A deep intronic variant in MME causes autosomal recessive Charcot-Marie-Tooth neuropathy through aberrant splicing.

BACKGROUND: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AIMS: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. METHODS: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. RESULTS: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. INTERPRETATION: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

Calcium-induced structural transitions are central to the folding, function, and processing of serratiopeptidase zymogen into mature form.

Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.

Targeting CK2 eliminates senescent cells and prolongs lifespan in Zmpste24-deficient mice.

Senescent cell clearance is emerging as a promising strategy for treating age-related diseases. Senolytics are small molecules that promote the clearance of senescent cells; however, senolytics are uncommon and their underlying mechanisms remain largely unknown. Here, we investigated whether genomic instability is a potential target for senolytic. We screened small-molecule kinase inhibitors involved in the DNA damage response (DDR) in Zmpste24-/- mouse embryonic fibroblasts, a progeroid model characterized with impaired DDR and DNA repair. 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), which specifically inhibits casein kinase 2 (CK2), was selected and discovered to preferentially trigger apoptosis in Zmpste24-/- cells. Mechanistically, inhibition of CK2 abolished the phosphorylation of heterochromatin protein 1α (HP1α), which retarded the dynamic HP1α dissociation from repressive histone mark H3K9me3 and its relocalization with γH2AX to DNA damage sites, suggesting that disrupting heterochromatin remodeling in the initiation of DDR accelerates apoptosis in senescent cells. Furthermore, feeding Zmpste24-deficient mice with TBB alleviated progeroid features and extended their lifespan. Our study identified TBB as a new class senolytic compound that can reduce age-related symptoms and prolong lifespan in progeroid mice.

GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Nardilysin in vascular smooth muscle cells controls blood pressure via the regulation of calcium dynamics.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Bacteroides fragilis toxin expression enables lamina propria niche acquisition in the developing mouse gut.

Bacterial toxins are well-studied virulence factors; however, recent studies have revealed their importance in bacterial niche adaptation. Enterotoxigenic Bacteroides fragilis (ETBF) expresses B. fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition. We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B. fragilis. LP entry by ETBF required BFT metalloprotease activity, and showed temporal restriction to the pre-weaning period, dependent on goblet-cell-associated passages. In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP. BFT expression increased goblet cell number and goblet-cell-associated passage formation. These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition, suggesting that a selective advantage conferred by a toxin may impact long-term host health.

Epidemiological characteristics of patients with Hutchinson-Gilford progeria syndrome and progeroid laminopathies in China.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) and progeroid laminopathies (PL) are extremely rare genetic diseases with extremely poor prognoses. This study aims to investigate the epidemiological and genotypic characteristics of patients with HGPS/PL in China. METHODS: Using a cross-sectional study design, general characteristics and genotypic data of 46 patients with HGPS/PL from 17 provinces in China were analyzed. RESULTS: Among the 46 patients with HGPS/PL, 20 patients are HGPS, and the rest are PL; the identified total prevalence of HGPS/PL is 1/23 million. Among 42 patients with gene reports, 3 carried compound heterozygous mutations in the ZMPSTE24 while the other 39 carried LMNA mutations. Among PL, LMNA c.1579 C > T homozygous mutation was the most common. The onset of classic genotype HGPS is skin sclerosis in the first month after birth. The primary clinical manifestations of PL patients include skin abnormalities, growth retardation, and joint stiffness. The median age of onset for PL was 12 (6,12) months. CONCLUSIONS: In China, the identified total prevalence of HGPS/PL is 1/23 million. 92.8% of the genetic mutations of HGPS/PL were located in LMNA, and the rest in ZMPSTE24. Most patients of HGPS/PL have skin abnormalities as the earliest manifestation. Compared to PL, the classic genotype HGPS starts earlier. IMPACT STATEMENT: Hutchinson-Gilford progeria syndrome (HGPS) and progeroid laminopathies (PL) are extremely rare genetic diseases with extremely poor prognoses. To date, there is a paucity of epidemiological data related to HGPS/PL in China. This study first examined the genotypic, phenotypic, and prevalence characteristics of 40-50% of the cases of HGPS/PL in mainland China through a collaborative international registry effort. In China, the identified total prevalence of HGPS/PL is 1/23 million. 92.8% of the genetic mutations of HGPS/PL are located in LMNA. LMNA c.1579 C > T homozygous mutations are the most common form of gene mutations among the Chinese PL population.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma.

OBJECTIVES: Data support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). METHODS: Quantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. RESULTS: We documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06-1.85) for CIMP-high and 2.14 (1.65-2.77) for MSI-high, but 1.02 (0.78-1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16-3.45) for CIMP-high and 2.86 (1.64-5.00) for BRAF mutation, but 1.09 (0.67-1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DISCUSSION: The tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

Expression dynamics indicate the involvement of SPG7 in the reproduction and spermiogenesis of Phascolosoma esculenta.

Spastic paraplegia 7 (SPG7) is an m-AAA protease subunit involved in mitochondrial morphology and physiology. However, its function in animal reproduction is yet to be evaluated. In this study, its molecular features, subcellular localization, and expression dynamics were investigated to analyze its potential function in the reproduction of male Phascolosoma esculenta, an economically important marine species in China. The full-length cDNA of P. esculenta spg7 (Pe-spg7) measures 3053 bp and encodes an 853-amino acid protein (Pe-SPG7). Pe-SPG7 includes two transmembrane domains, an AAA domain and a proteolytic domain. Amino acid sequence alignment revealed that SPG7 was conserved during evolution. The mRNA and protein expression of spg7 indicated its involvement in reproduction. Its expression was the highest in coelomic fluid, where spermatids develop, and it was significantly higher in the breeding stage than in the nonbreeding stage. SPG7 was mainly found in the mitochondria of spermatids in the coelomic fluid, indicating that it functions in this organelle in spermatids. Immunofluorescence experiments showed that SPG7 was expressed and colocalized in the mitochondria during spermiogenesis, suggesting its involvement in P. esculenta spermiogenesis. Therefore, SPG7 may participate in spermiogenesis by functioning in the mitochondria and regulate the reproduction of male P. esculenta. This study provided insights into the function of SPG7 in animal reproduction and P. esculenta gametogenesis.

DegS protease regulates antioxidant capacity and adaptability to oxidative stress environment in Vibrio cholerae.

Adaptation to oxidative stress is critical for survival of Vibrio cholerae in aquatic ecosystems and hosts. DegS activates the σE envelope stress response. We have previously revealed that DegS may be involved in regulating the oxidative stress response. In this study, we demonstrated that deletion of the degS gene attenuates the antioxidant capacity of V. cholerae. In addition, our results further revealed that the regulation of antioxidant capacity by DegS in V. cholerae could involve the cAMP-CRP complex, which regulates rpoS. XthA is an exonuclease that repairs oxidatively damaged cells and affects the bacterial antioxidant capacity. qRT-PCR showed that DegS, σE, cAMP, CRP, and RpoS positively regulate xthA gene transcription. XthA overexpression partially compensates for antioxidant deficiency in the degS mutant. These results suggest that DegS affects the antioxidant capacity of V.cholerae by regulating xthA expression via the cAMP-CRP-RpoS pathway. In a mouse intestinal colonization experiment, our data showed that V.cholerae degS, rpoE, and rpoS gene deletions were associated with significantly reduced resistance to oxidative stress and the ability to colonize the mouse intestine. In conclusion, these findings provide new insights into the regulation of antioxidant activity by V.cholerae DegS.

The farnesyl transferase inhibitor (FTI) lonafarnib improves nuclear morphology in ZMPSTE24-deficient fibroblasts from patients with the progeroid disorder MAD-B.

Several related progeroid disorders are caused by defective post-translational processing of prelamin A, the precursor of the nuclear scaffold protein lamin A, encoded by LMNA. Prelamin A undergoes farnesylation and additional modifications at its C-terminus. Subsequently, the farnesylated C-terminal segment is cleaved off by the zinc metalloprotease ZMPSTE24. The premature aging disorder Hutchinson Gilford progeria syndrome (HGPS) and a related progeroid disease, mandibuloacral dysplasia (MAD-B), are caused by mutations in LMNA and ZMPSTE24, respectively, that result in failure to process the lamin A precursor and accumulate permanently farnesylated forms of prelamin A. The farnesyl transferase inhibitor (FTI) lonafarnib is known to correct the aberrant nuclear morphology of HGPS patient cells and improves lifespan in children with HGPS. Importantly, and in contrast to a previous report, we show here that FTI treatment also improves the aberrant nuclear phenotypes in MAD-B patient cells with mutations in ZMPSTE24 (P248L or L425P). As expected, lonafarnib does not correct nuclear defects for cells with lamin A processing-proficient mutations. We also examine prelamin A processing in fibroblasts from two individuals with a prevalent laminopathy mutation LMNA-R644C. Despite the proximity of residue R644 to the prelamin A cleavage site, neither R644C patient cell line shows a prelamin A processing defect, and both have normal nuclear morphology. This work clarifies the prelamin A processing status and role of FTIs in a variety of laminopathy patient cells and supports the FDA-approved indication for the FTI Zokinvy for patients with processing-deficient progeroid laminopathies, but not for patients with processing-proficient laminopathies.

Prelamin A and ZMPSTE24 in premature and physiological aging.

As human longevity increases, understanding the molecular mechanisms that drive aging becomes ever more critical to promote health and prevent age-related disorders. Premature aging disorders or progeroid syndromes can provide critical insights into aspects of physiological aging. A major cause of progeroid syndromes which result from mutations in the genes LMNA and ZMPSTE24 is disruption of the final posttranslational processing step in the production of the nuclear scaffold protein lamin A. LMNA encodes the lamin A precursor, prelamin A and ZMPSTE24 encodes the prelamin A processing enzyme, the zinc metalloprotease ZMPSTE24. Progeroid syndromes resulting from mutations in these genes include the clinically related disorders Hutchinson-Gilford progeria syndrome (HGPS), mandibuloacral dysplasia-type B, and restrictive dermopathy. These diseases have features that overlap with one another and with some aspects of physiological aging, including bone defects resembling osteoporosis and atherosclerosis (the latter primarily in HGPS). The progeroid syndromes have ignited keen interest in the relationship between defective prelamin A processing and its accumulation in normal physiological aging. In this review, we examine the hypothesis that diminished processing of prelamin A by ZMPSTE24 is a driver of physiological aging. We review features a new mouse (LmnaL648R/L648R) that produces solely unprocessed prelamin A and provides an ideal model for examining the effects of its accumulation during aging. We also discuss existing data on the accumulation of prelamin A or its variants in human physiological aging, which call out for further validation and more rigorous experimental approaches to determine if prelamin A contributes to normal aging.

Clinical significance of Bacteroides fragilis as a potential prognostic factor in colorectal cancer.

INTRODUCTION: Bacteroides fragilis (B. fragilis) is considered to act in an anti-inflammatory manner on the intestinal tract. On the contrary, enterotoxigenic B. fragilis (ETBF), a subtype of B. fragilis, produces an enterotoxin (BFT; B. fragilis toxin), leading to asymptomatic chronic infections and colonic tumor formation. However, the impact of B. fragilis and ETBF on the clinical outcome of colorectal cancer (CRC) remains unclear. We aim to assess whether their presence affects the outcome in patients with CRC after curative resection. METHODS: We obtained 197 pairs of matched formalin-fixed paraffin-embedded samples from cancerous and adjacent non-cancerous tissues of patients with pathological stage (pstage) II and III CRC after curative resection. The presence of B. fragilis and ETBF were estimated using real-time polymerase chain reaction, and recurrence-free survival (RFS) and overall survival (OS) of the patients were analyzed. RESULTS: 16S rRNA for B. fragilis and bft DNA were detected in 120 (60.9%) and 12 (6.1%) of the 197 patients, respectively. B. fragilis-positive patients had better RFS than B. fragilis-negative patients, although that was not statistically significant. In subgroup analysis, better outcomes on RFS were observed in the presence of B. fragilis in pstage II and left-sided CRC. The association of B. fragilis positivity on OS was accentuated in the depth of T4 subgroup. No significant differences were observed in RFS and OS between ETBF and non-toxigenic B. fragilis. CONCLUSIONS: Our findings suggest that the presence of B. fragilis is associated with better outcomes in patients with pstage II and III CRC after curative resection.

Neurolysin Knockout Mice in a Diet-Induced Obesity Model.

Neurolysin oligopeptidase (E.C.3.4.24.16; Nln), a member of the zinc metallopeptidase M3 family, was first identified in rat brain synaptic membranes hydrolyzing neurotensin at the Pro-Tyr peptide bond. The previous development of C57BL6/N mice with suppression of Nln gene expression (Nln-/-), demonstrated the biological relevance of this oligopeptidase for insulin signaling and glucose uptake. Here, several metabolic parameters were investigated in Nln-/- and wild-type C57BL6/N animals (WT; n = 5-8), male and female, fed either a standard (SD) or a hypercaloric diet (HD), for seven weeks. Higher food intake and body mass gain was observed for Nln-/- animals fed HD, compared to both male and female WT control animals fed HD. Leptin gene expression was higher in Nln-/- male and female animals fed HD, compared to WT controls. Both WT and Nln-/- females fed HD showed similar gene expression increase of dipeptidyl peptidase 4 (DPP4), a peptidase related to glucagon-like peptide-1 (GLP-1) metabolism. The present data suggest that Nln participates in the physiological mechanisms related to diet-induced obesity. Further studies will be necessary to better understand the molecular mechanism responsible for the higher body mass gain observed in Nln-/- animals fed HD.

Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis.

OBJECTIVE: The aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. METHODS: ETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. RESULTS: Six different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). CONCLUSIONS: Our data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

An SPG7 mutation as a novel cause of monogenic progressive muscular atrophy.

BACKGROUND: Progressive muscular atrophy (PMA) is a rare adult-onset neurological disease that is characterized by isolated lower motor neuron degeneration. While it is still disputable whether PMA is a subtype of amyotrophic lateral sclerosis (ALS) or an isolated disorder, it is well-established as a clinically defined entity. About 5% of PMA cases are monogenic, and the implicated genes largely overlap with those causing monogenic ALS. CASE DESCRIPTION: Here we describe a 68-year-old female patient with progressive and asymmetric upper-limb weakness throughout an 18-month period, with muscle atrophy, dysphagia and slurring of speech. The lower limbs were unaffected, and there was no sign of upper motor neuron dysfunction. Comprehensive genetic testing for single nucleotide and copy-number variants revealed a pathogenic monoallelic variant c.1529C>T, p.(Ala510Val) in the SPG7 gene. DISCUSSION: Pathogenic biallelic SPG7 variants have been originally associated with hereditary spastic paraplegia, but other phenotypes are nowadays known to be linked to these variants, such as ALS. However, there is no report of this (or any) other SPG7 variant in association with PMA, whether it progressed to ALS or not. In conclusion, we present the first known case of PMA associated with a monoallelic SPG7 mutation.