Pathogenic Mutations Research Articles

Nanomechanical collective vibration of SARS-CoV-2 spike proteins.

The development of effective therapeutics against COVID-19 requires a thorough understanding of the receptor recognition mechanism of the SARS-CoV-2 spike (S) protein. Here the multidomain collective dynamics on the trimer of the spike protein has been analyzed using normal mode analysis (NMA). A common nanomechanical profile was identified in the spike proteins of SARS-CoV-2 and its variants. The profile involves collective vibrations of the receptor-binding domain (RBD) and the N-terminal domain (NTD), which may mediate the physical interaction process. Quantitative analysis of the collective modes suggests a nanomechanical property involving large-scale conformational changes, which explains the difference in receptor binding affinity among different variants. These results support the use of intrinsic global dynamics as a valuable perspective for studying the allosteric and functional mechanisms of the S protein. This approach also provides a low-cost theoretical toolkit for screening potential pathogenic mutations and drug targets.

Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases

Cell-based exvivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines exvivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

Clinical, Hormonal, and Genetic Spectrum of 46 XY Disorders of Sexual Development (DSD) Patients.

To evaluate the clinical,hormonaland genetic characteristics of 46XY disorders of sexual development (DSD) patients from South India. 46XY DSD patients with a provisional diagnosis of 17β-hydroxysteroid dehydrogenase 3 (17BHSD3) deficiency, 5 alpha-reductase type 2 deficiency (5ARD2) or partial androgen insensitivity syndrome (PAIS) based on clinical and hormonal analysis were included in this study. All the patients underwent detailed clinical and hormonal evaluations. Targeted next-generation sequencing for all three genes (AR, HSD17B3, and SRD5A2) in parallel was carried out for all the included patients and their parents. Based upon the clinical and hormonal analysis, among the 37 children with 46XY DSD in the present study, 21 children were diagnosed with 5ARD2, 10 with PAIS, and six with 17BHSD3 deficiency. However, genetic analysis revealed pathogenic mutations in nine patients - six in the AR gene, two in the SRD5A2 gene, and one in the HSD17B3 gene. The concordance rate between provisional hormonal and genetic diagnosis was only 22.2%. Two out of six subjects with AR gene variants were positive for somatic mosaicism. In the present study, a positive genetic diagnosis was detected in nine patients (24%), including five novel variants. In this study, mutations in the AR gene was the most reported. The authors did not find the testosterone: dihydrotestosterone (T: DHT) ratio to be an accurate hormonal diagnostic tool.

O18 Precision genome editing for targeted correction of pathogenic D50N mutation in keratitis–ichthyosis–deafness syndrome using CRISPR/Cas9 and homology-directed repair

Abstract Introduction and aims Keratitis–ichthyosis–deafness (KID) syndrome is an ectodermal disorder that causes blindness, skin inflammation and deafness. It is caused by autosomal dominant mutations in the gap junction beta 2 (GJB2) gene with 86% being a hotspot mutation on c.148G>A, resulting in the amino acid replacement (D50N) in its coding protein connexin 26 (Cx26). There is currently no curative treatment for KID syndrome. We aim to correct the hotspot mutation D50N using a genome-editing approach. Methods Keratinocytes generated from the patient with KID who had a D50N mutation (KID-KCs) were genetically corrected by homologous-directed repair/CRISPR-Cas9 using an electroporation approach. The correction efficiency was determined and analysed using Sanger sequencing and Synthego Inference of CRISPR Edits analysis. The off-target effects were confirmed by whole-exome sequencing [exome next-generation sequencing (NGS)]. Furthermore, the functional recovery following genome editing was assessed by GJB2 mRNA production, Cx26 protein expression, and trafficking and hemichannel activity. Results The correction efficiencies ranged from 95% to 100% with different doses of CRISPR-Cas9 and 8µg CRISPR-Cas9 was the most optimal concentration with the efficiency of 100% and approximately 0% insertion-deletion (INDEL). Fifty days after gene editing, the INDEL increased to only 2%, indicating a minimum residual CRISPR-Cas9 effect in edited cells. The exome-NGS analysis also showed no off-target effects in the outside target area. Functional studies showed that there was a significant increase in wildtype GJB2 mRNA expression and a reduction of mutant GJB2 expression (n = 5, P < 0.01). Increased Cx26 membranous localization in KID-KCs was also observed (n = 3, P < 0.01). Additionally, Cx26 hemichannel activity assessment using the neurobiotin assay showed a significant decrease in ‘leaky’ hemichannel in gene-edited KID-KCs compared with unedited cells (n = 3, P < 0.05). Conclusions We can genome-correct KID keratinocytes harbouring the mutation D50N with 100% editing efficiency, 0–2% INDEL, and undetectable off-target effects. This result indicates a promising gene therapy for KID syndrome with the hotspot mutation D50N.

P09 Next-generation sequencing: on-and off-target analysis of gene editing in fibroblasts from patients with recessive dystrophic epidermolysis bullosa

Abstract Introduction and aims Base editors (BEs) and prime editors (PEs) have proved to have advantages over traditional clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in correcting pathogenic mutations. We previously demonstrated 94.6% efficiency using adenine base editor (ABE)8e in correcting nonsense mutation (c.5047 C>T) in fibroblasts, which causes recessive dystrophic epidermolysis bullosa (RDEB). However, safety profile and cell-type-specific efficiencies of gene editing techniques have not been characterized. The aim of our research is to establish corrections of c.5047 C>T pathogenic variant in fibroblasts and compare off-target effects and efficiency of CRISPR-Cas9, BE and PE systems. Methods Primary fibroblasts harbouring heterozygous C>T pathogenic variant on exon 54 will be transfected with CRISPR-Cas9, ABE8e and PE using electroporation or lipofectamine. Both Sanger sequencing and next-generation sequencing (NGS) will be conducted to confirm pathogenic variant corrections. Galaxy platform which incorporates different NGS analysis tools will be used to analyse the NGS data. Subsequently, whole-exome sequencing and targeted amplicon sequencing will be used for off-target analysis. Results On-target efficiency for ABE8e to correct c.5047 C>T pathogenic variant in patient primary fibroblasts was determined first by Sanger sequencing showing 100% editing in contrast to 84% determined by NGS. Moving forward, we plan to correct the same pathogenic variant with PEs. Initially experiments are under way with PEmax mRNA and engineered PE guide RNAs (epegRNA) to compare different PEs. Furthermore, we will investigate the off-target effects of CRISPR-Cas9, ABE8e and PEs to edit RDEB primary fibroblasts using NGS Methods and conduct functional studies. Conclusions Here, we showed that the c.5047 C>T pathogenic variant in RDEB fibroblasts could be successfully edited using ABE8e. We demonstrated that NGS is required to precisely determine the editing efficacy. Taken together, we have established a pipeline for base editing in RDEB and we aim to achieve the same for prime editing in the future.

Clinical and Genetic Characteristics and Outcome in Patients with Neonatal Diabetes Mellitus from a Low Middle-Income Country.

Neonatal Diabetes Mellitus (NDM) is a disorder characterized by persistent, severe hyperglycemia presenting during the first 6 months of life. These disorders are rare and the incidence is approximately 1 in 90,000 live births. To describe the clinical presentation, molecular genetics and outcome of patients with NDM from a single paediatric endocrine center from a low middle income country. A retrospective study was conducted on patients diagnosed with NDM. Medical records were reviewed for demographic data and data on clinical, biochemical and genetic analysis. 96% of patients who underwent mutation analysis had pathogenic genetic mutations on Sanger sequencing. Permanent NDM (PNDM) was diagnosed in 19 patients with 3 of them having a syndromic diagnosis. The commonest mutation was found in KCNJ11 gene. Majority of the PNDM (63%) presented with severe diabetic ketoacidosis. All patients with Transient NDM (TNDM) remitted by 6 months of age. 47% of the cases with PNDM made a switch to sulfonylurea therapy with good glycemic control (glycosylated Haemoglobin A1C 6-7.5). Data from the Sri Lankan cohort is comparable with other populations. The majority of cases are due to KCNJ11 mutations resulting in PNDM.

CARDIAC AMYLOIDOSIS IN ELDERLY WITH POSITIVE SCINTIGRAPHY AND MONOCLONALITY: A DIAGNOSTIC DILEMMA

Abstract Background and Aims Clinical identification of elderly patients with both wild–type transthyretin amyloidosis (ATTR) and monoclonal gammopathy of unknown significance (MGUS) is increasing. For this reason, accurate amyloid histologic subtyping is crucial in case of concomitant positivity of bisphosphonate scintigraphy and monoclonal protein testing. We report the diagnostic work–up of cardiac amyloidosis in this specific patient subtype in our tertiary referral center. Methods Among 133 patients who received the diagnosis of cardiac amyloidosis from March 2021 to March 2023, 21 (16%) presented with an abnormality of both bisphosphonate scintigraphy and at least one of the serum monoclonal protein tests. Patients underwent abdominal fat biopsy/needle aspiration as the first diagnostic procedure. In case of negative results, an endomyocardial biopsy (EMB) was planned. Amyloid was identified at histology by Congo Red staining. Amyloid typing was obtained by immunohistochemistry using a recently developed amyloid antibody panel AmYkit (Amimed) applicable on fixed paraffin–embedded tissues in an automated platform. The results were compared with those obtained with standard immunohistochemistry. Results Sixteen of the 21 patients were older than 75 years (76%, 81±10 ys) with a grade 3 (62%) or 2 (33%) scintigraphy. A grade 1 positive scintigraphy was detected in one patient. Serum immunofixation revealed a previously unknown monoclonal protein peak in 78%, while 22% had a history of MGUS. None of the patients had hematologic disorders. TTR pathogenic mutation was detected in one patient. On pathologic examination, abdominal fat was negative for amyloid in all cases, while Congo Red staining was positive in all examined EMBs. Immunohistochemistry performed with standard antibodies was not able to discriminate between AL and ATTR, due to signal overlapping of variable intensity. Conversely, our preliminary results with AmYkit panel of antibodies demonstrate absence of signal overlapping in over 90% of cases, allowing accurate histologic discrimination between AL and TTR. Conclusion Based on our clinical and histologic observations, in elderly patients with concomitant positivity of both bisphosphonate scintigraphy and monoclonal protein testing, TTR is the most frequent type of cardiac amyloid. EMB is a useful diagnostic tool to confirm the clinical suspicion of amyloidosis. Accurate amyloid subtyping is granted by a more sensitive panel of antibodies.

Cascade genetic testing: an underutilized pathway to equitable cancer care?

The Precision Medicine Initiative was launched upon the potential of genomic information to tailor medical care. Cascade genetic testing represents a powerful application of precision medicine and involves the process of familial diffusion or the "cascade" of genomic risk information. When an individual (proband) is found to carry a cancer-associated germline pathogenic mutation, the information should be cascaded or shared with at-risk relatives. First degree relatives have a 50% likelihood of carrying the same cancer-associated mutation. This process of cascade testing offers at-risk relatives the opportunity for genetic testing and, for those who also carry the cancer-associated mutation, genetically targeted primary disease prevention through intensive cancer surveillance, chemoprevention and risk-reducing surgery, reducing morbidity and preventing mortality. Cascade testing has been designated by the Centers for Disease Control and Prevention as a Tier 1 genomic application for hereditary breast and ovarian cancer. In this manuscript we describe a cascade genetic testing and in particular focus on its potential to provide necessary care to medically underserved and vulnerable populations.

Correlation of serum DKK1 level with skeletal phenotype in children with osteogenesis imperfecta

PurposeWe aim to detect serum DKK1 level of pediatric patients with OI and to analyze its relationship with the genotype and phenotype of OI patients.MethodsA cohort of pediatric OI patients and age-matched healthy children were enrolled. Serum levels of DKK1 and bone turnover biomarkers were measured by enzyme-linked immunosorbent assay. Bone mineral density (BMD) was measured by Dual-energy X-ray absorptiometry. Pathogenic mutations of OI were detected by next-generation sequencing and confirmed by Sanger sequencing.ResultsA total of 62 OI children with mean age of 9.50 (4.86, 12.00) years and 29 healthy children were included in this study. The serum DKK1 concentration in OI children was significantly higher than that in healthy children [5.20 (4.54, 6.32) and 4.08 (3.59, 4.92) ng/mL, P < 0.001]. The serum DKK1 concentration in OI children was negatively correlated with height (r = − 0.282), height Z score (r = − 0.292), ALP concentration (r = − 0.304), lumbar BMD (r = − 0.276), BMD Z score of the lumbar spine and femoral neck (r = − 0.32; r = − 0.27) (all P < 0.05). No significant difference in serum DKK1 concentration was found between OI patients with and without vertebral compression fractures. In patients with spinal deformity (22/62), serum DKK1 concentration was positively correlated with SDI (r = 0.480, P < 0.05). No significant correlation was observed between serum DKK1 concentration and the annual incidence of peripheral fractures, genotype and types of collagen changes in OI children.ConclusionThe serum DKK1 level was not only significantly elevated in OI children, but also closely correlated to their skeletal phenotype, suggesting that DKK1 may become a new biomarker and a potential therapeutic target of OI.

A whole-exome sequencing study of patent foramen ovale: investigating genetic variants and their association with cardiovascular disorders.

Patent foramen ovale (PFO) has a genetic predisposition and is closely associated with cryptogenic stroke (CS), migraine, decompression sickness, and hypoxemia. Identifying PFO-related mutant genes through whole-exome sequencing (WES) can help in the early recognition of cardiovascular genetic risk factors, guide timely clinical intervention, and reduce the occurrence of cardiovascular events. We analyzed mutant genes from ClinVar and OMIM databases. WES was performed on 25 PFO patients from Zhejiang Provincial Hospital of Chinese Medicine. Pathogenicity of variants was evaluated using American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology. (AMP) guidelines. In ClinVar (4 Feb 2023), 113 coding gene mutations were found, including 83 associated with PFO. From OMIM (18 Apr 2023), 184 gene mutations were analyzed, with 110 mutant coding genes. WES identified pathogenic mutations in two of 25 PFO patients (8%). LDLR, SDHC, and NKX2-5 genes were linked to PFO and primarily involved in myocardial tissue function. NKX2-5 may play a crucial role in PFO development, interacting with NOTCH1, GATA4, MYH6, SCN5A signaling pathways regulating cardiomyocyte characteristics. We identified pathogenic mutations in LDLR, SDHC, and NKX2-5 genes, implying their role in PFO development. Functional enrichment analysis revealed NKX2-5's interaction with signaling pathways regulating cardiomyocyte function. These findings enhance our understanding of PFO's genetic basis, suggesting potential therapeutic targets for future research.

Detection of genetic mutations underlying early-onset systemic lupus erythematosus.

We aimed to investigate the presence of monogenic causes of systemic lupus erythematosus (SLE) in our early-onset SLE patients. Fifteen pediatric SLE cases who had early disease onset (≤6 years) were enrolled in this study. All patients fulfilled the Systemic Lupus International Collaborating Clinics (SLICC) criteria. Genomic DNA was used for whole exome sequencing (WES). Pathogenic variants were confirmed by Sanger sequencing. The median age at diagnosis of 15 early-onset SLE patients included in the study was 4 (2-6) years (F/M = 12/3). Significant gene mutations were detected in five of these patients (33.3%). Patients 1 and 2 with homozygous DNASE1L3 mutations [c.320+4_320+7del and G188A (c.563G>C) variants] had skin involvement and oral ulcers. One of them (patient 1) had arthritis and nephritis, and another (patient 2) had nonscarring alopecia and thrombocytopenia. They are currently clinically inactive but have positive serological findings. Patient 3 with homozygous pathogenic ACP5 mutation [G109R (c.325G>A) variant] had arthritis, nephritis, short stature, and skeletal dysplasia. Patient 4 with a heterozygote novel IFIH1 mutation [L809F (c.2425C>T) variant] had skin findings and leukopenia. Patient 5 with novel C1S variant [homozygous C147W (c.441C>G) variant] had marked skin findings, oral ulcers, nonscarring alopecia, pancytopenia, and low total hemolytic complement CH50 level. All patients have responded to the treatments and have low Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores, on therapy. Genetic causes should be investigated in early-onset SLE, for better management and genetic counseling. On the other hand, multicenter studies may help to further define genotype-phenotype associations.

Assessing the Impact of Novel BRCA1 Exon 11 Variants on Pre-mRNA Splicing.

Our study focused on assessing the effects of three newly identified BRCA1 exon 11 variants (c.1019T>C, c.2363T>G, and c.3192T>C) on breast cancer susceptibility. Using computational predictions and experimental splicing assays, we evaluated their potential as pathogenic mutations. Our in silico analyses suggested that the c.2363T>G and c.3192T>C variants could impact both splicing and protein function, resulting in the V340A and V788G mutations, respectively. We further examined their splicing effects using minigene assays in MCF7 and SKBR3 breast cancer cell lines. Interestingly, we found that the c.2363T>G variant significantly altered splicing patterns in MCF7 cells but not in SKBR3 cells. This finding suggests a potential influence of cellular context on the variant's effects. While attempts to correlate in silico predictions with RNA binding factors were inconclusive, this observation underscores the complexity of splicing regulation. Splicing is governed by various factors, including cellular contexts and protein interactions, making it challenging to predict outcomes accurately. Further research is needed to fully understand the functional consequences of the c.2363T>G variant in breast cancer pathogenesis. Integrating computational predictions with experimental data will provide valuable insights into the role of alternative splicing regulation in different breast cancer types and stages.

Clinical Case of Rare Genetic Disorder (Proud Syndrome) in a Child

Background. Proud syndrome is rare inherited disease with X-linked inheritance associated with mutations in the homeobox gene ARX. Typical clinical signs of this syndrome are severe mental retardation, intractable epilepsy, agenesis (dysgenesis) of corpus callosum. Less common features are genital abnormalities, microcephaly, facial dysmorphia, and skeletal malformations. Clinical case description. The article describes the clinical findings of Proud syndrome in girl A., admitted to Children’s City Clinical Hospital No. 1 in Nizhny Novgorod. The girl was born without asphyxia at term through natural vaginal delivery after the first uneventful pregnancy. Body weight at birth was 2600 g. The genealogical history is burdened by the presence of epilepsy in girl’s father relatives. There were no deviations in psychomotor skills development before disease onset. Hemi-convulsive seizures (switching sides) have appeared at the age of 6 months. These attacks had status course and were resistant to anticonvulsant therapy. Neuroimaging has revealed agenesis of corpus callosum. Regression of psychomotor development, new behavioral disorders (stereotypes and auto-aggression), hyperexcitability, and sleep disorders were observed in dynamics. The diagnosis of Proud syndrome was confirmed by identification of probably pathogenic mutation in the ARX gene (c.1111C&gt;T, p. Arg 371*64). The features of anticonvulsant management were demonstrated in the patient. Conclusion. This clinical case presents typical clinical picture of Proud syndrome. The disease is non-curable. Such patients should be administered with syndromic therapy: constant anticonvulsant therapy, correction of behavioral disorders (classes with specialist on mental defects), neurologist, epileptologist, psychiatrist observations. Parental examination is crucial ARX mutation search in order to determine the prognosis for further child-bearing (parents refused to perform genetic study at the time of article writing).

Columnar Cell Thyroid Carcinoma: A Heterogeneous Entity Demonstrating Overlap Between Papillary Thyroid Carcinoma and Follicular Neoplasms.

Columnar cell papillary thyroid carcinoma (CC-PTC) is a morphologic subtype of papillary thyroid carcinoma with a variable prognosis. It is characterized by neoplastic thyroid follicular-derived cells with pseudostratified columnar morphology arranged in papillary or follicular structures with supranuclear or subnuclear vacuoles. The molecular profile of this subtype has only recently come under scrutiny, with mixed results. The aim of this study is to further explore the morphologic, immunohistochemical, and genetic profile of CC-PTC, as well as to correlate these features with clinical outcomes. CC-PTC cases were identified from 3 institutions. Immunohistochemistry (ER, CDX2) and molecular testing (DNA and RNA sequencing) were performed. Clinicopathologic parameters and patient outcomes were recorded. Twelve cases (2006-2023) were identified, all in adults (age 45-91). Two presented with disease outside the thyroid gland (neck and mediastinum) and two presented with distant metastasis. Four were high-grade differentiated thyroid carcinomas (necrosis or mitoses), one of which died of disease. Four were noninvasive or minimally invasive, one of which locally recurred. Three patients had lymph node metastases. ER and CDX2 were positive in 73% and 50%, respectively. Pathogenic mutations were found in TERT promoter (n = 3), RAS (n = 2), ATM, NOTCH1, APC, and ESR1, along with cases bearing AGK::BRAF fusion (n = 1), BRAF VE1 expression (n = 1), and NF2 loss (n = 1). This study represents the largest molecularly defined cohort of non-oncocytic thyroid carcinomas with columnar cell morphology. These tumors represent a genetically and behaviorally heterogeneous group of neoplasms, some of which have RAS-like or follicular neoplasm-like genetics, some of which have BRAF-p.V600E-like or classic papillary thyroid carcinoma-like genetics, and some of which remain unclear. Noninvasive or minimally invasive tumors showed an indolent course compared to those with angioinvasion, gross extrathyroidal growth, or high-grade morphology. Consideration could be given to reclassification of this neoplasm outside of the subtyping of papillary thyroid carcinoma in light of its genetic diversity, distinct morphology, and clinical behavior more closely aligned with follicular thyroid neoplasms.

Diagnosis of Fabry Disease Using Alpha-Galactosidase A Activity or LysoGb3 in Blood Fails to Identify Up to Two Thirds of Female Patients.

Anderson-Fabry disease is a lysosomal storage disorder caused by mutations in the GLA gene, which encodes the enzyme α-galactosidase A. The GLA gene is located on the X-chromosome, causing an X-linked pathology: due to lyonization, female patients usually manifest a variable symptomatology, ranging from asymptomatic to severe phenotypes. The confirmation of the clinical diagnosis of Fabry disease, achieved by measuring α-galactosidase A activity, which is usually the first test used, shows differences between male and female patients. This assay is reliable in male patients with causative mutations in the GLA gene, in whom the enzymatic activity is lower than normal values; on the other hand, in female Fabry patients, the enzymatic activity is extremely variable between normal and pathological values. These fluctuations are also found in female patients' blood levels of globotriaosylsphingosine (LysoGb3) for the same reason. In this paper, we present a retrospective study conducted in our laboratories on 827 Fabry patients with causative mutations in the GLA gene. Our results show that 100% of male patients had α-galactosidase A activity below the reference value, while more than 70% of female patients had normal values. It can also be observed that almost half of the female patients with pathogenic mutations in the GLA gene showed normal values of LysoGb3 in blood. Furthermore, in women, blood LysoGb3 values can vary over time, as we show in a clinical case presented in this paper. Both these tests could lead to missed diagnoses of Fabry disease in female patients, so the analysis of the GLA gene represents the main diagnostic test for Fabry disease in women to date.

Epidemiology of rotavirus infection: the evolution of the pathogen and the success of vaccination

The review presents data on the spread of various rotavirus genotypes and its variability in the world and in Russia, which occur both due to natural causes and under the influence of vaccination against rotavirus infection, as well as on the transformation of clinical and epidemiological features of the course of this disease in children as a result of the pathogen mutations.

Outcomes of a universal germline screening program in a community urology practice.

The role of germline genetic testing in urologic oncology has expanded in recent years. However, implementation of genetic testing in community practices remains a challenge, often due to limited access to qualified genetics trained providers. In this study, we report outcomes of a universal germline screening program in a community urology practice. Between November 2021 and September 2022, all patients referred for urology clinic visits at Frederick Health (Frederick, MD, USA) were provided an online genetics screening questionnaire prior to the visit. Responses were compared against National Comprehensive Cancer Network (NCCN) criteria for germline testing. Those who met criteria were provided educational materials at the end of the questionnaire, and then counseled by a trained urologic oncologist (HC) in the clinic or referred to a genetic counselor prior to testing. Testing was performed with a 36-gene pan-cancer panel (CancerNext) or a 14-gene targeted prostate cancer panel (ProstateNext), with or without additional RNA analysis (RNAinsight) (Ambry Genetics, CA, USA). Demographic and clinical parameters, as well as genetic testing results, were retrospectively collected under IRB approval. In the study period, 765 patients were seen over 1370 clinic visits. Of these, 505 patients (66.0%) completed the screening questionnaire. The majority were completed via email (54.5%) with the remainder (45.5%) via text message. Of the patients who completed screening, 125/505 (24.7%) met NCCN criteria for germline testing. 58/125 patients (46.4%) who met criteria underwent germline testing, of whom 5/58 (8.6%) had distinct pathogenic mutations identified. These included actionable mutations in BRCA1, BRCA2, and CHEK2, as well as an additional pathogenic mutation in NBN. Variants of unknown significance were identified in 8/58 patients (13.8%) in 11 total genes. Challenges to implementation of this program included meeting institutional requirements for genetic testing consent, facilitating specimen collection in clinic, and integration of results into the electronic health record. Genetic risk assessment for high-risk individuals is feasible as part of a universal screening program in a community urology practice. Approximately 8% of tested patients were found to have pathogenic germline mutations, which is consistent with contemporary tertiary referral cohorts.

Evaluation of the outcomes of cochlear implant in children with auditory neuropathy

Objective: To investigate the auditory and speech abilities of children with congenital auditory neuropathy (AN) after cochlear implant (CI), and to analyze the role of genetic testing in predicting the postoperative outcomes of CI in AN patients. Methods: Fourteen children diagnosed with AN by audiological battery test and underwent CI surgery in Xijing Hospital of the Air Force Medical University from 2002 to 2021 were included in this study (9 males and 5 females), with an implantation age of (3.1±1.7) years (mean±standard deviation, the same as follows). The preoperative audiological results and deafness gene results were analyzed. Another 52 children with ordinary sensorineural hearing loss (SNHL) were selected as the control group (30 males and 22 females), with an implantation age of (2.2±0.9) years. The demographic factors such as age and gender were matched with those of the AN group. The modified Category Auditory Performance (CAP-Ⅱ) and Speech Intelligence Rate (SIR) were used to evaluate the development of postoperative auditory and speech abilities in two groups. The Mandarin Speech Test System was used to test the speech recognition rate of monosyllabic and disyllabic words and sentences. Matlab 2022 software was used to analyze the data. Results: The results of gene in 14 children with AN showed that 6 cases had OTOF gene mutations, 2 cases (siblings) were confirmed to have TNN gene mutations through whole exome sequencing, and the remaining 6 cases were not find any clear pathogenic gene mutations. All subjects underwent CI surgery with electrodes implanted into the cochlea smoothly, and there were no postoperative complications. After surgery, all AN children had improved auditory and speech abilities, but only 64% (9/14) of AN children with CI had auditory ability scores comparable to the control group of SNHL children (including 2 children with TNN gene mutations), and 36% (5/14) of AN children had lower scores than the control group of SNHL children.The average speech recognition rate of two children with TNN gene mutations was 86.5%, and of two children with OTOF gene mutations was 83.2%. Conclusions: AN children achieved varying degrees of auditory and speech abilities after CI, but the postoperative effects varied greatly. Some children achieved similar results as ordinary SNHL children, but there were still some children whose effects were worse than those of ordinary SNHL children. The postoperative efficacy of CI in two children with AN caused by TNN pathogenic genes were comparable to that of ordinary SNHL in children. Genetic testing had certain reference value for predicting the postoperative effect of CI in AN children.

Precise correction of a spectrum of β-thalassemia mutations in coding and non-coding regions by base editors

β-thalassemia/HbE results from mutations in the β-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of β-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of β-thalassemia mutations that were integrated into the genome as fragments ofHBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with β-thalassemia/HbE. These β-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional β-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe β0/βE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

A Novel Frameshift Mutation of HBB Causing Dominant β-Thalassemia in a Chinese Individual

We reported a rare β-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged β-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the β-globin predicted that the novel prolonged β-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.