Mutation Screening Research Articles

Analysis of low-density lipoprotein receptor gene mutations in a family with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common monogenic autosomal dominant disorder, primarily mainly caused by pathogenic mutations in the low-density lipoprotein receptor (LDLR) gene. Through phenotypic-genetic linkage analysis, two LDLR pathogenic mutations were identified in FH families: c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr). Whole exome sequencing was conducted on the proband with familial hypercholesterolemia to identify the target gene and screen for potential pathogenic mutations. The suspicious responsible mutation sites in 14 family members were analyzed using Sanger sequencing to assess genotype-phenotype correlations. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to evaluate LDLR mRNA and protein expression. In parallel, bioinformatics tools were employed to predict structural and functional changes in the mutant LDLR. Immunofluorescence analysis revealed no significant difference in the intracellular localization of the p.Gly343Ser mutation, whereas protein expression of the p.Ala627Thr mutation was decreased and predominantly localized in the cytoplasm. Western blotting has showed that protein expression levels of the mutant variants were markedly declined in both cell lysates and supernatants. Enzyme linked immunosorbent assay has demonstrated that LDLR protein levels in the supernatant of cell culture medium was not significant different from those of the wild-type group. However, LDLR protein levels in the cell lysate of both the Gly343Ser and Ala627Thr variants groups were significantly lower than those in the wild-type group. Bioinformatic predictions further suggested that these mutations may affect post-translational modifications of the protein, providing additional insight into the mechanisms underlying the observed reduction in protein expression. In this study, we identified two heterozygous pathogenic variants in the LDLR gene, c.G1027A (p.Gly343Ser) and c.G1879A (p.Ala627Thr), in a family with familial hypercholesterolemia. We also conducted preliminary investigations into the mechanisms by which these mutations contribute to disease pathology.

A Dual-Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase.

Industrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase-m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild-type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

A Dual‐Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase

AbstractIndustrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade‐off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate‐degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase‐m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild‐type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

Descriptive Analysis of Common Fusion Mutations in Papillary Thyroid Carcinoma in Hungary

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto’s disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto’s disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

Cost-saving approach with screening of selected variants in genetic diagnosis in Turkish pediatric familial Mediterranean fever patients: a single center longitudinal study.

The aim of this study was to investigate whether a short exon screening consisting of selected variants could confirm the diagnosis in patients with a preliminary diagnosis of familial Mediterranean fever (FMF), thus providing a cost-saving alternative to a comprehensive MEditerranean FeVer (MEFV) gene sequence analysis test. This observational study on pediatric patients focused on clinically suspected FMF cases without prior genetic analysis. Participants met the Turkish pediatric FMF criteria. They underwent short exon screening for M694V, M680I, V726A, and E148Q variants. Those who were heterozygous or negative on short exon screening received further MEFV gene sequence analysis. The study involved 1557 patients. Pathogenic variants in both alleles of the MEFV gene were found in 611 patients (39.2%), and a high-penetrance variant in heterozygosity or an E148Q variant on the other allele was found in 643 patients (41.3%). A further 189 patients (12.1%) had one or two E148Q variants. Short-exon screening was negative in 114 patients (7.6%). Of the 876 patients who underwent MEFV gene sequence analysis, additional variants were found in 72 of the 762 initially heterozygous patients. Of the 114 initially negative patients, 34 had homozygous or compound heterozygous variants, and 74 had heterozygous variants. Ultimately, only 6 patients yielded negative results in the MEFV gene sequence analysis. The short exon screening for common MEFV mutations offers a practical and cost-saving alternative to comprehensive MEFV gene sequence analysis in populations with a high prevalence of FMF.

RpoS activates formation of Salmonella Typhi biofilms and drives persistence in the gall bladder.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

5159 Bone Disease Related To PLS3 Gene Mutation

Abstract Disclosure: S. Saberi: None. N. Esfandiari: None. Background: A variety of genes play a role in the development of osteoporosis. One of these is PLS3, located on the X chromosome. PLS3 gene mutation leads to defective function of the protein Plastin 3. Plastin 3 functions in the binding and bundling of F-actin, a main component of the actin cytoskeleton. In the skeletal system the actin cytoskeleton regulates adaption to gravity and mechanical stress. Dysregulation of this gene is associated with diseases of the bone, including osteoporosis. Clinical Case: A 60-year-old man with a history of hearing loss since age 52, hypothyroidism diagnosed at age 57 presents to the Genetics Clinic after 2 grandsons were diagnosed with osteogenesis imperfecta. The mothers (his two daughters) of the two grandsons were found to be carriers of the PLS3 gene mutation. Because of their carrier status the patient was screened and a pathogenic partial gene deletion of exons 8 to 16 in the PLS3 gene was detected. The patient has a history of a right wrist fracture at age 16 in a fall from 8 feet height from a ladder, right leg fracture at age 45 when his leg got caught and twisted in a ladder rung, 5 rib fractures and shoulder fracture at age 53 in a fall from 3 feet into a utility hole. A DXA scan showed osteopenia in his left femoral neck (T-score -1.3), normal bone density in his total hip (T score -0.1) and lumbar spine (L1-L4 T-score -0.4). FRAX score for major osteoporotic fracture was 9.4% and hip fracture was 1.2%. Labs for secondary causes of osteoporosis were normal. Due to his gene mutation and fracture history a bisphosphonate was recommended.Clinical Lessons:Obtaining a thorough family history in people with fractures can determine the need for screening for gene mutations associated with osteoporosis. This can identify patients who need more aggressive management. Presentation: 6/2/2024

8042 A Rare Case of Papillary Thyroid Carcinoma in a Patient with Li-Fraumeni Syndrome

Abstract Disclosure: K.M. Bakhtawar: None. G. Jaiswal: None. Li-Fraumeni syndrome (LFS) is an autosomal dominant syndrome with a high lifetime probability of cancer at an early age. The spectrum of cancers primarily includes osteosarcoma, soft tissue sarcoma, breast cancer, brain tumors, leukemia, and Adenoid cystic carcinoma. Prior studies have estimated the cumulative cancer risk to be about 50% by age 40 and as high as 90% by age 60. As screening for TP53 mutations has improved and more patients are identified, the LFS cancer spectrum has expanded to include thyroid cancer. However, the occurrence of thyroid cancer in LFS has not been extensively assessed in the literature. A recent study examined cancer patients carrying the Brazilian p.R337H TP53 mutation which demonstrated that out of 101 patients, 11 had thyroid cancer (10.9%). In our case, a 45-year-old female with a past medical history of Li-Fraumeni syndrome, Von Willebrand disease, Pulmonary embolism, HER-2 positive invasive ductal carcinoma of the left breast status post chemotherapy, and bilateral mastectomy presented for evaluation of papillary thyroid carcinoma (PTC). An enlarged thyroid was initially identified on physical exam by the PCP with a follow-up thyroid ultrasound showing bilateral nodules. The right upper pole 1.9 x 1.3 x 1.6 cm nodule was biopsied revealing PCT. Following neck ultrasound demonstrated mildly enlarged lymph nodes on the right side, level 2A 1.5 cm and level 3 1.6 cm. The patient subsequently underwent total thyroidectomy and lymph node dissection. The pathology report revealed a 1.4 cm PTC in the upper right pole, a 0.9 cm PCT in the isthmus, and a 0.8 cm PTC in the left midpole. 4/62 lymph nodes were positive with the largest deposit of 1 cm. All margins were negative for carcinoma with no angioinvasion or lymphatic invasion. She was staged as T1bN1bM0. We are awaiting post-operative Thyroglobulin level to assess the need for Radioactive iodine. BRAFV600E and P53 mutation was identified. The patient carries known heterozygous mutation p.R1755H in the TP53 gene with an estimated lifetime cancer risk of 93%. This particular mutation has not been associated with thyroid cancer in prior studies. Although thyroid cancer is an uncommon manifestation of LFS, this case begs the importance of further studies to explore this association which may then lead to early screening and treatment of thyroid cancer in individuals with founder TP53 mutations. Presentation: 6/1/2024

7831 Unveiling Drug Resistance Mechanisms In Triple-Positive Breast Cancer: Insights From Long-Term Estrogen Deprivation Models And Innovative Mutation Detection Strategies

Abstract Disclosure: S. Bahnassy: None. H.S. Andrews: None. L. Jin: None. B.F. Kohrn: None. S. Tam: None. D. Mobin: None. Y.J. Dunn: None. M. Balachandran: None. M. Podar: None. W. He: None. M.D. McCoy: None. R.A. Beckman: Consulting Fee; Self; AstraZeneca, Boehringer Ingelheim. Other; Self; Chief Scientific Officer of OncoMind, LLC, which owns patents on dynamic precision medicine. R.B. Riggins: None. Compelling evidence from preclinical and clinical data suggests that triple-positive (TP; HER2+/HR+) breast cancer (BC) has lower response rates to HER2-targeted therapy and a higher risk of recurrence to endocrine therapy (ET) than HER2+/HR−. Molecular mechanisms underpinning drug resistance in TP-BC are poorly understood, thereby limiting our ability to improve patient outcomes and prevent metastatic progression. In our recent publication, we established and characterized long-term estrogen deprivation (LTED) ET-resistant (ETR) TP-BC models that mimic resistance to aromatase inhibitors without using genetic manipulation approaches. We reported intrinsic subtype shifts towards non-luminal phenotypes, metabolic reprogramming, and acquiring mutations in genes encoding for transcription factors and chromatin modifiers, as potential mechanisms of ETR in our LTED models. The emergence of drug-resistant mutator subclones under treatment-induced selective pressures, coupled with limitations in shallow-depth whole genome sequencing coverage, highlight an unmet need to develop a strategy for visually monitoring, capturing, and mapping resistant subclones through a mutational phenotypic screen, an approach offering valuable insights for optimizing treatment schedules. To address this, we developed and applied an innovative dual fluorescence mutation detection system to detect mutator cells expressing both mCherry and green fluorescence protein (GFP). First, we validated if our system is working properly. Compared to parental cells, transduced BT-474 and MDA-MB-361 LTEDs showed a drastic increase in GFP+ cells, suggesting the presence of resistant mutator cells. Consistent with the increase in GFP signal, TP-LTEDs showed increased mutation number and frequency in two Duplex Sequencing targeted capture panels (neutral loci and DNA polymerase-focused), used to perform ultra-deep, highly accurate sequencing. We then treated parental cells with single, dual, and triple combinations of anti-HER2, ET and CDK4/6 inhibitors to track the emergence of GFP+ mutator cells. Nine-day treatments with triple combinatorial therapies were best at inhibiting the growth of both BT-474 and MDA-MB-361 parental cell lines, compared to single- and double-agent targeted therapies. However, no GFP signal was detected within treated cells, suggesting that longer treatment durations are needed for drug-resistance mutator subclones to be discovered. Ongoing studies monitoring TP-drug resistance mutator subclones with long-term conventional targeted-therapies will improve our understanding of drug resistance mechanisms in this understudied subtype of BC. Importantly, these studies will enable dynamic precision medicine simulations to empower for a more tailored and effective therapeutic administration to prevent drug resistance and metastatic progression of TP-BC. Presentation: 6/3/2024

7831 Unveiling Drug Resistance Mechanisms in Triple-Positive Breast Cancer: Insights from Long-Term Estrogen Deprivation Models and Innovative Mutation Detection Strategies

Abstract Disclosure: S. Bahnassy: None. H.S. Andrews: None. L. Jin: None. B.F. Kohrn: None. S. Tam: None. D. Mobin: None. Y.J. Dunn: None. M. Balachandran: None. M. Podar: None. W. He: None. M.D. McCoy: None. R.A. Beckman: Consulting Fee; Self; AstraZeneca, Boehringer Ingelheim. Other; Self; Chief Scientific Officer of OncoMind, LLC, which owns patents on dynamic precision medicine. R.B. Riggins: None. Compelling evidence from preclinical and clinical data suggests that triple-positive (TP; HER2+/HR+) breast cancer (BC) has lower response rates to HER2-targeted therapy and a higher risk of recurrence to endocrine therapy (ET) than HER2+/HR−. Molecular mechanisms underpinning drug resistance in TP-BC are poorly understood, thereby limiting our ability to improve patient outcomes and prevent metastatic progression. In our recent publication, we established and characterized long-term estrogen deprivation (LTED) ET-resistant (ETR) TP-BC models that mimic resistance to aromatase inhibitors without using genetic manipulation approaches. We reported intrinsic subtype shifts towards non-luminal phenotypes, metabolic reprogramming, and acquiring mutations in genes encoding for transcription factors and chromatin modifiers, as potential mechanisms of ETR in our LTED models. The emergence of drug-resistant mutator subclones under treatment-induced selective pressures, coupled with limitations in shallow-depth whole genome sequencing coverage, highlight an unmet need to develop a strategy for visually monitoring, capturing, and mapping resistant subclones through a mutational phenotypic screen, an approach offering valuable insights for optimizing treatment schedules. To address this, we developed and applied an innovative dual fluorescence mutation detection system to detect mutator cells expressing both mCherry and green fluorescence protein (GFP). First, we validated if our system is working properly. Compared to parental cells, transduced BT-474 and MDA-MB-361 LTEDs showed a drastic increase in GFP+ cells, suggesting the presence of resistant mutator cells. Consistent with the increase in GFP signal, TP-LTEDs showed increased mutation number and frequency in two Duplex Sequencing targeted capture panels (neutral loci and DNA polymerase-focused), used to perform ultra-deep, highly accurate sequencing. We then treated parental cells with single, dual, and triple combinations of anti-HER2, ET and CDK4/6 inhibitors to track the emergence of GFP+ mutator cells. Nine-day treatments with triple combinatorial therapies were best at inhibiting the growth of both BT-474 and MDA-MB-361 parental cell lines, compared to single- and double-agent targeted therapies. However, no GFP signal was detected within treated cells, suggesting that longer treatment durations are needed for drug-resistance mutator subclones to be discovered. Ongoing studies monitoring TP-drug resistance mutator subclones with long-term conventional targeted-therapies will improve our understanding of drug resistance mechanisms in this understudied subtype of BC. Importantly, these studies will enable dynamic precision medicine simulations to empower for a more tailored and effective therapeutic administration to prevent drug resistance and metastatic progression of TP-BC. Presentation: 6/3/2024

The Long-Term Cure of Patients With Hereditary Medullary Thyroid Carcinoma: 40 Years of Follow-Up in a Single Center.

The cure rate of patients with hereditary medullary thyroid carcionoma (MTC) can be decisively improved by screening for elevated calcitonin (Ctn) levels and RET gene mutations in patients from families affected by multiple endocrine neoplasia type 2 (MEN2), followed by prophylactic thyroidectomy in persons with mutated RET genes. In this long-term observational study, we investigated whether postoperative cures are indeed maintained decades after the procedure. From 1979 to 2021, 277 patients with MEN2 who underwent thyroidectomy were observed postoperatively for 14.4 ± 10.3 years (mean, standard deviation). They were classified as either cured or not cured depending on the last measured serum Ctn level (cured, Ctn < 10 pg/mL or < 2 pg/mL; not cured, Ctn ≥ 10 pg/mL). Depending on their RET mutation status, they were categorized as moderate, high, or highest risk (121, 130, and 26 patients, respectively). 154 patients (55.6%) obtained a long-term cure (Ctn <10 pg/mL). The median age at surgery was 27, 14, and 4 years in patients at moderate, high, and highest risk. All 52 patients who had undergone prophylactic thyroidectomy before the age of 6 years, 9 years, or 6 months had a Ctn level below 2 pg/mL and were cured at the end of the follow-up period. In a multivariable analysis, prognostic factors for a long-term cure were a lower tumor stage and, by tendency, classification as belonging to the moderate as opposed to the highest-risk group. In patients receiving an early diagnosis of MEN2 via family screening, prophylactic thyroidectomy taking into account the RET mutation risk group can achieve a long-term cure of MTC with undetectable serum Ctn levels.

Catching the Silent Culprits: TERT Promoter Mutation Screening of Minimally Invasive Follicular and Oncocytic Thyroid Carcinoma in Clinical Practice.

De-escalation of thyroid cancer treatment is crucial to prevent overtreatment of indolent disease, but it remains important to identify clinically aggressive cases. TERT promoter mutations are molecular events frequently associated with high-risk thyroid tumors with poor outcomes and may identify cases at risk of dissemination. In various international guidelines, small minimally invasive follicular thyroid carcinoma and oncocytic thyroid carcinoma (miFTC/miOTC) are classified as low-risk lesions and are not recommended adjuvant treatment. Our study aimed to explore the association between size-based risk assessment and TERT promoter mutations. Between 2019 and May 2024, 84 miFTCs/miOTCs diagnosed at our department underwent digital droplet PCR analysis targeting TERT promoter mutational hotspots C228T and C250T in clinical routine. TERT promoter mutations were found in 10 out of 84 cases (11.9%). Mutated cases were pT1 (n = 1), pT2 (n = 3), or pT3 (n = 6). Patients with mutated tumors were older compared to patients with wild-type tumors (median age of 71years vs. 57years, p = 0.041). There were no significant differences regarding patient sex, tumor size, Ki-67 labeling index, or the presence of distant metastases. Notably, 30% of mutations displayed variant allele frequencies < 10%, possibly suggesting subclonal events. To conclude, TERT promoter mutations in miFTCs and miOTCs were associated with higher patient age and were often suspected to be subclonal. However, they did not affect clinical outcomes, possibly due to short follow-up. Reflex testing for this genetic alteration in miFTCs and miOTCs could be justified regardless of tumor size, though the clinical benefit remains uncertain.

56P Mutational screening of distal myopathy gene ACTN2 reveals an aggregation hot-spot in the actin-binding domain

56P Mutational screening of distal myopathy gene ACTN2 reveals an aggregation hot-spot in the actin-binding domain

Molecular Study of the Fukutin-Related Protein (FKRP) Gene in Patients from Southern Italy with Duchenne/Becker-like Phenotype.

Pathogenic variants localized in the gene coding for the Fukutin-Related Protein (FKRP) are responsible for Limb-Girdle Muscular Dystrophy type 9 (LGMDR9), Congenital Muscular Dystrophies type 1C (MDC1C), Walker-Warburg Syndrome (WWS), and Muscle-Eye-Brain diseases (MEBs). LGMDR9 is the fourth most common hereditary Limb Girdle Muscular Dystrophy in Italy. LGMDR9 patients with severe disease show an overlapping Duchenne/Becker phenotype and may have secondary dystrophin reduction on muscle biopsy. We conducted a molecular analysis of the FKRP gene by direct sequencing in 153 patients from Southern Italy (Calabria) with Duchenne/Becker-like phenotypes without confirmed genetic diagnosis. Mutational screening of the patients (112 men and 41 women, aged between 5 and 84 years), revealed pathogenic variants in 16 subjects. The most frequent variants identified were c.427C > A, p.R143S, and c.826C > A, p.L276I (NM_024301.5). The results obtained show that the Duchenne/Becker-like phenotype is frequently determined by mutations in the FKRP gene in our cohort and highlight the importance of considering LGMDR9 in the differential diagnosis of dystrophinopathies in Calabria. Finally, this study, which, to our knowledge, is the first conducted on Calabrian subjects, will contribute to the rapid identification and management of LGMDR9 patients.

A capillary electrophoresis-based assay for carrier screening of the hotspot mutations in the CYP21A2 gene

Molecular genetic analysis of the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene is challenging owing to the highly homologous with its pseudogene. A reliable approach for the large-scale population screening of CYP21A2 is required. This study aimed to establish and evaluate a capillary electrophoresis-based assay for hotspot mutation carrier screening of the CYP21A2 gene. A total of 22 different variants in the CYP21A2 gene were detected by a capillary electrophoresis-based assay consisting of single nucleotide primer extension (SNaPshot) and high-throughput ligation-dependent probe amplification (HLPA) in the Chinese population, and the results were validated by alternative methods. Among the 5376 subjects, 1.51 % (81/5376) individuals were identified as CYP21A2 pathogenic variant carriers, with a carrier rate of 1/66. A total of 11 different variants were identified, of which c.293-13A/C > G (33.33 %) was the most common variant, followed by c.844C > T (19.75 %), c.518T > A (19.75 %), and Del/Con (16.05 %). There was a 100 % concordance between capillary electrophoresis and alternative method results. Furthermore, a total of 63 individuals (1.17 %, 63/5376) carried the c.955C > T (p. Q319∗) variant, among which 61 (61/63, 96.83 %) had a duplicated CYP21A2 gene and are therefore not carriers of a CYP21A2 allele. In conclusion, the capillary electrophoresis-based assay is an accurate and effective approach for genotyping the CYP21A2 gene and has the potential for the large-scale population screening of CYP21A2.

Alzheimer's Disease Immunotherapy and Mimetic Peptide Design for Drug Development: Mutation Screening, Molecular Dynamics, and a Quantum Biochemistry Approach Focusing on Aducanumab::Aβ2-7 Binding Affinity.

Seven treatments are approved for Alzheimer's disease, but five of them only relieve symptoms and do not alter the course of the disease. Aducanumab (Adu) and lecanemab are novel disease-modifying antiamyloid-β (Aβ) human monoclonal antibodies that specifically target the pathophysiology of Alzheimer's disease (AD) and were recently approved for its treatment. However, their administration is associated with serious side effects, and their use is limited to early stages of the disease. Therefore, drug discovery remains of great importance in AD research. To gain new insights into the development of novel drugs for Alzheimer's disease, a combination of techniques was employed, including mutation screening, molecular dynamics, and quantum biochemistry. These were used to outline the interfacial interactions of the Aducanumab::Aβ2-7 complex. Our analysis identified critical stabilizing contacts, revealing up to 40% variation in the affinity of the Adu chains for Aβ2-7 depending on the conformation outlined. Remarkably, two complementarity determining regions (CDRs) of the Adu heavy chain (HCDR3 and HCDR2) and one CDR of the Adu light chain (LCDR3) accounted for approximately 77% of the affinity of Adu for Aβ2-7, confirming their critical role in epitope recognition. A single mutation, originally reported to have the potential to increase the affinity of Adu for Aβ2-7, was shown to decrease its structural stability without increasing the overall binding affinity. Mimetic peptides that have the potential to inhibit Aβ aggregation were designed by using computational outcomes. Our results support the use of these peptides as promising drugs with great potential as inhibitors of Aβ aggregation.

Rapid and direct detection of m6A methylation by DNAzyme-based and smartphone-assisted electrochemical biosensor

m6A methylation detection is crucial for understanding RNA functions, revealing disease mechanisms, guiding drug development and advancing epigenetics research. Nevertheless, high-throughput sequencing and liquid chromatography-based traditional methods still face challenges to rapid and direct detection of m6A methylation. Here we report a DNAzyme-based and smartphone-assisted electrochemical biosensor for rapid detection of m6A. We initially identified m6A methylation-sensitive DNAzyme mutants through site mutation screening. These mutants were then combined with tetrahedral DNA to modify the electrodes, creating a 3D sensing interface. The detection of m6A was accomplished by using DNAzyme to capture and cleave the m6A sequence. The electrochemical biosensor detected the m6A sequence at nanomolar concentrations with a low detection limit of 0.69 nM and a wide detection range from 10 to 104 nM within 60 min. As a proof of concept, the 3′-UTR sequence of rice was selected as the m6A analyte. Combined with a smartphone, our biosensor shows good specificity, sensitivity, and easy-to-perform features, which indicates great prospects in the field of RNA modification detection and epigenetic analysis.

Muscular dystrophy associated with the DMD gene in women

Dystrophinopathies are a spectrum of X-linked muscular disorders associated with pathogenic/likely pathogenic variants in the dystrophin gene (DMD). Typically, the condition develops in males, but cases of symptom manifestation have also been described in females. The review presents contemporary data on the manifestations of dystrophinopathies in women with pathogenic variants in the DMD gene, discussing the reasons for the varying degrees of symptom expression in carrier women with pathogenic/ likely pathogenic variants. It discusses the importance of mutation screening in the DMD gene for women presenting with muscular dystrophy symptoms and investigating carrier status in relatives of patients with Duchenne/Becker muscular dystrophy.

Corneal microstructural changes of precise CHST6 gene mutation: a case series

BackgroundMacular corneal dystrophy (MCD) is an inherited, autosomal recessive disorder of defective keratan sulfate (KS) metabolism. It is caused by the mutations in carbohydrate sulfotransferase 6 gene (CHST6) which is essential for the sulfation of KS. Unlike the western world, MCD is the most common corneal stromal dystrophy in India, especially in south Indian population; it could be due to high frequency of consanguineous marriages.Case presentationThis study presents the clinical findings of one North Indian MCD family, including 6 patients and 3 unaffected relatives. We used slit lamp examination and in vivo confocal microscopy for assessment. Mutation screening was performed with Sanger sequencing, and corneal structure was analyzed through histochemistry and immunohistochemistry. Our comparative findings revealed that all the patients identified with the deletion of major portion of CHST6 that included the Open Reading Frame (ORF). Although all the patients showed significantly reduced central corneal thickness (CCT-250 μm), a drastic decrease in stromal keratocyte count, and depletion of Bowman’s layer compared to controls.ConclusionsThis study first time revealed that MCD patients from one family with a deletion of major portion of CHST6 that included ORF leads to severe corneal morphological changes.

Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.