Modulation of biophysical properties of nucleocapsid protein in the mutant spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that 'packages' the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants ­ reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these 'intrinsically disordered' regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.

How mutations affect function.

A new study reveals how naturally occurring mutations affect the biophysical properties of nucleocapsid proteins in SARS-CoV-2.

Genotypic and Phenotypic Spectrum of maple syrup urine disease in Zhejiang of China.

BACKGROUND: Maple Syrup Urine Disease (MSUD) is an autosomal recessive metabolic disorder originating from defects in the branched-chain α-ketoacid dehydrogenase (BCKDH) complex encoded by BCKDHA, BCKDHB, and DBT. This condition presents a spectrum of symptoms and potentially fatal outcomes. Although numerous mutations in the BCKDH complex genes associated with MSUD have been identified, the relationship between specific genotypes remains to be fully elucidated. AIM: Our objective was to predict the pathogenicity of these genetic mutations and establish potential links between genotypic alterations and the clinical phenotypes of MSUD. DESIGN: Retrospective population-based cohort. METHODS: We analyzed 20 MSUD patients from the Children's Hospital at Zhejiang University School of Medicine (Hangzhou, China), recorded from January 2010 to May 2023. Patients' blood samples were collected by heel-stick through neonatal screening, and amino acid profiles were measured by tandem mass spectrometry. In silico methods were employed to assess the pathogenicity, stability, and biophysical properties. Various computation tools were utilized for assessment, namely PredictSNP, MAGPIE, iStable, Align GVGD, ConSurf and SNP effect. RESULTS: We detected 25 distinct mutations, including 12 novel mutations. The BCKDHB gene was the most commonly affected (53.3%) compared to the BCKDHA gene (20.0%) and DBT gene (26.7%). In silico webservers predicted all novel mutations were disease-causing. CONCLUSIONS: This study highlights the genetic complexity of MSUD and underscores the importance of early detection and intervention. Integrating neonatal screening with advanced sequencing methodologies is pivotal in ensuring precise diagnosis and effective management of MSUD, thereby significantly improving the prognosis for individuals afflicted with this condition.

Characteristics and outcomes associated with sarcomere mutations in patients with hypertrophic cardiomyopathy: A systematic review and meta-analysis.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an inherited heart disease that can lead to sudden cardiac death. Impact of genetic testing for the prognosis and treatment of patients with HCM needs to be improved. We conducted a systematic review and meta-analysis to investigate the characteristics and outcomes associated with sarcomere genotypes in index patients with HCM. METHODS: A systematic search was conducted in Medline, Embase, and Cochrane Library up to Dec 31, 2023. Data on clinical characteristics, morphological and imaging features, outcomes and interventions were collected from published studies and pooled using a random-effects meta-analysis. RESULTS: A total of 30 studies with 10,825 HCM index patients were included in the pooled analyses. The frequency of sarcomere genes in HCM patients was 41%. Sarcomere mutations were more frequent in women (p < 0.00001), and were associated with lower body mass index (26.1 ± 4.7 versus 27.5 ± 4.3; p = 0.003) and left ventricular ejection fraction (65.7% ± 10.1% vs. 67.1% ± 8.6%; p = 0.03), less apical hypertrophy (6.5% vs. 20.1%; p < 0.0001) and left ventricular outflow tract obstruction (29.1% vs. 33.2%; p = 0.03), greater left atrial volume index (43.6 ± 21.1 ml/m2 vs. 37.3 ± 13.0 ml/m2; p = 0.02). Higher risks of ventricular tachycardia (23.4% vs. 14.1%; p < 0.0001), syncope (18.3% vs. 10.9%; p = 0.01) and heart failure (17.3% vs. 14.6%; p = 0.002) were also associated with sarcomere mutations. CONCLUSIONS: Sarcomere mutations are more frequent in women, and are associated with worse clinical characteristics and poor outcomes.

Domain-specific phenotypes in LINS1-related disorder-A Chinese family with the Q92X variant and literature review.

LINS1 is the human homolog of the Drosophila segment polarity gene that encodes an essential regulator of the wingless/Wnt signaling. By 2011, only seven pedigrees (16 patients) with eight causative variants in LINS1 gene have been reported. These cases mainly presented with infancy-/child-onset neurodevelopmental disorders, facial dysmorphia, and other clinical features, and a wide spectrum of clinically distinct phenotypes were also manifested. In our study, two brothers in a family were admitted and diagnosed with child-onset movement disorders, slight intellectual disability, psychological symptoms, eye problems, urinary and bowel dysfunction, mitral value prolapse, and Q-T prolongation. By exome sequencing, we identified a nonsense homozygous pathogenic variant (LINS1: c.274C > T (p.Q92X)), which had been reported in a case diagnosed with intellectual disability and psychiatric disorders (such as schizophrenia and anxiety). Compared with this case, the clinical features of our cases were distinct. In particular, our cases displayed unusual features of heart and blood system. Furthermore, the genotype-phenotype relationship analysis suggested that distinct phenotypes presented in cases carrying variants in different domains of the LINS1 gene. In conclusions, our findings suggest the high clinical variations in the LINS1 variants-related disorders. Moreover, the Q92X might be a recurrent variant in Hans of Southern China.

Significant but partial lipoprotein lipase functional loss caused by a novel occurrence of rare LPL biallelic variants.

BACKGROUND: Lipoprotein lipase (LPL) plays a crucial role in triglyceride hydrolysis. Rare biallelic variants in the LPL gene leading to complete or near-complete loss of function cause autosomal recessive familial chylomicronemia syndrome. However, rare biallelic LPL variants resulting in significant but partial loss of function are rarely documented. This study reports a novel occurrence of such rare biallelic LPL variants in a Chinese patient with hypertriglyceridemia-induced acute pancreatitis (HTG-AP) during pregnancy and provides an in-depth functional characterization. METHODS: The complete coding sequences and adjacent intronic regions of the LPL, APOC2, APOA5, LMF1, and GPIHBP1 genes were analyzed by Sanger sequencing. The aim was to identify rare variants, including nonsense, frameshift, missense, small in-frame deletions or insertions, and canonical splice site mutations. The functional impact of identified LPL missense variants on protein expression, secretion, and activity was assessed in HEK293T cells through single and co-transfection experiments, with and without heparin treatment. RESULTS: Two rare LPL missense variants were identified in the patient: the previously reported c.809G > A (p.Arg270His) and a novel c.331G > C (p.Val111Leu). Genetic testing confirmed these variants were inherited biallelically. Functional analysis showed that the p.Arg270His variant resulted in a near-complete loss of LPL function due to effects on protein synthesis/stability, secretion, and enzymatic activity. In contrast, the p.Val111Leu variant retained approximately 32.3% of wild-type activity, without impacting protein synthesis, stability, or secretion. Co-transfection experiments indicated a combined activity level of 20.7%, suggesting no dominant negative interaction between the variants. The patient's post-heparin plasma LPL activity was about 35% of control levels. CONCLUSIONS: This study presents a novel case of partial but significant loss-of-function biallelic LPL variants in a patient with HTG-AP during pregnancy. Our findings enhance the understanding of the nuanced relationship between LPL genotypes and clinical phenotypes, highlighting the importance of residual LPL function in disease manifestation and severity. Additionally, our study underscores the challenges in classifying partial loss-of-function variants in classical Mendelian disease genes according to the American College of Medical Genetics and Genomics (ACMG)'s variant classification guidelines.

Genetic and clinical characterization of familial renal glucosuria.

Background: Familial renal glucosuria (FRG) is a hereditary disorder caused by variants in SLC5A2 encoding sodium-glucose cotransporter 2 (SGLT2). In this study, we aimed to characterize proximal tubule solute transport, glucagon secretion and the genotype-phenotype relationship in FRG patients. Methods: We sequenced SLC5A2 and PDZK1IP1 in 21 FRG patients and measured the renal threshold of glucose (RTG) in 15 patients. We built an open-source online calculator of RTG, evaluated the proximal tubule transport of amino acid, uric acid and phosphate, and explored glucagon secretion after glucose ingestion in FRG patients. Results: We identified 12 novel SLC5A2 variants (G484D, R564W, A212S, c.574+1G>C, W649*, S592Cfs*6, Q579*, Y339*, V39F, G491E, A464E and G360D) in our cohort and yielded 111 SLC5A2 variants from literature review. RTG in our cohort ranged from 1.0 to 9.2 mmol/L. Patients with two SLC5A2 variants had lower RTG (3.9 vs 6.2 mmol/L) and higher 24-h urinary glucose excretion (24hUG) than single-variant carriers (291.0 vs 40.0 mmol/1.73 m2). Patients with homozygous missense or in-frame indels had mean 24hUG of 457.2 mmol/1.73 m2, comparable to those with homozygous truncating variants (445.0 mmol/1.73 m2) and significantly more than those with homozygous splicing variants (196.6 mmol/1.73 m2). Patients with homozygous missense variants involving conservative residues (582.0 mmol/1.73 m2) had more 24hUG than those with variants at non-conservative residues (257.6 mmol/1.73 m2). Four out of 14 tested patients had mild aminoaciduria. The RTG of FRG patients had no significant correlation to phosphate reabsorption but a potential negative correlation to the fractional excretion of uric acid. Postprandial suppression of glucagon secretion was absent in most FRG patients. Conclusions: We built a comprehensive map showing the impact of SLC5A2 variant type and variant location on glucosuria severity. Our results highlighted the role of key residues in maintaining the transport function of SGLT2 and the functional link between glucosuria and reabsorption of amino acid and uric acid in FRG patients.

Alignment-based Protein Mutational Landscape Prediction: Doing More with Less.

The wealth of genomic data has boosted the development of computational methods predicting the phenotypic outcomes of missense variants. The most accurate ones exploit multiple sequence alignments, which can be costly to generate. Recent efforts for democratizing protein structure prediction have overcome this bottleneck by leveraging the fast homology search of MMseqs2. Here, we show the usefulness of this strategy for mutational outcome prediction through a large-scale assessment of 1.5M missense variants across 72 protein families. Our study demonstrates the feasibility of producing alignment-based mutational landscape predictions that are both high-quality and compute-efficient for entire proteomes. We provide the community with the whole human proteome mutational landscape and simplified access to our predictive pipeline.

Systematic Approach Revealed SERPING1 Splicing-Affecting Variants to be Highly Represented in the Czech National HAE Cohort.

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare and life-threatening condition characterized by recurrent localized edema. We conducted a systematic screening of SERPING1 defects in a cohort of 207 Czech patients from 85 families with C1-INH-HAE. Our workflow involved a combined strategy of sequencing extended to UTR and deep intronic regions, advanced in silico prediction tools, and mRNA-based functional assays. This approach allowed us to detect a causal variant in all families except one and to identify a total of 56 different variants, including 5 novel variants that are likely to be causal. We further investigated the functional impact of two splicing variants, namely c.550 + 3A > C and c.686-7C > G using minigene assays and RT-PCR mRNA analysis. Notably, our cohort showed a considerably higher proportion of detected splicing variants compared to other central European populations and the LOVD database. Moreover, our findings revealed a significant association between HAE type 1 missense variants and a delayed HAE onset when compared to null variants. We also observed a significant correlation between the presence of the SERPING1 variant c.-21 T > C in the trans position to causal variants and the frequency of attacks per year, disease onset, as well as Clinical severity score. Overall, our study provides new insights into the genetic landscape of C1-INH-HAE in the Czech population, including the identification of novel variants and a better understanding of genotype-phenotype correlations. Our findings also highlight the importance of comprehensive screening strategies and functional analyses in improving the C1-INH-HAE diagnosis and management.

Genetic and geographic influence on phenotypic variation in European sarcoidosis patients.

Introduction: Sarcoidosis is a highly variable disease in terms of organ involvement, type of onset and course. Associations of genetic polymorphisms with sarcoidosis phenotypes have been observed and suggest genetic signatures. Methods: After obtaining a positive vote of the competent ethics committee we genotyped 1909 patients of the deeply phenotyped Genetic-Phenotype Relationship in Sarcoidosis (GenPhenReSa) cohort of 31 European centers in 12 countries with 116 potentially disease-relevant single-nucleotide polymorphisms (SNPs). Using a meta-analysis, we investigated the association of relevant phenotypes (acute vs. sub-acute onset, phenotypes of organ involvement, specific organ involvements, and specific symptoms) with genetic markers. Subgroups were built on the basis of geographical, clinical and hospital provision considerations. Results: In the meta-analysis of the full cohort, there was no significant genetic association with any considered phenotype after correcting for multiple testing. In the largest sub-cohort (Serbia), we confirmed the known association of acute onset with TNF and reported a new association of acute onset an HLA polymorphism. Multi-locus models with sets of three SNPs in different genes showed strong associations with the acute onset phenotype in Serbia and Lublin (Poland) demonstrating potential region-specific genetic links with clinical features, including recently described phenotypes of organ involvement. Discussion: The observed associations between genetic variants and sarcoidosis phenotypes in subgroups suggest that gene-environment-interactions may influence the clinical phenotype. In addition, we show that two different sets of genetic variants are permissive for the same phenotype of acute disease only in two geographic subcohorts pointing to interactions of genetic signatures with different local environmental factors. Our results represent an important step towards understanding the genetic architecture of sarcoidosis.

Frameshift coding sequence variants in the LPL gene: identification of two novel events and exploration of the genotype-phenotype relationship for variants reported to date.

BACKGROUND: Lipoprotein lipase (LPL) is the rate-limiting enzyme for triglyceride hydrolysis. Homozygous or compound heterozygous LPL variants cause autosomal recessive familial chylomicronemia syndrome (FCS), whereas simple heterozygous LPL variants are associated with hypertriglyceridemia (HTG) and HTG-related disorders. LPL frameshift coding sequence variants usually cause complete functional loss of the affected allele, thereby allowing exploration of the impact of different levels of LPL function in human disease. METHODS: All exons and flanking intronic regions of LPL were Sanger sequenced in patients with HTG-related acute pancreatitis (HTG-AP) or HTG-AP in pregnancy. Previously reported LPL frameshift coding sequence variants were collated from the Human Gene Mutation Database and through PubMed keyword searching. Original reports were manually evaluated for the following information: zygosity status of the variant, plasma LPL activity of the variant carrier, disease referred for genetic analysis, patient's age at genetic analysis, and patient's disease history. SpliceAI was employed to predict the potential impact of collated variants on splicing. RESULTS: Two novel rare variants were identified, and 53 known LPL frameshift coding sequence variants were collated. Of the 51 variants informative for zygosity, 30 were simple heterozygotes, 12 were homozygotes, and 9 were compound heterozygotes. Careful evaluation of the 55 variants with respect to their clinical and genetic data generated several interesting findings. First, we conclude that 6-7% residual LPL function could significantly delay the age of onset of FCS and reduce the prevalence of FCS-associated syndromes. Second, whereas a large majority of LPL frameshift coding sequence variants completely disrupt gene function through their "frameshift" nature, a small fraction of these variants may act wholly or partly as "in-frame" variants, leading to the generation of protein products with some residual LPL function. Third, we identified two candidate LPL frameshift coding sequence variants that may retain residual function based on genotype-phenotype correlation or SpliceAI-predicted data. CONCLUSIONS: This study reported two novel LPL variants and yielded new insights into the genotype-phenotype relationship as it pertains to LPL frameshift coding sequence variants.

Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene (ARSA) and categorized into three subtypes according to age of onset. The functional effect of most ARSA mutants remains unknown; better understanding of the genotype-phenotype relationship is required to support newborn screening (NBS) and guide treatment. RESULTS: We collected a patient data set from the literature that relates disease severity to ARSA genotype in 489 individuals with MLD. Patient-based data were used to develop a phenotype matrix that predicts MLD phenotype given ARSA alleles in a patient's genotype with 76% accuracy. We then employed a high-throughput enzyme activity assay using mass spectrometry to explore the function of ARSA variants from the curated patient data set and the Genome Aggregation Database (gnomAD). We observed evidence that 36% of variants of unknown significance (VUS) in ARSA may be pathogenic. By classifying functional effects for 251 VUS from gnomAD, we reduced the incidence of genotypes of unknown significance (GUS) by over 98.5% in the overall population. CONCLUSIONS: These results provide an additional tool for clinicians to anticipate the disease course in MLD patients, identifying individuals at high risk of severe disease to support treatment access. Our results suggest that more than 1 in 3 VUS in ARSA may be pathogenic. We show that combining genetic and biochemical information increases diagnostic yield. Our strategy may apply to other recessive diseases, providing a tool to address the challenge of interpreting VUS within genotype-phenotype relationships and NBS.

Case report: A novel STXBP1 splice variant and the landscape of splicing-involved STXBP1-related disorders.

STXBP1 variants are one of the most common genetic causes of neurodevelopmental disorders and epilepsy, wherein STXBP1-related disorders are characterized by neurodevelopmental abnormalities in 95% and seizures in 89% of affected patients. However, the spectrums of both genotype and phenotype are quite wide and diverse, with a high baseline variability even for recurrent STXBP1 variants. Until now, no clear genotype-phenotype correlations have been established and multiple disease mechanisms have been proposed for STXBP1-related disorders. Without an ascertained disease cause for many cases of STXBP1 variants, it is challenging to manage this disease in an effective manner and current symptom-based treatments are focused on seizure control only, which has a minimal impact on global development. A novel STXBP1 canonical splice variant, NM_001032221.4:c.578+2T>C, was reported in this study, together with detailed documentation of disease manifestations and treatment management. Further RNA expression analysis revealed abnormal intron retention and possible production of truncated STXBP1 proteins as a likely pathogenic mechanism. More importantly, the landscape of previously understudied STXBP1 splice variants and functional investigations was assessed for the first time to provide a context for the discussion of the complicated genotype-phenotype relationship of STXBP1-related disorders. Future cases of this disorder and a deeper mechanism-based understanding of its pathogenic cause are required for precision medicine and better disease management.

Polycyclic aromatic hydrocarbon (PAH) biodegradation capacity revealed by a genome-function relationship approach.

BACKGROUND: Polycyclic aromatic hydrocarbon (PAH) contamination has been a worldwide environmental issue because of its impact on ecosystems and human health. Biodegradation plays an important role in PAH removal in natural environments. To date, many PAH-degrading strains and degradation genes have been reported. However, a comprehensive PAH-degrading gene database is still lacking, hindering a deep understanding of PAH degraders in the era of big data. Furthermore, the relationships between the PAH-catabolic genotype and phenotype remain unclear. RESULTS: Here, we established a bacterial PAH-degrading gene database and explored PAH biodegradation capability via a genome-function relationship approach. The investigation of functional genes in the experimentally verified PAH degraders indicated that genes encoding hydratase-aldolase could serve as a biomarker for preliminarily identifying potential degraders. Additionally, a genome-centric interpretation of PAH-degrading genes was performed in the public genome database, demonstrating that they were ubiquitous in Proteobacteria and Actinobacteria. Meanwhile, the global phylogenetic distribution was generally consistent with the culture-based evidence. Notably, a few strains affiliated with the genera without any previously known PAH degraders (Hyphomonas, Hoeflea, Henriciella, Saccharomonospora, Sciscionella, Tepidiphilus, and Xenophilus) also bore a complete PAH-catabolic gene cluster, implying their potential of PAH biodegradation. Moreover, a random forest analysis was applied to predict the PAH-degrading trait in the complete genome database, revealing 28 newly predicted PAH degraders, of which nine strains encoded a complete PAH-catabolic pathway. CONCLUSIONS: Our results established a comprehensive PAH-degrading gene database and a genome-function relationship approach, which revealed several potential novel PAH-degrader lineages. Importantly, this genome-centric and function-oriented approach can overcome the bottleneck of conventional cultivation-based biodegradation research and substantially expand our current knowledge on the potential degraders of environmental pollutants.

The genetic architecture of behavioral canalization.

Behaviors are components of fitness and contribute to adaptive evolution. Behaviors represent the interactions of an organism with its environment, yet innate behaviors display robustness in the face of environmental change, which we refer to as 'behavioral canalization'. We hypothesize that positive selection of hub genes of genetic networks stabilizes the genetic architecture for innate behaviors by reducing variation in the expression of interconnected network genes. Robustness of these stabilized networks would be protected from deleterious mutations by purifying selection or suppressing epistasis. We propose that, together with newly emerging favorable mutations, epistatically suppressed mutations can generate a reservoir of cryptic genetic variation that could give rise to decanalization when genetic backgrounds or environmental conditions change to allow behavioral adaptation.

Genomic and phenotypic signatures of bacteriophage coevolution with the phytopathogen Pseudomonas syringae.

The rate and trajectory of evolution in an obligate parasite is critically dependent on those of its host(s). Adaptation to a genetically homogeneous host population should theoretically result in specialization, while adaptation to an evolving host population (i.e., coevolution) can result in various outcomes including diversification, range expansion, and/or local adaptation. For viruses of bacteria (bacteriophages, or phages), our understanding of how evolutionary history of the bacterial host(s) impacts viral genotypic and phenotypic evolution is currently limited. In this study, we used whole genome sequencing and two different metrics of phage impacts to compare the genotypes and phenotypes of lytic phages that had either coevolved with or were repeatedly passaged on an unchanging (ancestral) strain of the phytopathogen Pseudomonas syringae. Genomes of coevolved phages had more mutations than those of phages passaged on a constant host, and most mutations were in genes encoding phage tail-associated proteins. Phages from both passaging treatments shared some phenotypic outcomes, including range expansion and divergence across replicate populations, but coevolved phages were more efficient at reducing population growth (particularly of sympatric coevolved hosts). Genotypic similarity correlated with infectivity profile similarity in coevolved phages, but not in phages passaged on the ancestral host. Overall, while adaptation to either host type (coevolving or ancestral) led to divergence in phage tail proteins and infectivity patterns, coevolution led to more rapid molecular changes that increased bacterial killing efficiency and had more predictable effects on infectivity range. Together, these results underscore the important role of hosts in driving viral evolution and in shaping the genotype-phenotype relationship.

X-linked retinoschisis: mutation spectrum and genotype-phenotype relationship in an Italian pediatric cohort.

BACKGROUND: X-linked juvenile retinoschisis (×LRS) is an X-linked vitreoretinal degenerative disease that consists of variable phenotypes ranging from severe early-onset defects to subtle abnormalities diagnosed in elderly patients. XLRS is caused by a loss of function of the protein Retinoschisin (RS1), which is essential to preserve retinal integrity and function of photoreceptor-bipolar synapse. The literature data so far mostly agree on the absence of a clear genotype-phenotype correlation in XLRS. We reviewed clinical and molecular characteristics of a cohort of Italian pediatric XLRS patients to assess the presence of a correlation between genotype and phenotype severity. MATERIALS AND METHODS: We retrospectively examined clinical and genetic features of a cohort of 27 XLRS patients. In this study we included patients with a diagnosis of XLRS confirmed by fundus photography, spectral domain optical coherence tomography, and molecular analysis and with an onset of less than 10 years of age. We sorted RS1 variants according to their effect of RS1 structure and function in three separate groups. RESULTS: According to previous studies, we did not observe a conclusive genotype-phenotype correlation in our cohort; nevertheless, we noticed that patients harboring RS1 variants leading to RS1-secreted mutants show a more homogeneous phenotype, with an overall good visual acuity, compared to the other two groups. CONCLUSIONS: Our data support the hypothesis that secretion profile of RS1 could influence the severity of the phenotype. More extensive and functional studies are needed to acquire notions in view of the opportunity of gene replacement therapy for XLRS patients.

Genotype-Phenotype Relationship among 785 Unrelated White Women with Inherited Congenital Factor VII Deficiency: A Three-Center Database Study.

BACKGROUND: Congenital factor VII (FVII) deficiency, a rare bleeding disorder resulting from mutations in the F7 gene with autosomal recessive inheritance, exhibits clinical heterogeneity that lacks a strong correlation with FVII:C levels. The objective of this study was to discern genetic defects and assess their associations with the clinical phenotype in a substantial cohort comprising 785 white women exhibiting FVII:C levels below the age-dependent cut-off percentage. PATIENTS AND METHODS: Individuals with verified inherited factor VII deficiency underwent i) genotyping using the Sanger method and multiplex ligation-dependent probe amplification (MLPA) to identify F7 mutations, including common polymorphic variants. Additionally, they were ii) categorized based on clinical bleeding scores (BS). Thrombophilic variants and blood groups were also determined in the study participants. RESULTS: The probands in this study encompassed both asymptomatic individuals (referred for a laboratory investigation due to recurrent prolonged prothrombin time; n = 221) and patients who manifested mild, moderate, or severe bleeding episodes (n = 564). The spectrum of bleeding symptoms included epistaxis, gum bleeding, gastrointestinal bleeding, hematuria, postoperative bleeding, and gynecologic hemorrhage. The median ISTH bleeding score (BS) recorded within a two-year period prior to the work-up was 2 (0-17). Notably, this score was significantly higher in symptomatic women compared to their asymptomatic counterparts (3 versus 0; p < 0.001). The corresponding PBAC score before hormonal treatment stood at 225 (5-1200), exhibiting a positive correlation with the ISTH BS (rho = 0.38; p = 0.001). Blood group O was more prevalent in symptomatic women compared to asymptomatic individuals (58 versus 42%; p = 0.01). Among the 329 women (42%), known and novel mutations in the F7 gene, encompassing coding regions, exon/intron boundaries, and the promoter region, were identified, while common polymorphisms were detected in 647 subjects (95%). Logistic regression analysis, adjusted for clinical and laboratory data (including blood group, FVII activity, the presence of F7 gene mutations and/or polymorphisms, thrombophilia status, and additional factor deficiencies) revealed that older age at referral (increase per year) (odds/95% CI: 1.02/1.007-1.03), the presence of blood group O (odds/95% CI: 1.9/1.2-3.3), and the coexistence of further bleeding defects (odds/95% CI: 1.8/1.03-3.1) partially account for the differences in the clinical bleeding phenotype associated with FVII deficiency. CONCLUSION: The clinical phenotype in individuals with FVII deficiency is impacted by factors such as age, blood group, and the concurrent presence of other bleeding defects.

Computational approaches for predicting variant impact: An overview from resources, principles to applications.

One objective of human genetics is to unveil the variants that contribute to human diseases. With the rapid development and wide use of next-generation sequencing (NGS), massive genomic sequence data have been created, making personal genetic information available. Conventional experimental evidence is critical in establishing the relationship between sequence variants and phenotype but with low efficiency. Due to the lack of comprehensive databases and resources which present clinical and experimental evidence on genotype-phenotype relationship, as well as accumulating variants found from NGS, different computational tools that can predict the impact of the variants on phenotype have been greatly developed to bridge the gap. In this review, we present a brief introduction and discussion about the computational approaches for variant impact prediction. Following an innovative manner, we mainly focus on approaches for non-synonymous variants (nsSNVs) impact prediction and categorize them into six classes. Their underlying rationale and constraints, together with the concerns and remedies raised from comparative studies are discussed. We also present how the predictive approaches employed in different research. Although diverse constraints exist, the computational predictive approaches are indispensable in exploring genotype-phenotype relationship.

Novel LRP6 Mutations Causing Non-Syndromic Oligodontia.

The process of tooth formation is a series of reciprocal interactions between the ectoderm and mesoderm, and it is believed that many genetic factors are involved in this complex process. More than a dozen genes have been identified in non-syndromic tooth agenesis; however, the genetic etiology underlying tooth agenesis is not fully understood yet. In this study, we identified two novel LRP6 mutations in two non-syndromic oligodontia families. Both probands had 16 and 17 missing teeth in their permanent dentition. Mutational analysis identified a de novo frameshift mutation by a 1-bp insertion in exon 9 (NM_002336.2: c.1870dupA, p.(Met624Asnfs*29)) and a splicing donor site mutation in intron 8 (c.1762+2T>C). An in vitro splicing assay confirmed the deletion of exon 8, and the deletion would result in a frameshift. Due to the premature termination codons introduced by the frameshift, both mutant transcripts would be degraded by nonsense-mediated mRNA decay, resulting in haploinsufficiency.