Frameshift Research Articles

Four novel mutations identified in the COL4A3, COL4A4 and COL4A5 genes in 10 families with Alport syndrome

BackgroundAlport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient.MethodsWe screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R).ResultsWe identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same.ConclusionsOur research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research.

Novel TMPRSS6 variants and their impact on iron-refractory iron deficiency anaemia in pregnancy: A North Indian genotype phenotype study.

Iron-refractory iron deficiency anaemia (IRIDA) is a rare autosomal recessive disorder, distinguished by hypochromic microcytic anaemia, low transferrin levels and inappropriately elevated hepcidin (HEPC) levels. It is caused by mutations in TMPRSS6 gene. Systematic screening of 500 pregnant women with iron deficiency anaemia having moderate to severe microcytosis with no other causes of anaemia were enrolled to rule out oral iron refractoriness. It identified a final cohort of 10 (2.15% prevalence) individuals with IRIDA phenotype. Haematological and biochemical analysis revealed significant differences between iron responders and iron non-responders, with iron non-responders showing lower haemoglobin, red blood cell count, serum iron and serum ferritin levels, along with elevated HEPC (9.47 ± 2.75 ng/mL, p = 0.0009) and erythropoietin (4.58 ± 4.07 µ/mL, p = 0.0196) levels. Genetic sequencing of the TMPRSS6 gene in this final cohort identified 10 novel variants, including seven missense and three frame-shift mutations, with four missense variants showing high functional impact defining the IRIDA phenotype. Structural analysis revealed significant damage caused by two variants (p.L83R and p.S235R). This study provides valuable insights into IRIDA among pregnant women in the Indian subcontinent, unveiling its underlying causes of unresponsiveness, genetic mechanisms and prevalence. Furthermore, research collaboration is essential to validate these findings and develop effective treatments.

Analysis of Phenotypes Associated with Deficiency of PAX6 Haplotypes in Chinese Aniridia Families.

To examine the clinical phenotype and genetic deficiencies present in Chinese aniridia families with PAX6 haplotype deficiency. A comprehensive questionnaire and ophthalmological assessments were administered to both affected patients and unaffected relatives. The clinical feature analysis included the evaluation of visual acuity, intraocular pressure, slit-lamp anterior segment examination, fundus photography, and spectral domain optical coherence tomography. To identify the mutation responsible for aniridia, targeted next-generation sequencing was used as a beneficial technique. A total of 4 mutations were identified, consisting of two novel frameshift mutations (c.314delA, p.K105Sfs*33 and c.838_845dup AACACACC, p.S283Tfs*85), along with two recurring nonsense mutations (c.307C>T, p.R103X and c.619A>T, p.K207*). Complete iris absence, macular foveal hypoplasia, and nystagmus were consistent in these PAX6 haplotype-deficient Chinese aniridia families, while corneal lesions, cataracts, and glaucoma exhibited heterogeneity both among the families and within the same family. In our study, two novel PAX6 mutations associated with aniridia were identified in Chinese families, which expanded the phenotypic and genotypic spectrum of PAX6 mutations. We also analyzed the clinical characteristics of PAX6 haplotype deficiency in Chinese aniridia families.

Dissecting the sequential evolution of a selfish mitochondrial genome in Caenorhabditis elegans.

Mitochondrial genomes exist in a nested hierarchy of populations where mitochondrial variants are subject to genetic drift and selection at each level of organization, sometimes engendering conflict between different levels of selection, and between the nuclear and mitochondrial genomes. Deletion mutants in the Caenorhabditis elegans mitochondrial genome can reach high intracellular frequencies despite strongly detrimental effects on fitness. During a mutation accumulation (MA) experiment in C. elegans, a 499 bp deletion in ctb-1 rose to 90% frequency within cells while significantly reducing fitness. During the experiment, the deletion-bearing mtDNA acquired three additional mutations in nd5, namely two single insertion frameshift mutations in a homopolymeric run, and a base substitution. Despite an additional fitness cost of these secondary mutations, all deletion-bearing molecules contained the nd5 mutations at the termination of the MA experiment. The presence of mutant mtDNA was associated with increased mtDNA copy-number. Variation in mtDNA copy-number was greater in the MA lines than in a wildtype nuclear background, including a severe reduction in copy-number at one generational timepoint. Evolutionary replay experiments using different generations of the MA experiment as starting points suggests that two of the secondary mutations contribute to the proliferation of the original ctb-1 deletion by unknown mechanisms.

Lack of nuclear localization of the Creb3l1 transcription factor causes defects in caudal fin bifurcation in zebrafish Danio rerio.

The formation of normal bone and bone healing require the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after fracture. Creb3l1 undergoes regulated intra-membrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. To expand our understanding of Creb3l1 role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish D. rerio caused a frame-shift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ doesn't regulate transcription, and the creb3l1TA+/TA+ fish animals are creb3l1 transcriptional nulls. We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased capacity for regeneration, and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway, and document a novel role of Creb3l1 in tissue patterning.

Mutations in embB406 Are Associated with Low-Level Ethambutol Resistance in Canadian Mycobacterium tuberculosis Isolates.

In Mycobacterium tuberculosis, molecular predictions of ethambutol resistance rely primarily on the detection of mutations within embB. However, discordance between embB406 mutations and gold standard phenotypic drug sensitivity testing (DST) questions the significance of embB406 mutations used in molecular DST. This study tabulates embB mutations found in Canadian M. tuberculosis isolates and evaluates the impact of specific mutations on ethambutol resistance. The National Reference Centre for Mycobacteriology culture collection (n = 2796) was screened for isolates with embB mutations. Phenotypic DST was performed on the BACTEC™ MGIT™ 960 at ethambutol concentrations of 2-5 μg/mL. Whole genome sequencing was used for drug resistance predictions, phylogenomics and single nucleotide polymorphism analysis. Detection of resistance-associated embB mutations corresponded to a positive predictive value of 64.3%, negative predictive value of 99.2%, 98.7% specificity, and 73.3% sensitivity compared to phenotypic DST. Two embB406 mutation subtypes (Gly406Asp, Gly406Ala) were found among 16 isolates, of which 12 were sensitive at 5 µg/mL ethambutol with variable resistance between 2-4 µg/mL. A novel frameshift mutation in regulator embR (Gln258fs) was found in nine isolates. Mutations in embB406 were associated with low-level ethambutol resistance undetectable at the recommended critical concentration (5 μg/mL). These novel mutations may exacerbate variability in ethambutol resistance.

Characterization of Clinical Phenotypes in Congenital Myasthenic Syndrome Associated with the c.1327delG Frameshift Mutation in CHRNE Encoding the Acetylcholine Receptor Epsilon Subunit.

Congenital myasthenic syndromes (CMS) are a group of rare but often treatable inherited disorders of neuromuscular transmission characterized by fatigable skeletal muscle weakness. In this paper we present the largest phenotypic analysis to date of a cohort of patients carrying the pathogenic variant c.1327delG in the CHRNE gene, leading to CHRNE-CMS. This study aims to identify the phenotypic variability in CMS associated with c.1327delG mutation in the CHRNE gene. Disease specific symptoms were assessed using specific standardized tests for autoimmune myasthenia (Quantitative Myasthenia Gravis score) as well as patient-reported scales for symptom severity. Evaluated clinical manifestations included ocular symptoms (ophthalmoparesis and ptosis), bulbar weakness, axial muscle weakness, proximal and distal muscle weakness, and respiratory function. Patients were allocated into three groups according to clinical impression of disease severity: mild, moderate, and severe. We studied 91 Bulgarian Roma patients, carrying the same causative homozygous CHRNE c.1327delG mutation. Bulbar weakness was present in patients throughout all levels of severity of CHRNE-CMS in this study. However, difficulties in eating and swallowing are more prominent characteristics in the moderate and severe clinical phenotypes. Diplopia and ptosis resulting from fatigue of the extraocular muscles were permanent features regardless of disease severity or age. Levels of axial, proximal and distal muscle weakness were variable between disease groups. The statistical analysis showed significant differences between the patients in the three groups, emphasizing a possible variation in symptom manifestation in the evaluated patient population despite the disease originating from the same genetic mutation. Impairment of respiratory function was more prominent in severely affected patients, which might result from loss of compensatory muscle function in those individuals. Results from our study indicate significant phenotypic heterogeneity leading to mild, moderate, or severe clinical manifestation in CHRNE-CMS, despite the genotypic homogeneity.

Metabolic remodeling and calcium handling abnormality in induced pluripotent stem cell-derived cardiomyocytes in dilated phase of hypertrophic cardiomyopathy with MYBPC3 frameshift mutation

Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca2+ imaging showed increased abnormal Ca2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

O-259 Seminal plasma cell-free RNA to predict testicular biopsy sperm retrieval outcome in azoospermic men

Abstract Study question Is there a way to assess seminal plasma of NOA men to predict a successful sperm retrieval at testicular biopsy? Summary answer Multi-omics approach in seminal plasma of NOA men provides reliable prediction on the spermatogenic activity within the seminiferous tubule. What is known already Microdissection testicular sperm extraction (m-TESE) in NOA men successfully retrieved spermatozoa in about 60% of the cases. While several algorithms have been investigated aiming at estimating spermatogenic progress such as age, serum FSH, inhibin B, and genetics, there is no specific assay that can do so reliably. Even histopathology is often inconclusive while equally invasive. Recently, epigenetic analysis on testicular biopsy specimens has shown a differential gene expression in relation to the origin of azoospermia and spermatogenic function (Cheung, et al, 2024, in press). Study design, size, duration Over a 4-year period, twenty-eight men were deemed azoospermic following several failed extensive semen analyses. Transcriptomic analysis was performed by RNAseq on seminal fluid and the specific gene expression were evaluated and compared. In addition, DNAseq and proteomics were carried out to confirm our findings. These patients then underwent micro-TESE by a single surgeon and were divided into two cohorts based on whether spermatozoa was successfully retrieved or not. Participants/materials, setting, methods RNA and DNA were isolated from seminal plasma using a commercial kit and sequenced by Illumina HiSeq3000/4000 platform. Differentially expressed genes (DEGs) were assessed and compared to a fertile donor control using DESeq2 and Gene Ontology analysis. An absolute log2fold change of > 1 and a P-value of < 0.0005 were considered statistically significant. Gene mutations were detected by CLC Genomic Server 9.0. Main results and the role of chance Transcriptomic in 28 ejaculates from iNOA men browsed 21,855 genes against a fertile donor. Of 3,505 imbalanced genes, 12 were consistently imbalanced in 12 men, while 19 genes were imbalanced in remaining 16. Among the 2 cohorts, 8 common imbalanced genes were found. Particularly TPTE2, a testis-specific gene that regulates spermatogenesis, was overexpressed in 10/12 men (OE-12-cohort), while underexpressed in the remainder 16 (UE-16-cohort). DNAseq showed that TPTE2 was conserved in the OE-12-cohort, while exhibiting a frameshift mutation in the UE-16-cohort. Most interestingly, NEU1, a gene involved in acrosome development and fertilization, was found to be consistently and concurrently overexpressed in the OE-12-cohort, while being convserly underexpressed in all the men in the UE-16 cohort. DNAseq confirmed the NEU1 gene exhibited a synonymous mutation in the OE-12 and a frameshift mutation in the UE-16-cohort. About 1-2 months after the DEG analysis was completed, all of the patients underwent m-TESE. The 12 men from the OE-12-cohort all had successful retrieval of spermatozoa at m-TESE, conversely the 16 men from the UE-16-cohort all consistently failed to yield spermatozoa at testicular surgery in spite of extensive search for several hours and by multiple embryologists. Moreover, histopathology confirmed spermatogenic status in both OE-12&UE-16-cohorts. Limitations, reasons for caution Using non-invasive RNAseq and DNAseq on the seminal plasma has allowed us to identify DEGs that may be used to predict whether a patient with iNOA would have a successful or failed sperm retrieval with micro-TESE. However, these results are preliminary and should be further validated in larger study cohort. Wider implications of the findings Transcriptomics on the ejaculates of men with iNOA represents a noninvasive tool to detect presence of residual spermatogenesis. If confirmed by further studies, this precision medicine method may be utilized in order to properly counsel patients regarding their future prognosis. Trial registration number Not applicable

P-794 Unraveling the function of GATA2 and GATA3 in mouse blastocyst formation via CRISPR/Cas9 gene editing

Abstract Study question What is the effect of the CRISPR/Cas9 mediated knock-out (KO) of Gata2 and Gata3 on mouse preimplantation development/blastocyst formation? Summary answer Efficient generation of Gata3/Gata2 KO mouse embryos was achieved. Gata3 and Gata2 KO embryos could form blastocysts, suggesting a possible dispensability for blastocyst formation. What is known already GATA2 and GATA3 are important trophectoderm markers in mouse and human embryos. Prior studies, involving RNAi knockdown or conditional KO of Gata3, demonstrated high rates of morula arrest of Gata3-/- embryos, indicating its essential role in blastocyst formation. However, conflicting results showed embryonic lethality in Gata3-/- embryos only at 11-12 dpf. Besides, conditional KO of Gata2 suggested its dispensability in blastocyst formation, as Gata2-/- embryos could form blastocysts. It is of interest to validate these findings and potential functional redundancy between GATA2 and GATA3 in mouse embryos, by generating CRISPR/Cas9-mediated single and double KOs of Gata2 and Gata3. Study design, size, duration Upon assessment of the on-/ off-target effects of CRISPR/Cas9 components in mouse embryonic stem cells (mESC), CRISPR/Cas9 ribonucleoprotein (RNP) complexes were electroporated in mouse zygotes. Suitable control groups (non-targeted and scrambled targeted (inactive crRNA)) were incorporated. Embryos were genotyped by next generation sequencing (NGS), to assess the effect of gene KO on embryo morphology. Additionally, the expression pattern of Gata2 and Gata3 was examined in wild type and CRISPR/Cas9 targeted embryos, with immunofluorescent staining (IFS). Participants/materials, setting, methods Following CRISPR/Cas9 mediated targeting of Gata2 and Gata3, mouse embryos were cultured and morphologically monitored until day 4.5, alongside relevant control groups (scrambled, media control). Subsequently, DNA was extracted, and embryos were genotyped by PCR amplification of the region surrounding the target site, followed by NGS. IFS of mouse embryos at different developmental stages (E2.5, E3.0, E3.5 and E4.5) was performed to assess expression patterns and localization of both Gata2 and Gata3 throughout development. Main results and the role of chance Following the design of two gRNAs targeting either Gata2 or Gata3, on-target editing efficiency was tested in mESCs. The selected Gata2-targeting design resulted in a frameshift efficiency of 75%, whereas the Gata3-targeting design resulted in a frameshift efficiency of 78%. No off-target effects were observed in Gata2 and Gata3 targeted mESCs when examining in silico predicted off-target sites through PCR and NGS. Furthermore, 51% of Gata3 targeted and 41% of Gata2 targeted mouse embryos contained frameshift mutations in all cells. Notably, the majority of Gata3-/- (93%, n = 22) and Gata2-/- (94%, n = 16) embryos developed to blastocysts, similar to media control and scrambled groups. No statistically significant difference between Gata3-/- (n = 24, p = 0.326) and Gata2-/- (n = 17, p = 0.978) embryo morphology and scrambled (n = 96) embryo morphology was detected. IFS of control embryos (n = 16) showed that nuclear expression of GATA2 precedes nuclear expression of GATA3, indicating that GATA2 might control the expression of Gata3. Following IFS of Gata3 targeted embryos (n = 4), no nuclear signal for both GATA2 and GATA3 was observed, indicating that GATA3 could regulate the expression of Gata2. Furthermore, we created 100% double KO mouse embryos for Gata2 and Gata3 in two ways, enabling comprehensive exploration of functional redundancy in future studies. Limitations, reasons for caution Mosaicism (>1 genotype present in one embryo), off-target effects and chromosomal arrangements pose challenges regarding CRISPR/Cas9 germline genome editing. Zygote-stage delivery of RNP complexes is used to address mosaicism, and off-target effects are screened in mESCs via NGS. Future efforts aim to increase the sample sizes for stronger support. Wider implications of the findings Our goal is to identify the molecular pathways underlying unsuccessful implantation in mouse and human embryos. Utilizing CRISPR/Cas9 to generate KO embryos enhances our understanding of key molecular mechanisms, potentially shedding light on pregnancy failure due to failure of trophectoderm specification, formation or maintenance. Trial registration number not applicable

P-076 Role of N-DRC complex in male fertility

Abstract Study question There are many causes of human infertility, what’s the role of Nexin-dynein regulatory complex (N-DRC) in male fertility? Summary answer Many gene mutants in N-DRC components have been identified in infertile men, which affect the assembly of N-DRC complex and cause defects of sperm. What is known already human infertility affects 10–15% of couples, asthenozoospermia accounting for 18% of infertile men and is a common male infertility phenotype. Nexin-dynein regulatory complex (N-DRC) is a large protein complex regulating device in the flagellum and connects adjacent doublets of microtubules (DMTs). Some N-DRC components were reported to be associated with human disease, such as Primary ciliary dyskinesia (PCD). In recent years, more and more studies reported mutants in subunits of N-DRC are related to male infertility. For example, DRC1, DRC5 and DRC3 mutant were identified in patients displayed multiple morphological abnormalities of the flagella (MMAF) and obstructive azoospermia. Study design, size, duration We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Besides, we summarized the parameters of semen and ultrastructure of sperm flagella in these patients. Participants/materials, setting, methods We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Main results and the role of chance N-DRC is a highly conserved structural complex located in the axoneme of the flagellum and cilium, containing at least 11 different subunits (DRC1∼DRC11). Patients who were diagnosed as MMAF and male infertility carrying homozygous DRC1 mutants. TEM analyses of the patients’ sperm revealed the flagellar axoneme structure is disordered. A frameshift mutant of DRC5 was identified in an infertile male with asthenozoospermia, but the patient’s problem of infertility could be successfully solved by vitro Fertilization (IVF). A recent study identified two bi-allelic DRC3 frameshift variants in two unrelated patients, one patient displayed MMAF and the other present obstructive azoospermia. The sperm flagella function of DRC5 deletion mice was abrogated but flagellum ultrastructure remained normal and the mice behaved as asthenozoospermia and sterile. DRC3, DRC7 and DRC9 knockout mice exhibit MMAF and male infertility, and in DRC7 KO mice, the axoneme of sperm is disorganized and it was difficult to detect the ‘9 + 2’ microtubule structure. DRC1 mutant mice on the C57BL/6 background died before puberty, KO DRC6 in mice neither has no obvious effects on sperm motility function nor destroy the “9 + 2” arrangement of microtubules in cilia, and male mice was fertile. Limitations, reasons for caution More samples of MMAF and asthenozoospermia men to sequence N-DRC gene, and track their assisted reproductive outcomes. Wider implications of the findings The conclusions in our study uncover certain mechanism of N-DRC deficient in MMAF and asthenozoospermia phenotype, which could provide a new basis for diagnosis of male infertility. Trial registration number not applicable

Novel PATL2 variants cause female infertility with oocyte maturation defect.

Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Stretching the social protection system beyond capacity: tensions between governance capacity and conceptualisations of elder care in nursing homes in Indonesia

PurposeAgeing is a global concern that poses many challenges for governments, particularly in relation to the pressing issue of how to provide adequate social protection for the increasing number of elderly. Alongside rapid social and demographic transformation, Indonesia is especially challenged by the increasing number of elderly in need of formal care that is stretching the capacities of government and necessitates improvements in the social protection system. This study examines governance capacity in nursing homes and offers recommendations for improving the social protection system.Design/methodology/approachThe research presents a qualitative case study of government aged care centres in East Java and the experiences of residents and staff of three centres managed by the Provincial Social Service of East Java. The case study is based on ethnographic fieldwork, semi-structured interviews with 32 informants comprising directors, managers, staff and elderly residents of nursing homes.FindingsThe research finds that while social protection is in place, governance capacities at the provincial and local level are thinly stretched. The research highlights the tensions between existent governance capacities – in terms of mobility, decision-making, implementation and established local structures of governance and management – and the inability to meet the increasing demands for government elder care services and resources amidst broader societal transformations such as shifts in cultural framing of familial care of elderly. The findings point to the importance of understanding the dynamic relationship between governance capacity and the shifting terrains of elderly care due to societal transformation.Originality/valueThe research brings attention to the pivotal role that governance capacity plays in shaping the experiences and challenges of local government level elder care provision and the growing need for elder care in the face of an ageing population and shifts in kinship and family elder care arrangements. Elder care is not simply about addressing basic needs but also the quality and dignity of elderly and this is not easy to address where social protection systems are stretched beyond capacity. Recommendations are provided for enhancing government policy and expanding the scope for building cross-sectorial partnerships.

Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content (AC) and resistant starch content (RSC) of maize kernel starch, we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I (SBEI) and starch branching enzyme IIb (SBEIIb). A frameshift mutation in SBEI (E1, a nucleotide insertion in exon 6) led to plants with higher RSC (1.07 %), lower hundred-kernel weight (HKW, 24.71 ± 0.14 g), and lower plant height (PH, 218.50 ± 9.42 cm) compared to the wild type (WT). Like the WT, E1 kernel starch had irregular, polygonal shapes with sharp edges. A frameshift mutation in SBEIIb (E2, a four-nucleotide deletion in exon 8) led to higher AC (53.48 %) and higher RSC (26.93 %) than that for the WT. E2 kernel starch was significantly different from the WT regarding granule morphology, chain length distribution pattern, X-ray diffraction pattern, and thermal characteristics; the starch granules were more irregular in shape and comprised typical B-type crystals. Mutating SBEI and SBEIIb (E12) had a synergistic effect on RSC, HKW, PH, starch properties, and starch biosynthesis-associated gene expression. SBEIIa, SS1, SSIIa, SSIIIa, and SSIIIb were upregulated in E12 endosperm compared to WT endosperm. This study lays the foundation for rapidly improving the starch properties of elite maize lines

DIAPH1 mutations predict a favorable outcome for de novo MDS

DIAPH1, a member of the formins family and a Rho effector, was found to be involved in thrombocytopoiesis, and the process of MDS in mice with unknown pathogenesis. In this study, we reported a preliminary study about the heterogeneity in the clinical features and outcomes of DIAPH1 mutations in MDS. DIAPH1 frameshift mutations were identified in 20 out of 88 MDS patients, including 11 frameshift mutations locating at 140892588 ∼ 141000567 (5q31.3), which causes structure changes at FH1 domain. DIAPH1 mutated cases were correlated with lower megakaryocyte dysplasia in lower-risk patients (IPSS-M score < 0) at first diagnosis (P = 0.031), and higher megakaryocyte counts pre-transplant (P = 0.033). The megakaryopoiesis-related genes: GP1BA and SETBP1 mutation were positively and negatively associated with DIAPH1 mutation, respectively. DIAPH1 mutated cases showed superior overall survival of all patients (P = 0.037) and low-risk cohorts (P = 0.049). In conclusion, we found DIAPH1 frameshift mutations are implicated in megakaryopoiesis of MDS and correlated with superior prognosis.

Clinical phenotypic and genetic analysis of syndrome families with EYA1 gene variants

Objective:To investigate the clinical phenotype of a family with branchio-oto syndrome （BOS） and to explore the genetic etiology of the syndrome in this family. Methods:Clinical data were collected from a child diagnosed with BOS and his family members. Genomic DNA was extracted from peripheral blood of the proband and his family members. Whole-exome sequencing was performed, and the mutation sites were verified and analyzed by Sanger sequencing. Results:The family consists of two generations with four members, three of whom exhibit the phenotype. Two members have hearing loss and bilateral preauricular fistulas and bilateral branchial cleft fistulas. One member has bilateral preauricular fistulas and bilateral branchial cleft fistulas. All of which were in line with the clinical diagnosis of gill ear syndrome, the inheritance mode of the family was autosomal dominant inheritance, genetic testing showed that all members of the family had c. 1744delC（p. L592Cfs*47） mutation in the EYA1 gene, while unaffected members have the wild-type allele at this locus. This mutation is a frameshift mutation, which results in the early appearance of the stop codon, and has not been reported so far. According to ACMG guidelines, the variant was preliminarily determined to be suspected pathogenic. Conclusion:The newly discovered EYA1c. 1744delC（p. L592Cfs*47） mutation in this family is the pathogenic mutant gene of the patients in this family, which further expands the mutation spectrum of EYA1 gene, gives us a new understanding of the disease, and provides an important reference for clinical diagnosis and genetic counseling.

Rewinding the Ratchet: Rare Recombination Locally Rescues Neo-W Degeneration and Generates Plateaus of Sex-Chromosome Divergence.

Natural selection is less efficient in the absence of recombination. As a result, nonrecombining sequences, such as sex chromosomes, tend to degenerate over time. Although the outcomes of recombination arrest are typically observed after many millions of generations, recent neo-sex chromosomes can give insight into the early stages of this process. Here, we investigate the evolution of neo-sex chromosomes in the Spanish marbled white butterfly, Melanargia ines, where a Z-autosome fusion has turned the homologous autosome into a nonrecombining neo-W chromosome. We show that these neo-sex chromosomes are likely limited to the Iberian population of M. ines, and that they arose around the time when this population split from North-African populations, around 1.5 million years ago. Recombination arrest of the neo-W chromosome has led to an excess of premature stop-codons and frame-shift mutations, and reduced gene expression compared to the neo-Z chromosome. Surprisingly, we identified two regions of ∼1 Mb at one end of the neo-W that are both less diverged from the neo-Z and less degraded than the rest of the chromosome, suggesting a history of rare but repeated genetic exchange between the two neo-sex chromosomes. These plateaus of neo-sex chromosome divergence suggest that neo-W degradation can be locally reversed by rare recombination between neo-W and neo-Z chromosomes.

An intricate balancing act: Upstream and downstream frameshift co-regulatory elements.

Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against Covid-19. During ribosomal translation, a fraction of elongating ribosomes slips by one base in the 5 ' direction and enters a new reading frame for viral protein synthesis. Any interference with this process profoundly affects viral replication and propagation. For Covid-19, two RNA sites associated with ribosomal frameshifting for SARS-CoV-2 are positioned on the 5 ' and 3 ' of the frameshifting residues. Although much attention has been on the 3 ' frameshift element (FSE), the 5 ' stem-loop (attenuator hairpin, AH) can play a role. The formation of AH has been suggested to occur as refolding of the 3 ' RNA structure is triggered by ribosomal unwinding. However, the attenuation activity and the relationship between the two regions are unknown. To gain more insight into these two related viral RNAs and to further enrich our understanding of ribosomal frameshifting for SARS-CoV-2, we explore the RNA folding of both 5 ' and 3 ' regions associated with frameshifting. Using our graph-theory-based modeling tools to represent RNA secondary structures, "RAG" (RNA-As-Graphs), and conformational landscapes to analyze length-dependent conformational distributions, we show that AH coexists with the 3-stem pseudoknot of the 3 ' FSE (graph 3_6 in our dual graph notation) and alternative pseudoknot (graph 3_3) but less likely with other 3 ' FSE alternative folds (such as 3-way junction 3_5). This is because an alternative length-dependent Stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. In addition, we design four mutants for long lengths that stabilize or disrupt AH, AS1 or FSE pseudoknot to illustrate the deduced AH/AS1 roles and favor the 3_5, 3_6 or stem-loop. These mutants further show how a strengthened pseudoknot can result from a weakened AS1, while a dominant stem-loop occurs with a strengthened AS1. These structural and mutational insights into both ends of the FSE in SARS-CoV-2 advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting a sequence of length-dependent folds, which in turn define potential therapeutic intervention techniques involving both elements. Our work also highlights the complexity of viral landscapes with length-dependent folds, and challenges in analyzing these multiple conformations.

Allosteric effects of EF-G domain I mutations inducing ribosome frameshifting revealed by multiplexed force spectroscopy.

Ribosome translocation catalyzed by elongation factor G (EF-G) is a critical step in protein synthesis where the ribosome typically moves along the mRNA by three nucleotides at each step. To investigate the mechanism of EF-G catalysis, it is essential to precisely resolve the ribosome motion at both ends of the mRNA, which, to our best knowledge, is only achieved with the magnetic-based force spectroscopy developed by our groups. Here, we introduce a novel multiplexed force spectroscopy technique that, for the first time, offers single-nucleotide resolution for multiple samples. This technique combines multiple acoustic force generators with the smallest atomic magnetometer designed for biological research. Utilizing this technique, we demonstrate that mutating EF-G at the GTP binding pocket results in the ribosome moving only two nucleotides on both ends of the mRNA, thereby compromising ribosome translocation. This finding suggests a direct link between GTP hydrolysis and ribosome translocation. Our results not only provide mechanistic insights into the role of GTP binding pocket but also illuminate how allosteric mutations can manipulate translocation. We anticipate broader applications of our technique in the ribosome field, leveraging its high efficiency and single-nucleotide resolution.

P53 protein expression patterns associated with TP53 mutations in breast carcinoma

PurposeThe importance of a TP53 mutation has been demonstrated in several tumor types, including breast cancer (BC). However, the accuracy of p53 protein expression as a predictor of gene mutation has not been well studied in BC. Therefore, we evaluated p53 protein expression associated with TP53 mutations in breast cancers from 64 patients.MethodsTP53 mutation was examined using next-generation sequencing (NGS). p53 protein expression was examined using immunohistochemistry (IHC).ResultsAmong the 64 BCs, 55% demonstrated abnormal expression patterns including 27% overexpression, 22% null, 6% equivocal with 45% having a wild-type pattern. A TP53 mutation was present in 53% (34/64) of tumors including 30% (19/64) demonstrating a missense mutation, 11% (7/64) with a frameshift mutation, 11% (7/64) with a nonsense mutation, and 3% (1/64) with a splice site mutation. Abnormal expression of p53 protein was present in 33 of 34 (97%) tumors carrying a TP53 mutation; conversely, a wild-type pattern was present in 28 of 30 (93%) tumors without a detectable mutation (p < 0.0001). The majority of BCs with a p53 IHC overexpression pattern (15/17, 88%) contained a missense TP53 mutation; while the majority of BCs with a null pattern (12/14, 86%) contained a truncating mutation (p < 0.0001). The BCs with a null pattern are associated with a high Nottingham histological grade and a triple-negative phenotype when compared to those demonstrating overexpression (p < 0.05).ConclusionThese findings suggest that p53 IHC can be a potential surrogate for TP53 mutations in BC. Different p53 expression patterns may correlate with specific TP53 genetic mutations in BC.