Heterozygous Deletion Research Articles

Ancient gene introgression promotes biodiversity of Nymphaea nouchali in Hainan under environmental changes

AbstractHybridization is common in the plant kingdom. Under the influence of global climate change, an increasing number of hybrid species have experienced significant shifts from the geographical distribution of their parents, making it more difficult to detect ancient gene flows. Nymphaea nouchali is a rare aquatic flower native to Hainan Province. Owing to habitat destruction and human interference, the natural range of N. nouchali has gradually reduced to the point of extinction. To explore how hybrid populations form using RAD‐seq with population genetic analysis, it is shown that there are heterozygous deletions and inbreeding in N. nouchali and a number of ancestral components in Nymphaea lotus, low of genetic differentiation, and little gene flow between N. nouchali and N. lotus, and ancient introgression between ancestors may have been more frequent than gene exchange after the origin of the species. Accordingly, the ancient N. nouchali genotypes were gradually infiltrated by N. lotus genes and their forms changed in Hainan. Potential distribution pattern analysis also predicted the possible distribution of N. lotus in Hainan. This lays a foundation for exploring the natural genetic mechanisms of Nymphaea species formation and evolution.

Clinical characteristics and prognostic analysis of CDKN2A/2B gene in pediatric acute lymphoblastic leukemia: a retrospective case-control study

ABSTRACT In this retrospective case-control study involving 424 pediatric patients diagnosed with Pediatric Acute Lymphoblastic Leukemia (ALL), the investigation focused on analyzing the clinical characteristics and prognosis associated with the Cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) gene. Treatment and evaluation followed the South China Children's Leukemia Group-ALL-2016 protocol (SCCLG-ALL-2016). Among the cohort, 92 patients (21.7%) exhibited CDKN2A/2B gene deletions, with 11.1% homozygous and 10.6% heterozygous deletions. Notably, ALL patients that do have CDKN2A/2B gene deletions tended to present at an older age (P = 0.001), demonstrate hepatosplenomegaly on palpation (P < 0.001), and exhibit a higher incidence of Central nervous system leukemia (CNSL) (P = 0.037) and T-ALL (P = 0.007). A significant correlation was observed between ALL that does have CDKN2A/2B gene deletions and ETV6::RUNX1-positive (8.7% vs. 19.3%, P = 0.017) and IKZF1 gene deletions (20.7% vs. 8.4%, P = 0.001). Survival analysis of 392 patients revealed no significant differences in 5-year relapse, Overall survival (OS), or Event-free survival (EFS) between ALL that does/ does not have CDKN2A/2B gene deletions. Subgroup analysis highlighted poorer prognosis among hepatosplenomegaly patients in the CDKN2A/2B gene deletion group, with a 5-year EFS of 81.8%, 95%CI (0.695–0.963), P = 0.05. Hepatosplenomegaly emerged as the most significant prognostic factor for EFS [HR = 2.306, 95%CI (1.192–4.461), P = 0.013]. Cox regression analyses identified covariates influencing prognosis, ALL with the CDKN2A/2B gene showing no significant impact on outcomes. In conclusion, while ALL that does have CDKN2A/2B gene deletions is associated with certain clinical characteristics and genetic aberrations, they did not significantly impact OS or EFS. Furthermore, subgroup analysis revealed a potential prognostic role of ALL that does have CDKN2A/2B deletions presenting with hepatosplenomegaly on palpation, emphasizing the importance of comprehensive risk stratification in treatment decision-making for this subgroup.

A new GRN variant in logopenic variant primary progressive aphasia: a case report and literature review

ABSTRACT The majority of genetic Primary progressive aphasia (PPA) patients harbor mutations in the granulin (GRN) gene. The present case showed impaired performances in single-word retrieval in spontaneous speech and naming, and repetition. Head MRI revealed marked lateral atrophy in the left parietal cortex. A diagnosis of logopenic variant PPA (lvPPA) was established. Genetic analysis showed a heterozygous 10-bp frameshift deletion in exon 4 of the GRN gene (NM_002087.4), leading to transformation of cysteine into alanine at amino acid 92 and creation of a premature stop codon at position 161. This patient represented a rare case of GRN-associated lvPPA. A new mutation site was detected in exon 4 of GRN gene.

Genetically confirmed Charcot–Marie–Tooth disease type 2A manifesting with postural tremor: a case report

BackgroundCharcot–Marie–Tooth disease is a spectrum of inherited disorders characterized by both motor and sensory manifestations, which include prominent distal muscle weakness, foot deformities (pes cavus and hammer toes), and sensory deficits. Postural tremor as a manifestation of Charcot–Marie–Tooth is seldom present, except in a variant of Charcot–Marie–Tooth subtype 1 (Roussy–Levy syndrome), and its presence often results in a diagnostic dilemma.Case presentationWe present a 34-year-old Eritrean man who came to our hospital with a complaint of tremors of the hands of 6 months duration. Associated with this, he had difficulty walking and weakness of the distal extremities bilaterally, prominently involving the lower limbs. The patient denied a family history of such illness. Physical examination revealed distal muscle weakness (4+/5 on upper limbs, while 3/5 on lower limbs bilaterally), pes cavus deformity, absent ankle reflexes, and mild vibratory sensory loss. We noted a postural tremor that attenuated when the patient assumed an anatomic position. The tremor was limited to the hands. Nerve conduction study of upper and lower limbs showed moderate to severe motor axonal and demyelinating polyneuropathy (axonal > demyelinating), suggestive of mixed axonal and demyelinating hereditary polyneuropathy. Subsequently, genetic testing revealed copy number changes (heterozygous deletion) on the MPZ and MFN2, while the PMP22 gene showed ambiguous copy number changes (decrease) on exons 2 and 3. Tying the clinical, electrophysiologic, and genetic findings, consideration of Charcot–Marie–Tooth subtype 2A with postural tremor was made. Subsequently, the patient was managed with regular physiotherapy and an anxiolytic resulting in minimal symptom improvement.ConclusionThe present case describes a 34-year-old male patient with Charcot–Marie–Tooth subtype 2A presenting with neuropathic postural tremor, which is a rare presentation of a common hereditary polyneuropathy. This case highlights the fact that tremors can be associated with peripheral neuropathy syndromes, and a high index of suspicion is needed to rightly diagnose our patients.

Empagliflozin improves pressure-overload-induced cardiac hypertrophy by inhibiting the canonical Wnt/β-catenin signaling pathway.

Empagliflozin (EMPA) is an SGLT-2 inhibitor that can control hyperglycemia. Clinical trials have indicated its cardio-protective effects against cardiac remodeling in diabetes or non-diabetes patients. However, the underlying molecular mechanisms of EMPA's cardio-protective effects remain elusive. We evaluated whether the EMPA attenuated the pressure-overload-induced cardiac hypertrophy by inhibiting the Wnt/β-catenin pathway. Furthermore, the effects of the EMPA on a mouse model of transverse aortic constriction (TAC) induced cardiac hypertrophy was also evaluated. Mice were administrated with 0.5% CMC-Na as a vehicle or EMPA (10mg/kg/day, daily, throughout the study) by intragastric gavage. The in vivo echocardiography and histologic morphological analyses revealed that EMPA attenuated TAC-induced cardiac hypertrophy. Moreover, it also ameliorated TAC-induced cardiac fibrosis and decreased the cell size of the cardiomyocytes in isolated adult cardiomyocytes. Molecular mechanism analysis revealed that the EMPA reduced the TAC-induced enhanced expression of the Wnt/β-catenin pathway in vivo. For in vitro assessments, isolated neonatal rat cardiomyocytes (NRCMs) were treated with Angiotensin II (AngII) and EMPA; the results showed that in the absence of EMPA, the expression of the Wnt/β-catenin pathway was enhanced. In the trans-genetic heterozygous β-catenin deletion mice, EMPA attenuated TAC-induced cardiac remodeling by reducing the Wnt/β-catenin pathway. In addition, molecular docking analysis indicated that EMPA interacts with FZD4 to inhibit the TAC and AngII induced Wnt/β-catenin pathway in cardiomyocytes. Our study illustrated that EMPA might directly interact with FZD4 to inhibit the TAC and AngII-induced activation of the Wnt/β-catenin pathway to attenuate the adverse cardiac remodeling.

Recruitment of chromatin remodelers by XIST B-repeat region is variably dependent on HNRNPK.

X-chromosome inactivation is triggered by the long non-coding RNA XIST, whose structure is characterized by tandem repeats that modularly recruit different proteins and chromatin remodelers. Previously, we reported that the addition of the mouse PID region to a transgene with human repeat regions A, F and E (miniXIST; 5.1kb) enabled binding of HNRNPK and also enabled the induction of silencing and recruitment of H3K27me3, UbH2A and H4K20me1, but only partially. As the 680bp PID region enabled so many features of inactivation, we hypothesized that augmenting the PID with more mouse or human sequences rich in CCC motifs would allow us to design a short transgene which was as effective as Full XIST. Three new transgenes using the A, F and E human domains as a backbone were tested for ability to induce silencing and heterochromatic mark recruitment. The all human-derived BhB-BhB transgene (4.9kb) was as good as our previous miniXIST, suggesting that these domains are the human equivalent of the mouse PID region. A PID-PID transgene (5.8kb) was not statistically different from Full XIST and could be potentially used for chromosome therapy. Adding BhB to PID (BhB-PID, 5.4kb) had an intermediate efficacy compared to the other two transgenes, suggesting that the most important component for silencing and heterochromatic mark recruitment is the number of CCC motifs, not the species of origin. Finally, we created a heterozygous HNRNPK deletion and observed a disproportionate impact on HNRNPK and UbH2A recruitment to XIST, reflecting complex roles for the PID and HNRNPK in X-chromosome inactivation.

Unexpected Findings of Duchenne Muscular Dystrophy in Prenatal Screening of Chromosome Abnormality Based on Cell-Free Fetal DNA.

This study aims to assess the feasibility of detecting and diagnosing Duchenne muscular dystrophy (DMD) during prenatal screening for chromosome abnormalities using cell-free fetal DNA extracted from peripheral blood samples of pregnant women. Two pregnant women identified as high risk through noninvasive prenatal testing (NIPT) underwent amniocentesis to obtain fetal cells. Subsequent fetal chromosomal karyotyping was conducted, and genomic DNA from fetal cells was extracted for copy number variation sequencing (CNV-Seq) analysis, complemented by multiplex ligation-dependent probe amplification (MLPA) to detect deletions or duplications within the DMD gene. NIPT results for the two samples indicated potential abnormalities involving chromosomes 21 and 18. However, karyotype analysis of the fetuses revealed no abnormalities. CNV-Seq identified deletions of 0.28 and 0.18 Mb within chromosome Xp21.1, encompassing the DMD gene, in each fetus. In family 1, MLPA results indicated a maternal heterozygous deletion spanning exons 12 to 41 in the DMD gene, while the fetus exhibited deletions in exons 12 to 41. In family 2, MLPA results confirmed normal DMD gene status in the pregnant woman's peripheral blood genomic DNA but revealed a fetal deletion spanning exons 48 to 52. Both fetuses were diagnosed with DMD and subsequently underwent termination. Abnormalities identified through NIPT necessitate further invasive prenatal diagnostic procedures. For cases involving chromosomal microdeletions or microduplications, a combination of karyotyping and CNV-Seq testing is essential for comprehensive diagnosis. NIPT followed by CNV-Seq may offer insights into large exon deletions within the DMD gene in specific instances. · NIPT results can offer valuable insights into the deletion and duplication of DMD gene for the fetus.. · It's crucial to notice unexpected findings in NIPT.. · A combination of karyotyping and CNV-Seq testing is essential for comprehensive diagnosis..

2q33 Deletions Underlying Syndromic and Non-syndromic CTLA4 Deficiency.

CTLA4 deficiency is an inborn error of immunity (IEI) due to heterozygosity for germline loss-of-function variants of the CTLA4 gene located on chromosome 2q33.2. CTLA4 deficiency underlies pleiotropic immune and lymphoproliferation-mediated features with incomplete penetrance. It has been identified in hundreds of patients but copy number variants (CNVs) have been reported in only 12 kindreds, including nine which displayed large 2q33.1-2q33.2 deletions encompassing CTLA4. We conducted a nationwide study in France to identify patients with 2q33 deletions encompassing CTLA4. We investigated the clinical and immunological phenotypes and genotypes of these patients. We identified 12 patients across six unrelated kindreds with clinical immunodeficiency. Neurological features were recorded in three patients, including one with syndromic neurodevelopmental disorder. Single-nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) array analysis, and targeted high-throughput sequencing revealed five different heterozygous 2q33 deletions of 26 kilobases to 7.12 megabases in size and encompassing one to 41 genes.We identified a contiguous gene syndrome (CGS) due to associated KLF7 deficiency in a kindred with a neurodevelopmental phenotype. Deletions within the 2q33 region encompassing CTLA4 are rare and not extensively explored, and are probably underdiagnosed in cytogenetic practice. A literature review identified 14 different CGS loci including at least one gene responsible for an IEI. The deletions involved in IEIs should be systematically delimited, to facilitate screening for CGS.

Exploring 17q12 Deletion Syndrome: A Case Report of Neurodevelopmental, Endocrine, and Genital Anomalies

Objective − 17q12 deletion syndrome is a rare genetic disease characterized by neurodevelopmental disorders, genital and renal abnormalities, and maturity-onset diabetes of the young type 5 (MODY5).Case Report − This case report details the case of a 13-year-old female with moderate intellectual disability, Mayer-Rokitansky-Küster-Hauser syndrome, multicystic dysplastic kidneys, and MODY5. Genetic testing revealed a 1.52-megabase heterozygous deletion on chromosome 17q12, encompassing the HNF1B and LHX1 genes, which was found to be inherited from the mother. Conclusion − This case underscores the importance of early genetic testing and multidisciplinary approach in managing the multisystemic manifestations of 17q12 deletion syndrome. Early diagnosis and appropriate management are crucial for improving the outcomes and quality of life for affected individuals.

CRISPR-Cas9 mediated knockout of NDUFS4 in human iPSCs: A model for mitochondrial complex I deficiency

Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4-related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD+ modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo, these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction.

PATH-32. THE PROGNOSTIC IMPACT OF CDKN2A/B HETEROZYGOUS DELETIONS IN IDH-MUTANT GLIOMA

Abstract BACKGROUND Homozygous deletions of CDKN2A/B have been shown to be a prognosticator for an unfavorable prognosis in gliomas, but the role of heterozygous CDKN2A/B deletions remains less well understood. The aim of this study was to assess the prognostic impact of CDKN2A/B heterozygous deletions in IDH-mutant low-grade astrocytoma and oligodendroglioma. METHODS Tissue samples diagnosed as astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3 were collected from the archive of the Institute of Neuropathology in Heidelberg. DNA methylation analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples. Evaluation of the CDKN2A/B locus was performed by visual inspection of copy-number plots derived from methylation-array data for each case. Heterozygous and homozygous losses were assessed in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan–Meier method. RESULTS A total of 334 low-grade glioma cases were identified. Those cases included 173 astrocytomas and 161 oligodendrogliomas. Heterozygous deletions in CDKN2A/B (37/173 in astrocytomas, 15/161 in oligodendrogliomas) did not confer worse survival outcomes compared to CDKN2A/B wildtype cases in neither low grade astrocytoma (p= 0.2556) nor oligodendroglioma (p= 0.2760), regardless of CNS WHO grade, which was further validated on a subgroup of astrocytoma, IDH mutant CNS WHO 4 cases (p= 0.1680). CONCLUSION In summary, our study suggests that heterozygous CDKN2A/B deletions do not confer worse survival rates with respect to OS and PFS in astrocytoma, IDH-mutant and oligodendroglioma, IDH-mutant, 1p/19q co-deleted CNS WHO grade 2 and 3.

PATH-04. THE PROGNOSTIC IMPACT OF CDKN2A/B HETEROZYGOUS DELETIONS IN MENINGIOMA- INSIGHTS OF A MULTICENTER ANALYSIS

Abstract BACKGROUND Meningiomas are one of the most common primary brain tumors. Homozygous deletions of CDKN2A/B have reliably been shown to confer an increased risk of progression. However, the role of heterozygous CDKN2A/B deletions remains less clear. MATERIAL AND METHODS DNA methylation data, copy-number information and mutation data were generated on a multicenter cohort of meningiomas for a total of 970 samples. The CDKN2A/B locus was determined manually for each individual case in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan–Meier method. RESULTS A total of 970 meningioma cases were identified with 30 homozygous and 114 heterozygous CDKN2A/B deletion cases, 826 cases had balanced CDKN2A/B. Heterozygous CDKN2A/B deletions in general did not confer an increased risk of progression compared to CDKN2A/B balanced cases (p= 0.074). However, segmental heterozygous losses of 9p, encompassing CDKN2A/B, yielded a significantly worse prognosis when compared to meningiomas CDKN2A/B-balanced (p= 0.002) and in contrast to focal heterozygous deletions (p= 0.523). Segmental heterozygous 9p deletions encompassing CDKN2A/B were significantly more frequently associated with a higher amount of copy number variations (CNVs) than focal losses. CONCLUSION Segmental heterozygous CDKN2A/B deletions do confer an increased risk of progression, but focal heterozygous CDKN2A/B deletions do not. The signal for segmental deletions may not be locus-specific but just one representation of the generally instable genome of aggressive meningiomas.

PATH-56. LOSS OF HETEROZYGOSITY OF CDKN2A/B DEFINES AN AGGRESSIVE SUBSET OF RECURRENT, HIGH-GRADE MENINGIOMAS

Abstract INTRODUCTION Loss of heterozygosity (LOH) occurs when one gene allele is lost, leaving the cell with a single functional copy. This genetic alteration drives tumor progression by disabling critical regulatory pathways. Currently, CDKN2A/B homozygous loss is a criterion for WHO Grade 3 meningiomas. However, the impact of CDKN2A/B LOH and other copy-number alterations remains unclear. This study investigates the effects of LOH in the CDKN2A/B tumor suppressor gene on progression-free survival (PFS) and overall survival (OS) in meningiomas. METHODS The study included adult patients with sporadic primary or recurrent meningiomas who underwent resection and whole exome sequencing. LOH and copy-number alterations were confirmed on visual inspection of b-allele frequency and mean ratio graphs. Samples of unsuitable quality for evaluation were excluded. CDKN2A/B copy number and LOH were associated with PFS and OS. RESULTS The sample included 333 meningiomas (64.6% female [n=215], 18.8% recurrent meningiomas [n=61], and 50 cases of postoperative recurrence [15%]). CDKN2A/B was intact in 88.5% of samples (n=295), while 38 samples (11.4%) exhibited CDKN2A/B LOH. This included homozygous deletions (n=2, 5.2%), heterozygous deletions (n=11, 28.9%), amplifications (n=12, 31.6%), and copy-neutral LOH (n=13, 34.2%). All copy-number alterations were associated with an increased risk of recurrence compared to CDKN2A/B intact samples, so CDKN2A/B LOH was used for subsequent analysis. CDKN2A/B LOH was more frequently associated with prior recurrence (22.9% vs. 8.8%; p=0.004), prior radiation (46% vs. 15.6%; p&amp;lt;0.0001), and WHO Grade 2 and 3 tumors (63.2% vs. 42.7%; p=0.02). CDKN2A/B LOH was associated with worse PFS (HR 3.97 [1.3-11.9]; p=0.01) and worse OS (HR 2.9 [1.03-8.2]; p=0.0429) only in recurrent, high-grade meningiomas, independent of the fraction of the genome altered, mutation count, and Simpson grade. Postoperative radiation therapy did not improve PFS (HR 3.24 [1.06-9.85]; p=0.038). CONCLUSION CDKN2A/B LOH is enriched in high-grade, recurrent meningiomas and confers worse PFS and OS despite postoperative radiotherapy. CDKN2A/B LOH is a high-risk biomarker that predicts tumor behavior and patient outcome.

Genetic Analysis of Heterotaxy in a Consanguineous Cohort.

Heterotaxy (HTX) is a group of clinical conditions with a shared pathology of dislocation of one or more organs along the left-right axis. The etiology of HTX is tremendously heterogeneous spanning environmental factors, chromosomal aberrations, mono/oligogenic variants, and complex inheritance. However, in the vast majority of cases, the etiology of HTX remains elusive. Here, we sought to describe the yield of genetic analysis and spectrum of variants in HTX in our highly consanguineous population. Twenty-four affected individuals, from 19 unrelated families, were consecutively recruited. Genetic analysis, with exome sequencing, genome sequencing, or multigene panel, detected 9 unique variants, 7 of which were novel, in 8 genes known to be implicated in autosomal recessive form of HTX (C1orf127, CCDC39, CIROP, DNAAF3, DNAH5, DNAH9, MMP21, and MNS1) providing a yield of 42.1%. Of note, 7 of the 9 variants were homozygous, while 2 were inherited in compound heterozygosity, including a heterozygous CNV deletion. A search for candidate genes in negative cases did not reveal a plausible variant. Our work demonstrates a relatively high yield of genetic testing in HTX in a consanguineous population with an enrichment of homozygous variants. The significant genetic heterogeneity observed herewith underscores the complex developmental mechanisms implicated in the pathogenesis of HTX and supports adopting a genome-wide analysis in the diagnostic evaluation of HTX.

Multi-Omics Approach Reveals Genes and Pathways Affected in Miller-Dieker Syndrome.

Miller-Dieker syndrome (MDS) is a rare neurogenetic disorder resulting from a heterozygous deletion of 26 genes in the MDS locus on human chromosome 17. MDS patients often die in utero and only 10% of those who are born reach 10years of age. Current treatments mostly prevent complications and control seizures. A detailed understanding of the pathogenesis of MDS through gene expression studies would be useful in developing precise medical approaches toward MDS. To better understand MDS at the molecular level, we performed RNA sequencing on RNA and mass spectrometry on total protein isolated from BJ (non-MDS) cells and GM06097 (MDS) cells, which were derived from a healthy individual and an MDS patient, respectively. Differentially expressed genes (DEGs) at the RNA and protein levels involved genes associated with phenotypic features reported in MDS patients (CACNG4, ADD2, SPTAN1, SHANK2), signaling pathways (GABBR2, CAMK2B, TRAM-1), and nervous system development (CAMK2B, BEX1, ARSA). Functional assays validated enhanced calcium signaling, downregulated protein translation, and cell migration defects in MDS. Interestingly, overexpression of methyltransferase-like protein 16 (METTL16), a protein encoded in the MDS locus, restored defects in protein translation, phosphorstates of mTOR (mammalian target of rapamycin) pathway regulators, and cell migration in MDS cells. Although DNA- and RNA-modifying enzymes were among the DEGs and the intracellular SAM/SAH ratio was eightfold lower in MDS cells, global nucleoside modifications remained unchanged. Thus, this study identified specific genes and pathways responsible for the gene expression changes, which could lead to better therapeutics for MDS patients.

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Identification and Characterization of Novel FSHR Copy Number Variations Causing Premature Ovarian Insufficiency.

Follicle stimulating hormone (FSH) is a key pituitary gonadotropic hormone implicated in human fertility and is crucial for folliculogenesis and recruitment of new antral follicles. Variations in its receptor, FSHR, can lead to diverse reproductive phenotypes including ovarian hyperstimulation syndrome (OHSS) and premature ovarian insufficiency (POI). This study reports a novel case of FSHR-related ovarian insufficiency in a patient with primary amenorrhea, subnormal AMH levels, and delayed puberty. Genetic exploration revealed two compound heterozygous intragenic deletions of FSHR. Specifically, the patient inherited a maternally derived deletion spanning exons 5-10 and a paternally derived deletion involving exons 3-6. Through chromosomal microarray analysis (CMA), exome sequencing, long-range PCR, and Sanger sequencing, we characterized the breakpoints and confirmed the compound heterozygous deletions. The findings reveal a complete loss of function of both FSHR alleles, contributing to the patient's POI phenotype. This case emphasizes the complexity of genotype-phenotype correlations in FSHR-related disorders and the role of CNVs in POI phenotypes. Although these events are rare, our results advocate for the inclusion of CNV detection in the diagnostic workup of POI to ensure accurate diagnosis and better patient management.

Phenotypic complexities of rare heterozygous neurexin-1 deletions.

Given the large number of genes significantly associated with risk for neuropsychiatric disorders, a critical unanswered question is the extent to which diverse mutations --sometimes impacting the same gene-- will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1 +/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, in order to achieve individualized restoration of NRXN1 isoform repertoires by increasing wildtype, or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.

Development of a Tagmentation-Based Next-Generation Sequencing Clinical Assay as an Alternative to Capillary Electrophoresis-Based Sequencing.

Next-generation sequencing (NGS) technology enables sample multiplexing for interrogation of multiple regions of interest (ROI). Leveraging this, together with access to affordable NGS platforms, we explored the practicality of moving capillary electrophoresis (CE), noncapillary electrophoresis and single-gene testing to NGS. In this work, we evaluated the iSeq 100's capacity to validate 89 samples at once. Genomic DNA was extracted from 89 archival samples of varying specimen types. Polymerase chain reaction (PCR) was done with in house primers, library preparation with the Nextera XT Library Preparation Kit and cleaning up with paramagnetic beads. The sequencing was performed on one Illumina iSeq 100 flow cell. With our workflow, 88 out of 89 samples were accurately sequenced with variant alleles identified. One sample of the 88 samples was initially discordant because the primers used were in a heterozygous deletion region. Upon redesigning of primers, the sample proved concordant. The iSeq-Nextera workflow proved accurate. However, variant allele frequencys generated by the Nextera are not precise.

Novel bi-allelic DNAH3 variants cause oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a widespread cause of male infertility. One of the usual clinical manifestations of OAT is multiple morphological abnormalities of the sperm flagella (MMAF), which are frequently associated with mutations and defects in the dynein family. However, the relationship between the newly identified Dynein Axonemal Heavy Chain 3 (DNAH3) mutation and oligonasthenospermia in humans has not yet been established. Whole exome sequencing, pathogenicity analysis, and species conservation analysis of mutation sites were conducted on two patients from different unrelated families with DNAH3 mutations. We identified representative mutation sites and predicted the protein structure following these mutations. The sperm characteristics of the two patients with DNAH3 mutations were verified using Papanicolaou staining, scanning electron microscopy, and transmission electron microscopy. Additionally, mRNA and protein levels were assessed through RT-qPCR and Western blotting. The biallelic mutations in the first progenitor included a heterozygous deletion and insertion, c.6535_6536 delinsAC (to infect mutation (p.Asp2179Thr), and stop codon premutation, c.3249G > A (p.Trp1083Ter). In Family II, the patient (P2) harbored a DNAH3 heterozygous missense mutation, c. 10439G> A(p.Arg3480Gln), along with a stop codon premutation, (c.10260G > A; p.Trp3420Ter). Patients with premature termination of transcription or translation due to DNAH3 mutations exhibit OAT phenotypes, including fibrous sheath dysplasia and multiple tail malformations. We identified the representative sites after mutation, predicted the protein structure, and assessed changes in the protein levels of DNAH3 and related genes following mutations. Notably,a significant reduction in DNAH3 protein expression was validated in these patients. We may explore in the future how DNAH3 affects sperm motility and quality through regulatory mechanisms involving protein structural changes. Novel biallelic mutations in DNAH3, especially those resulting in a premature stop codon, may alter protein expression, structure, and active site, leading to spermatogenic failure and potentially inducing OAT. The discovery of new mutations in DNAH3 may be the key to the diagnosis and treatment of OAT.