Where Morphological and Molecular Classifications Meet: The Role of p53 Immunohistochemistry in the Prognosis of Low-Risk Endometrial Carcinoma (GLAMOUR Study).

No prospective study has validated molecular classification to guide adjuvant treatment in endometrial cancer (EC), and not even retrospective data are present for patients with morphological low-risk EC. We conducted a retrospective, multicenter, observational study including 370 patients with low-risk endometrioid EC to evaluate the incidence and prognostic role of p53 abnormal expression (p53abn) in this specific subgroup. Among 370 patients, 18 had abnormal expressions of p53 (4.9%). In 13 out of 370 patients (3.6%), recurrences were observed and two were p53abn. When adjusting for median follow-up time, the odds ratio (OR) for recurrence among those with p53abn versus p53 wild type (p53wt) was 5.23-CI 95% 0.98-27.95, p = 0.053. The most common site of recurrence was the vaginal cuff (46.2%). One recurrence occurred within the first year of follow-up, and the patient exhibited p53abn. Both 1-year and 2-year DFS rates were 94.4% and 100% in the p53abn and p53wt groups, respectively. One patient died from the disease and comprised p53wt. No difference in OS was registered between the two groups; the median OS was 21.9 months (16.4-30.1). Larger multicenter studies are needed to tailor the treatment of low-risk EC patients with p53abn. Performing molecular classification on all EC patients might be cost-effective, and despite the limits of our relatively small sample, p53abn patients seem to be at greater risk of recurrence, especially locally and after two years since diagnosis.

Human mtDNA-Encoded Long ncRNAs: Knotty Molecules and Complex Functions.

Until a few decades ago, most of our knowledge of RNA transcription products was focused on protein-coding sequences, which were later determined to make up the smallest portion of the mammalian genome. Since 2002, we have learnt a great deal about the intriguing world of non-coding RNAs (ncRNAs), mainly due to the rapid development of bioinformatic tools and next-generation sequencing (NGS) platforms. Moreover, interest in non-human ncRNAs and their functions has increased as a result of these technologies and the accessibility of complete genome sequences of species ranging from Archaea to primates. Despite not producing proteins, ncRNAs constitute a vast family of RNA molecules that serve a number of regulatory roles and are essential for cellular physiology and pathology. This review focuses on a subgroup of human ncRNAs, namely mtDNA-encoded long non-coding RNAs (mt-lncRNAs), which are transcribed from the mitochondrial genome and whose disparate localisations and functions are linked as much to mitochondrial metabolism as to cellular physiology and pathology.

Early Quality of Life Assessment After Segmental Colorectal Resection for Deep Infiltrating Endometriosis.

STUDY OBJECTIVE: Endometriosis is a benign condition afflicting women of reproductive age that significantly impacts their quality of life (QoL). Given its debilitating symptoms and prevalence, it is essential to define its proper management. In this study, we have assessed patient-reported outcomes among women having undergone segmental colorectal resection for deep infiltrating endometriosis. Any correlation between preoperative nutritional status and overall postoperative complications has also been analyzed. STUDY DESIGN: Prospective observational study. SETTING: Public medical center. PATIENTS: One hundred forty consecutive patients that had undergone segmental colorectal resection for DIE between November 2020 and October 2021 at IRCCS Sacro Cuore Don Calabria Hospital of Negrar of Valpolicella (Verona, Italy). INTERVENTIONS: Patient-reported outcomes were measured using data collected from the MD Anderson Symptom Inventory for gastrointestinal surgery patients and Euro-QoL Group EQ-5D-5L (EQ-5Q-5L) questionnaires, which were administered preoperatively (T0), at discharge (T1) and at 4 to 6 weeks after surgery (T2). Nutritional status was examined through the Mini Nutritional Assessment Short form and Prognostic Nutritional Index. MEASUREMENTS AND MAIN RESULTS: A significant improvement in the EQ-5Q-5L and MDASI-GI scores was noted between T0 and T2 (p <. 001 and p <. 001, respectively.) No statistically significant differences were found in scores at T2 between patients who had experienced postoperative complications and those who had not. No statistically significant association was observed between the presence of malnutrition and overall postoperative complications and their severity. CONCLUSION: This study confirms, through patient-reported outcomes, the pivotal role of surgery in improving the QoL at 4 to 6 weeks of women affected by endometriosis who have previously been unresponsive to medical therapy.

"Things Have Changed"-Laparoscopic Cytoreduction for Advanced and Recurrent Ovarian Cancer: The Experience of a Referral Center on 108 Patients.

OBJECTIVE: To report the feasibility of laparoscopic cytoreduction surgery for primary and recurrent ovarian cancer in a select group of patients. METHODS: A retrospective analysis was conducted on a cohort of patients with FIGO stage IIIA-IV advanced ovarian cancer who underwent laparoscopic primary debulking surgery (PDS), interval debulking surgery (IDS), or secondary debulking surgery (SDS) between June 2008 and January 2020. The primary endpoint was achieving optimal cytoreduction, defined as residual tumor less than 1 cm. Secondary endpoints included evaluating surgical complications and long-term survival, assessed at three-month intervals during the initial two years and then every six months. RESULTS: This study included a total of 108 patients, among whom, 40 underwent PDS, 44 underwent IDS, and 24 underwent SDS. Optimal cytoreduction rates were found to be 95.0%, 97.7%, and 95.8% for the PDS, ISD, and SDS groups, respectively. Early postoperative complications (<30 days from surgery) occurred in 19.2% of cases, with 7.4% of these cases requiring reintervention. One patient died following postoperative respiratory failure. Late postoperative complications (<30 days from surgery) occurred in 9.3% of cases, and they required surgical reintervention only in one case. After laparoscopic optimal cytoreduction with a median follow-up time of 25 months, the overall recurrence rates were 45.7%, 38.5%, and 39.3% for PDS, ISD, and SDS, respectively. The three-year overall survival rates were 84%, 66%, and 63%, respectively, while the three-year disease-free survival rates were 48%, 51%, and 71%, respectively. CONCLUSIONS: Laparoscopic cytoreduction surgery is feasible for advanced ovarian cancer in carefully selected patients, resulting in high rates of optimal cytoreduction, satisfactory peri-operative morbidity, and encouraging survival outcomes. Future studies should focus on establishing standardized selection criteria and conducting well-designed investigations to further refine patient selection and evaluate long-term outcomes.

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.

"A Space Odyssey" on Laparoscopic Segmental Rectosigmoid Resection for Deep Endometriosis: A Seventeen-year Retrospective Analysis of Outcomes and Postoperative Complications among 3050 Patients Treated in a Referral Center.

STUDY OBJECTIVE: To evaluate the feasibility of laparoscopic rectosigmoid resection for bowel endometriosis (RSE), reporting surgical and short-term postoperative outcomes in a consecutive large series of patients. DESIGN: A retrospective cohort study. SETTING: Third-level national referral center for deep endometriosis (DE). PATIENTS: 3050 patients with symptomatic RSE requiring surgical treatment. INTERVENTIONS: Nerve-sparing laparoscopic resection for RSE perfomed by a multidisciplinary team. After collecting intraoperative surgical characteristics, postoperative complications were collected by evaluating the risk factors associated with their onset. MEASUREMENTS AND MAIN RESULTS: Clavien-Dindo IIIb postoperative complications were noted in 13.1% of patients, with anastomotic leakage and rectovaginal fistula accounting for 3.0% and 1.9%, respectively. Postoperative bladder impairment was observed in 13.9% of patients during hospital discharge but spontaneously decreased to 4.5% at the first evaluation after 30 days, alongside a statistically significant change towards global symptom improvement. Multivariate analyses were done to identify the risk factors for segmental bowel resection in terms of occurrence of postoperative major complications. Ultralow (≤5 cm from the anal verge), low rectal anastomosis (<8 cm, >5 cm), parametrectomy, vaginal resection, and previous surgeries seemed more related to anastomotic leakage, rectovaginal fistula, and bladder retention. CONCLUSIONS: Laparoscopic rectosigmoid resection for RSE seems an effective and feasible procedure. The surgical complication rate is not negligible but could be reduced by implementing a multidisciplinary approach, an endless improvement in nerve-sparing techniques and surgical anatomy, as well as technological enhancements. Real future challenges will be to reduce the time for the first diagnosis of DE and the likelihood of surgical indications.

Surgical Outcomes and Complications of Laparoscopic Hysterectomy for Endometriosis: A Multicentric Cohort Study.

STUDY OBJECTIVE: To investigate the postoperative morbidity of laparoscopic hysterectomy (LH) for endometriosis/adenomyosis in terms of operative outcomes and complications. DESIGN: Retrospective multicentric cohort study. SETTING: Eight European minimally invasive referral centers. PATIENTS: Data from 995 patients with pathologically confirmed endometriosis and/or adenomyosis who underwent LH without concomitant urological and/or gastroenterological procedures from January 2010 to December 2020. INTERVENTIONS: Total LH. MEASUREMENTS AND MAIN RESULTS: Demographic patients' characteristics, surgical outcomes, and intraoperative and postoperative complications were evaluated. We considered major postoperative surgical-related complications, any grade 2 or more events (Clavien-Dindo score) that occurred within 30 days from surgery. Univariate analysis and multivariable models fit with logistic regression were used to estimate the adjusted odds ratio (OR) and corresponding 95% confidence interval (CI) for major complications. Median age at surgery was 44 years (28-54), and about half of them (505, 50.7%) were on medical treatment (estro-progestins, progestin, or Gonadotropin hormone-releasing hormone-analogues) at the time of surgery. In association with LH, posterior adhesiolysis was performed in 387 (38.9%) cases and deep nodule resection in 302 (30.0%). Intraoperative complications occurred in 3% of the patients, and major postoperative complications were registered in 93 (9.3%). The multivariable analysis showed an inverse correlation between the occurrence of Clavien-Dindo >2 complications and age (OR 0.94, 95% CI 0.90-0.99), while previous surgery for endometriosis (OR 1.62, 95% CI 1.01-2.60) and intraoperative complications (OR 6.49, 95% CI 2.65-16.87) were found as predictors of major events. Medical treatment at the time of surgery has emerged as a protective factor (OR 0.50, 95% CI 0.31-0.81). CONCLUSION: LH for endometriosis/adenomyosis is associated with non-negligible morbidity. Knowing the factors associated with higher risks of complications might be used for risk stratification and could help clinicians during preoperative counseling. The administration of estro-progestin or progesterone preoperatively might reduce the risks of postoperative complications following surgery.

"Vitreous Hemorrhage and Long-Lasting Priapism After COVID-19 m-RNA Based Vaccine: A Case Report".

PURPOSE: To report a case of possible multi-district thromboembolic event involving the eye of a patient with several cardiovascular risk factors, following a second inoculation of SARS-CoV-2 m-RNA based vaccine. CASE-REPORT: A 60-year-old man presented with blurred vision in the left eye lasting 1 month but started within 24 hours from the 2nd dose of BNT162b2 vaccine inoculation. He also reported a long-lasting but self-limiting priapism which started about 4 h after the vaccination. Patient's medical history included: acute lymphoblastic leukemia, treated with chemotherapy and HLA-identical sibling donor transplant 18 months earlier; subsequent cytomegalovirus posterior outer retinal necrosis (PORN) resolved with antiviral treatment; type II diabetes and erectile dysfunction. Ocular examination of the affected eye revealed vitreous hemorrhage which limited the observation of details of the fundus. After a 2-week follow-up without any clinical improvement, parsplana vitrectomy (PPV) with cataract extraction was performed. Surgical aspiration of a large preretinal hemorrhage revealed intraretinal flame-shaped hemorrhages and some cotton wool spots. Further intraoperative examination and post-operative fluorescein angiography excluded the rhegmatogenaous and the neovascular origin of the intraocular bleeding. CONCLUSIONS: Due to the several predisposing factors such as diabetes, aspirin assumption, history of blood dyscrasia and infectious retinitis, the relationship between the acute intraocular bleeding and the BNT162b2 inoculation remains difficult to ascertain in this patient. However, the occurrence of lasting priapism and vitreous hemorrhage within 24 h from the vaccination is a critical event which deserves to be mentioned.

Heat can treat: long-term follow-up results after uterine-sparing treatment of adenomyosis with radiofrequency thermal ablation in 60 hysterectomy candidate patients.

BACKGROUND: Adenomyosis may induce pelvic pain, abnormal uterine bleeding or bulk symptoms. If hormonal treatment proves ineffective or contraindicated, hysterectomy may be necessary. For patients who desire to conserve the uterus despite severe symptomatology, uterine-sparing techniques have been introduced. Radiofrequency thermal ablation (RFA) consists of the local application of high temperature to eliminate diseased tissue, applied recently for adenomyosis treatment. The objective of the study was to analyze the efficacy of RFA for avoiding hysterectomy in patients with adenomyosis-related symptoms. METHODS: This is a single-center, retrospective cohort study performed in a referral center for endometriosis. The study population consisted of all consecutive patients who underwent Radiofrequency thermal ablation (RFA) treatment as an alternative to hysterectomy for adenomyosis between March 2011 and June 2019 in our institution. RFA was performed using laparoscopic access. To evaluate the impact of RFA treatment on symptoms, follow-up findings were compared to preoperative symptomatology using the ten-point visual analog scale (VAS) for pain assessment. RESULTS: Sixty patients were included in the study, 39 of them (65%), underwent a concomitant surgery for endometriosis in association to RFA. On a long-term follow-up (mean 56 months (range 10-115, SD 29), hysterectomy was performed in 8 patients (13%). The mean VAS score before vs after surgery was 7.4 vs 3.3 for dysmenorrhea, 3.7 vs 0.3 for dyschezia, 4.7 vs 0.7 for dyspareunia, and 4.0 vs 1.4 for chronic pelvic pain, being significantly reduced after RFA for all these pain components (p < 0.0001 in every case). Thirty-one patients (52%) suffered from AUB before RFA, this symptom persisted in 10 patients (16%) during follow-up (p < 0.001). Bulk symptoms were present in 16 patients (27%) and disappeared after RFA in all cases. CONCLUSIONS: RFA allows for hysterectomy avoidance in most cases. It leads to marked improvements in pain symptomatology, uterine bleeding and bulk symptoms.

"The Sword in the Stone": radical excision of deep infiltrating endometriosis with bowel shaving-a single-centre experience on 703 consecutive patients.

BACKGROUND: Laparoscopic segmental bowel resection, disc excision and rectal shaving are described as surgical options for the treatment of bowel endometriosis, but the gold standard has not yet established. The aim of the study is to investigate the efficacy of the laparoscopic bowel shaving technique in terms of pain symptomatology and to analyse early and late postoperative complications. METHODS: Retrospective cohort study of a series of 703 consecutive patients treated between January 2014 and December 2019 in a tertiary care referral centre. All patients underwent laparoscopic bowel shaving with concomitant radical excision of DIE. RESULTS: Bilateral posterolateral parametrectomy and ureterolysis were performed, respectively, in 314 (44.7%) and 318 cases (45.2%). A radical hysterectomy was performed in 107 cases (82.9%). Postoperative complications were infrequent: 17 patients required a reoperation (2.4%) and in this subgroup we registered 2 rectovaginal fistulas (0.3%), 4 patients received blood transfusion (0.6%), 12 patients (1.7%) experienced postoperative fever, 6 patients experienced impaired bladder voiding (0.9%) after 6 months. Median follow-up was 14 months. The study reported good clinical and surgical results, with a regression of symptoms (p < 0.0001) and an overall rate of recurrence of 6.5%. Clinical and instrumental criteria of bowel endometriosis relapse were exclusively detected in 5 patients (0.8%). Eleven patients (1.7%) with relapsed endometriosis were reoperated. CONCLUSIONS: Bowel shaving is a feasible and valuable surgical procedure. It is only the last step of a complex surgery which is aimed to minimize the residual quote of infiltrating nodule and requires a multidisciplinary team to achieve optimal treatment preoperatively, intraoperatively and postoperatively.

Evaluation of the impact of cataract surgery on cognitive function in very elderly patients: a prospective, observational study.

AIM: To evaluate the impact of cataract surgery on cognitive function in very elderly patients (≥ 85 years). METHODS: A prospective, nonrandomized, comparative study of very elderly patients (≥ 85 years), and elderly patients (≥ 65 < 85 years) scheduled for first time cataract surgery. Cognitive function, quality of life (QoL), best corrected visual acuity (BCVA), endothelial cell count (ECC), and central corneal thickness (CCT) were assessed at 90 days before surgery (T0), at surgery (T1), and at 30 (T2) and 90 days (T3) after surgery. Macular thickness (MT) was evaluated at T2. Six-item cognitive impairment test (6CIT) was used to evaluate cognitive function while the Catquest-9SF test was used to assess QoL. The confusion assessment method (CAM) was used to evaluate the presence of delirium episodes at within 48 h from T1. RESULTS: A total of 78 patients (very elderly n = 24, elderly n = 54) were enrolled; average age 80.3 ± 6.2 years old. Significant improvements for cognitive function, BCVA, QoL (p < 0.01) during the observational period were observed among the cohort. A greater improvement in cognitive function was observed in the very elderly patients between T1 and T2 (p = 0.02), while there was no difference in BCVA and QoL between the groups. There were no significant differences in ECC and CCT changes between the groups. No delirium episodes or significant macular abnormalities were registered. CONCLUSIONS: Cataract surgery in very elderly patients seems to offer a greater improvement in cognitive function as compared to elderly patients, and it is associated with a significant improvement in QoL, without any incidence of postoperative delirium.

Subcellular Localization of Fad1p in Saccharomyces cerevisiae: A Choice at Post-Transcriptional Level?

FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms-that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3' region of FAD1 mRNA was analyzed by 3'RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3'UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3'UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3'UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.

Mitochondria: From Physiology to Pathology.

Over the past decade, the role of mitochondria has extended beyond those tasks for which these organelles are historically known [...].

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.

Total laparoscopic vs. conventional open abdominal nerve-sparing radical hysterectomy: clinical, surgical, oncological and functional outcomes in 301 patients with cervical cancer.

OBJECTIVE: Total laparoscopic nerve-sparing radical hysterectomy (TL-NSRH) has been considered a promising approach, however, surgical, clinical, oncological and functional outcomes have not been systematically addressed. We present a large retrospective multi-center experience comparing TL-NSRH vs. open abdominal NSRH (OA-NSRH) for early and locally-advanced cervical cancer, with particular emphasis on post-surgical pelvic function. METHODS: All consecutive patients who underwent class C1-NSRH plus bilateral pelvic + para-aortic lymphadenectomy for stage IA2-IIB cervical cancer at 4 Italian gynecologic oncologic centers (Negrar, Varese, Bologna, Avellino) were enrolled. Patients were divided into TL-NSRH and OA-NSRH groups and were investigated with preoperative questionnaires on urinary, rectal and sexual function. Postoperatively, patients filled a questionnaire assessing quality of life, taking into account sexual function and psychological status. Oncological outcomes were analyzed using Kaplan-Meyer method. RESULTS: 301 consecutive patients were included in this study: 170 in the TL-NSRH group and 131 in the OA-NSRH group. Patients in the OA-NSRH group were more likely to experience urinary incontinence and (after 12-months follow-up) urinary retention. No patient in the TL-NSRH group vs. 5 (5.5%) in the OA-NSRH group had complete urinary retention (at the >24-month follow-up [p=0.02]). A total of 20 (11.8%) in the TL-NSRH and 11 (8.4%) patients in the OA-NSRH had recurrence of disease (p=0.44) and 14 (8.2%) and 9 (6.9%) died of disease during follow-up, respectively (p=0.83). CONCLUSION: Our study shows that TL-NSRH is feasible, safe and effective and conjugates adequate radicality and improvement in post-operative functional outcomes. Oncological outcomes of laparoscopic procedures deserve further investigation.

Messenger RNA delivery to mitoribosomes - hints from a bacterial toxin.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome.

Biallelic Mutations in MTPAP Associated with a Lethal Encephalopathy.

BACKGROUND: A homozygous founder mutation in MTPAP/TENT6, encoding mitochondrial poly(A) polymerase (MTPAP), was first reported in six individuals of Old Order Amish descent demonstrating an early-onset, progressive spastic ataxia with optic atrophy and learning difficulties. MTPAP contributes to the regulation of mitochondrial gene expression through the polyadenylation of mitochondrially encoded mRNAs. Mitochondrial mRNAs with severely truncated poly(A) tails were observed in affected individuals, and mitochondrial protein expression was altered. OBJECTIVE: To determine the genetic basis of a perinatal encephalopathy associated with stereotyped neuroimaging and infantile death in three patients from two unrelated families. METHODS: Whole-exome sequencing was performed in two unrelated patients and the unaffected parents of one of these individuals. Variants and familial segregation were confirmed by Sanger sequencing. Polyadenylation of mitochondrial transcripts and de novo synthesis of mitochondrial proteins were assessed in patient's fibroblasts. RESULTS: Compound heterozygous p.Ile428Thr and p.Arg523Trp substitutions in MTPAP were recorded in two affected siblings from one family, and a homozygous p.Ile385Phe missense variant identified in a further affected child from a second sibship. Mitochondrial poly(A) tail analysis demonstrated shorter posttranscriptional additions to the mitochondrial transcripts, as well as an altered expression of mitochondrial proteins in the fibroblasts of the two siblings compared with healthy controls. CONCLUSION: Mutations in MTPAP likely cause an autosomal recessive perinatal encephalopathy with lethality in the first year of life.

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.