault syndrome type 2 with neurologic symptoms.

Perrault syndrome represents a genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and ovarian dysfunction in females. Causative genes include HARS2, HSD17B4, CLPP, C10orf2, and LARS2. Some patients with Perrault syndrome exhibit neurologic features including learning disability, cerebellar ataxia, and peripheral neuropathy and are classified as type 2 and are clinically separate from those without neurological symptoms other than a hearing loss (type 1). To date, all reported patients with LARS2 mutations (15 patients in 8 families) have been classified as type 1. Here, we report female siblings with biallelic mutations in LARS2, p.Glu294Lys, and p.Thr519Met, who were classified as type 2. The proposita developed progressive sensorineural hearing loss at 18 months and pervasive developmental disorder at 8 years, with repetitive behavior, insistence on sameness, attention deficit, tic, irritability, and an ataxic gait. At age 15 years, she was diagnosed as having primary amenorrhea with elevated FSH and LH and a decreased estradiol; ultrasound and magnetic resonance imaging examinations revealed a small uterus and no detectable ovaries. The proposita's younger sister presented with neonatal sensorineural hearing loss and a mild delay in motor and speech development. She was diagnosed as having primary amenorrhea with endocrinologic and radiographic findings that were comparable to those of her sister. She had difficulty with reading comprehension, and had trouble with open-ended test questions at 12 years of age. We concluded that Perrault syndrome patients with LARS2 mutations are at risk for neurologic problems, despite previous notions otherwise.

An Application of NGS for Molecular Investigations in Perrault Syndrome: Study of 14 Families and Review of the Literature.

Perrault syndrome (PS) is a rare autosomal recessive condition characterized by deafness and gonadic dysgenesis. Recently, mutations in five genes have been identified: C10orf2, CLPP, HARS2, HSD17B4, and LARS2. Probands included are presented with sensorineural deafness associated with gonadic dysgenesis. DNA was sequenced using next-generation sequencing (NGS) with a panel of 35 deafness genes including the five Perrault genes. Exonic variations known as pathogenic mutations or detected with <1% frequency in public databases were extracted and subjected to segregation analysis within each family. Both mutations and low coverage regions were analyzed by Sanger sequencing. Fourteen female index patients were included. The screening in four cases has been extended to four family members presenting with PS phenotype. For four unrelated patients (28.6%), causative mutations were identified: three homozygous mutations in C10orf2, CLPP, and HARS2, and one compound heterozygous mutation in LARS2. Three additional heterozygous mutations in LARS2 and HSD17B4 were found in three independent familial cases. All these missense mutations were verified by Sanger sequencing. Familial segregation analyses confirmed the molecular diagnosis in all cases carrying biallelic mutations. Because of NGS, molecular analysis confirmed the clinical diagnosis of PS in 28.6% of our cohort and four novel mutations were found in four Perrault genes. For the unsolved cases, exome sequencing should be performed to search for a sixth unknown PS gene.

Novel Cases of Non-Syndromic Hearing Impairment Caused by Pathogenic Variants in Genes Encoding Mitochondrial Aminoacyl-tRNA Synthetases.

Dysfunction of some mitochondrial aminoacyl-tRNA synthetases (encoded by the KARS1, HARS2, LARS2 and NARS2 genes) results in a great variety of phenotypes ranging from non-syndromic hearing impairment (NSHI) to very complex syndromes, with a predominance of neurological signs. The diversity of roles that are played by these moonlighting enzymes and the fact that most pathogenic variants are missense and affect different domains of these proteins in diverse compound heterozygous combinations make it difficult to establish genotype-phenotype correlations. We used a targeted gene-sequencing panel to investigate the presence of pathogenic variants in those four genes in cohorts of 175 Spanish and 18 Colombian familial cases with non-DFNB1 autosomal recessive NSHI. Disease-associated variants were found in five cases. Five mutations were novel as follows: c.766C>T in KARS1, c.475C>T, c.728A>C and c.1012G>A in HARS2, and c.795A>G in LARS2. We provide audiograms from patients at different ages to document the evolution of the hearing loss, which is mostly prelingual and progresses from moderate/severe to profound, the middle frequencies being more severely affected. No additional clinical sign was observed in any affected subject. Our results confirm the involvement of KARS1 in DFNB89 NSHI, for which until now there was limited evidence.

Diagnostic value of lncRNAs LINC00152 and LARS2-AS1 and their regulatory roles in macrophage immune response in tuberculosis.

OBJECTIVES: To determine the usefulness of LINC00152 and LARS2-AS1 as potential biomarkers for latent tuberculosis (LTB) and active tuberculosis (ATB), as well as their effect on Mycobacterium (Mtb) infection. METHODS: The expression levels of LINC00152 and LARS2-AS1 in the health, patients with LTB and ATB were detected by qRT-PCR. The ROC curves were constructed to show their potential as biomarkers. The intracellular survival assays for Mtb and the levels of immune-related cytokines were determined to discover the effect of LINC00152 and LARS2-AS1 on Mtb infection. The relationships of miR-485-5p with LINC00152 and LARS2-AS1 were explored. RESULTS: LINC00152 and LARS2-AS1 levels were significantly elevated in patients with ATB and LTB, and Mtb-infected macrophages. LINC00152 and LARS2-AS1 can distinguish the LTB from the health and ATB from LTB. LARS2-AS1 and LINC00152 knock-down reduced the intracellular Mtb survival and induced cellular immune response after Mtb challenge. miR-485-5p was a targeting miRNA for LINC00152 and LARS2-AS1. CONCLUSIONS: LINC00152 and LARS2-AS1 can be considered as potential biomarkers for tuberculosis disease. LINC00152 and LARS2-AS1 have anti-Mtb effects.

Lin28a induced mitochondrial dysfunction in human granulosa cells via suppressing LARS2 expression.

Granulosa cells surround the oocytes as a component of ovarian follicles and produce sex steroids and growth factors to support oocyte development. Mitochondria is one of the multiple factors regulating granulosa cell function by modulating bioenergetic pathways and maintaining cells' metabolic needs. Lin28a was reported to regulate the primordial germ cell development in the ovary and affect the fertility rate in females. However, whether Lin28a modulated mitochondria function in granulosa cells to regulate steroidogenesis remains a further exploration. In this study, we utilized immortal human granulosa cells (HGrC1) to overexpress or suppress the protein level of Lin28a. Results showed that overexpression of Lin28a could decrease the estrogen level, ATP content, mitochondrial membrane potential and Glutathione (GSH) level, while silencing Lin28a caused the opposite effect. Further, we found that overexpression of LARS2, a mitochondrial leucyl-tRNA synthetase, could increase the estrogen level, ATP content, mitochondrial membrane potential and GSH level while silencing LARS2 caused adverse results. Overexpression of LARS2 reversed Lin28a-induced estrogen downregulation and mitochondrial dysfunction. Moreover, overexpression of LARS2 increased the mRNA level of Pgc1α and Nrf2, which were involved in mitochondrial biogenesis. Besides, Lin28a could directly bind to the mRNA of Lars2 to suppress its translation. Taken together, overexpression of Lin28a caused mitochondrial dysfunction in granulosa cells via suppressing LARS2 expression. This study can provide new insights into how Lin28a regulates mitochondrial function in granulosa cells and influences female fertility.

Comprehensive Characterization of RNA-Binding Proteins in Colon Adenocarcinoma Identifies a Novel Prognostic Signature for Predicting Clinical Outcomes and Immunotherapy Responses Based on Machine Learning.

BACKGROUND: RNA-binding proteins (RBPs) are crucial factors that function in the posttranscriptional modification process and are significant in cancer. OBJECTIVE: This research aimed for a multigene signature to predict the prognosis and immunotherapy response of patients with colon adenocarcinoma (COAD) based on the expression profile of RNA-binding proteins (RBPs). METHODS: COAD samples retrieved from the TCGA and GEO datasets were utilized for a training dataset and a validation dataset. Totally, 14 shared RBP genes with prognostic significance were identified. Non-negative matrix factorization clusters defined by these RBPs could stratify COAD patients into two molecular subtypes. Cox regression analysis and identification of 8-gene signature categorized COAD patients into high- and low-risk populations with significantly different prognosis and immunotherapy responses. RESULTS: Our prediction signature was superior to another five well-established prediction models. A nomogram was generated to quantificationally predict the overall survival (OS) rate, validated by calibration curves. Our findings also indicated that high-risk populations possessed an enhanced immune evasion capacity and low-risk populations might benefit immunotherapy, especially for the joint combination of PD-1 and CTLA4 immunosuppressants. DHX15 and LARS2 were detected with significantly different expressions in both datasets, which were further confirmed by qRTPCR and immunohistochemical staining. CONCLUSION: Our observations supported an eight-RBP-related signature that could be applied for survival prediction and immunotherapy response of patients with COAD.

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease.

In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial translation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype.

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA.

Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.

Perrault syndrome: Clinical report and retrospective analysis.

BACKGROUND: Perrault syndrome (PRLTS4; OMIM# 615300) is a rare autosomal recessive disorder and only a few cases have been reported worldwide. We report a Chinese female characterized by sensorineural hearing loss and premature ovarian insufficiency. METHODS: We evaluated audiological, endocrine, and ultrasound examinations and examined the genetic causes using whole-exome sequencing. We reviewed the literature to discuss the pathogenesis, genotype-phenotype correlation, treatment, and prevention of PRLTS4. RESULTS: Bioinformatic analysis revealed compound heterozygous mutations in the LARS2 gene, c.880G>A (p.Glu294Lys), and c.2108T>C (p.Ile703Thr) which is a novel missense mutation, co-segregated in this family. Taken together, the patient was clinically diagnosed as PRLTS4. The literature review showed that the phenotype for PRLTS4 varies widely, but the sensorineural hearing loss, increased gonadotropin levels, and amenorrhea occurred frequently. All reported mutations are highly conserved in mammals based on conservation analysis, and there is a mutation hotspot for PRLTS4. CONCLUSION: This study expanded the mutation spectrum of LARS2 and is the first report of PRLTS4 in a Chinese family. Genetic testing plays an important role in early diagnosis of syndromic deafness and clinical genetic evaluation is essential to guide prevention.