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'Candidatus Phytoplasma solani' (CPs), a phytoplasma endemic to the Euro-Mediterranean basin is a causative agent of several plant diseases, including the grapevine yellows disease "bois noir" (BN). As different CPs strains have been shown to have different ecological reservoirs and pathways for spread, the genetic characterization of CPs strains is a prerequisite, and better control of BN relies on the identification of reservoir plants. The variability of the phytoplasma genotypes involved in the BN pathosystem in Croatian vineyards was assessed by a multilocus sequence typing (MLST) approach. The genotyping was performed on selected grapevine, wild plants, and insects collected within the eleven years of national survey conducted in all Croatian viticultural regions. The extensive tuf, secY, stamp, and vmp1 genes-based MLST analyses revealed two new genotypes for stamp and vmp1 genes, designated as ST59 and V28, respectively, and overall identified 28 different CPs MLST genotypes. The prevalent MLST genotype in grapevine CPsSqt21 (S6/ST6/V18/tuf-b2) was widespread in nine counties across Uplands, Slavonia, and Danube wine regions and was affiliated to the known vector Hyalesthes obsoletus and to Urtica dioica. The other two most frequent genotypes were the U. dioica-associated CPsSqt28 (S39/ST46/V3/tuf-a) and the C. arvensis-associated CPsSqt2 (S1/ST9/V4/tuf-b1). CPs of different vmp1 genotypes was also detected in Cixius wagneri specimens originating from different parts of Croatia. In addition, CPs was detected in several Dichtyophara europaea insects and in two new potential plant reservoirs Ailanthus altissima and Robinia pseudoacacia. The substantial number of found MLST genotypes indicates the presence of several independent epidemiological cycles and is certainly a consequence of a unique geographical position of Croatia, bridging the different eco-climatic areas of central and south-eastern Europe. [ABSTRACT FROM AUTHOR]

Abstract 'Candidatus Phytoplasma solani’ (CPs), a phytoplasma endemic to the Euro-Mediterranean basin is a causative agent of several plant diseases, including the grapevine yellows disease “bois noir” (BN). As different CPs strains have been shown to have different ecological reservoirs and pathways for spread, the genetic characterization of CPs strains is a prerequisite, and better control of BN relies on the identification of reservoir plants. The variability of the phytoplasma genotypes involved in the BN pathosystem in Croatian vineyards was assessed by a multilocus sequence typing (MLST) approach. The genotyping was performed on selected grapevine, wild plants, and insects collected within the eleven years of national survey conducted in all Croatian viticultural regions. The extensive tuf, secY, stamp, and vmp1 genes-based MLST analyses revealed two new genotypes for stamp and vmp1 genes, designated as ST59 and V28, respectively, and overall identified 28 different CPs MLST genotypes. The prevalent MLST genotype in grapevine CPsSqt21 (S6/ST6/V18/tuf-b2) was widespread in nine counties across Uplands, Slavonia, and Danube wine regions and was affiliated to the known vector Hyalesthes obsoletus and to Urtica dioica. The other two most frequent genotypes were the U. dioica-associated CPsSqt28 (S39/ST46/V3/tuf-a) and the C. arvensis-associated CPsSqt2 (S1/ST9/V4/tuf-b1). CPs of different vmp1 genotypes was also detected in Cixius wagneri specimens originating from different parts of Croatia. In addition, CPs was detected in several Dichtyophara europaea insects and in two new potential plant reservoirs Ailanthus altissima and Robinia pseudoacacia. The substantial number of found MLST genotypes indicates the presence of several independent epidemiological cycles and is certainly a consequence of a unique geographical position of Croatia, bridging the different eco-climatic areas of central and south-eastern Europe.

A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the N-glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin's origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants. [ABSTRACT FROM AUTHOR]

Migraine is a severe, debilitating neurovascular disorder. Hemiplegic migraine (HM) is a rare and debilitating neurological condition with a strong genetic basis. Sequencing technologies have improved the diagnosis and our understanding of the molecular pathophysiology of HM. Linkage analysis and sequencing studies in HM families have identified pathogenic variants in ion channels and related genes, including CACNA1A, ATP1A2, and SCN1A, that cause HM. However, approximately 75% of HM patients are negative for these mutations, indicating there are other genes involved in disease causation. In this review, we explored our current understanding of the genetics of HM. The evidence presented herein summarises the current knowledge of the genetics of HM, which can be expanded further to explain the remaining heritability of this debilitating condition. Innovative bioinformatics and computational strategies to cover the entire genetic spectrum of HM are also discussed in this review. [ABSTRACT FROM AUTHOR]

Background: Biotinidase deficiency (BTD) is a rare autosomal recessive metabolic disease, which develops neurological symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. The clinical features and mutation analysis of Pakistani children with BTD deficiency have rarely been described. Herein, for the first time, we report the clinical features, BTD gene mutations and biochemical analysis of seven symptomatic children with BTD deficiency from Pakistan. Methods: Seven suspected BTD‐deficient patients who presented abnormal organic acid profiles and clinical features were subjected to Sanger sequencing to identify pathogenic mutations in the BTD gene. The results were analyzed by Mutation Surveyor Software. Results: All seven patients exhibited common biotinidase deficiency symptoms including hypotonia, developmental delay and seizures. Biochemical analysis shows marked excretion of 3‐hydroxy isovalerate in all cases, followed by 3‐hydroxy propionate and methyl citrate. Sanger sequencing revealed one frame‐shift mutation, c.98_104delinsTCC (p.Cys33Phefs), and two missense mutations, c.1612C>A (p.Arg538Ser) and c.1330G>C (p.Asp444His). All mutations were in the homozygous state and classified as pathogenic in published studies and mutation databases. Conclusions: This study has validated the BTD variants as the underlying cause of biotinidase deficiency in which molecular testing of BTD is supported by urinary organic acid analysis and clinical diagnosis. Secondly, the strength of the local availability of this test in Pakistan will paved the way for the neonatal screening of biotinidase deficiency. [ABSTRACT FROM AUTHOR]

A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the N-glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin’s origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants.

Abstract Objectives Allele counts of sequence variants obtained by whole genome sequencing (WGS) often play a central role in interpreting the results of genetic and genomic research. However, such variant counts are not readily available for individuals in the Danish population. Here, we present a dataset with allele counts for sequence variants (single nucleotide variants (SNVs) and indels) identified from WGS of 8,671 (5,418 females) individuals from the Danish population. The data resource is based on WGS data from three independent research projects aimed at assessing genetic risk factors for cardiovascular, psychiatric, and headache disorders. To enable the sharing of information on sequence variation in Danish individuals, we created summarized statistics on allele counts from anonymized data and made them available through the European Genome-phenome Archive (EGA, https://identifiers.org/ega.dataset:EGAD00001009756 ) and in a dedicated browser, DanMAC5 (available at www.danmac5.dk ). The summary level data and the DanMAC5 browser provide insight into the allelic spectrum of sequence variants segregating in the Danish population, which is important in variant interpretation. Data description Three WGS datasets with an average coverage of 30x were processed independently using the same quality control pipeline. Subsequently, we summarized, filtered, and merged allele counts to create a high-quality summary level dataset of sequence variants.

Introduction: Genetic and environmental factors play a significant role in the development of Cardiovascular Disease (CVD). With advancements in molecular techniques, it is now possible to study genes that may provide evidence of the role of genetic factors in Coronary Artery Disease (CAD). Genetic variants of the Apo A-1 gene might play an important role in regulating lipid profiles and their alteration, leading to the subsequent development of Myocardial Infarction (MI). Aim: The aim of this study was to evaluate the association between C+83T sequence variations in the first intron of the Apo A-1 gene and lipid variables in patients with MI. Materials and Methods: A case-control study was conducted at the Department of Biochemistry in association with the Department of Cardiology, Kilpauk Medical College Hospital, Chennai, Tamil Nadu, India, from October 2013 to March 2014. The study included 52 MI cases and 52 age-, gender-, and risk factor-matched controls. Fasting venous blood samples were collected from each patient, and routine investigations, lipid profile assessments, and polymorphic studies were carried out. Allele frequencies between cases and controls were compared using the Chi-square test. Analysis of Variance (ANOVA) was performed to determine the association between fasting serum lipid variables and allele distribution. Results: Both study groups were matched for age, gender, and risk factors such as alcohol consumption, smoking, Hypertension (HT), and Diabetes Mellitus (DM). The results were compared and found to be statistically insignificant. Genotype analysis between the groups disclosed that the TT homozygous genotype was more prevalent in cases, while the ‘CC’ genotype was more prevalent in controls, and the CT genotype was approximately equal in both cases and controls. The genotype difference between cases and controls was statistically significant (p-value=0.006). The ‘T’ allele frequency was higher among cases (0.36) compared to controls (0.13), while the frequency of the ‘C’ allele was higher among controls (0.86) compared to cases (0.63). In the CC genotype, there was a higher mean value of Apo A-1 and a lower ApoB/Apo A-1 ratio compared to the TT genotype. It was observed that carriers of the C allele and T allele showed no statistical difference in lipid variables except for High-density Lipoprotein (HDL). Conclusion: This study found a significantly increased T allele frequency in cases compared to controls, suggesting that the presence of the T allele in the C+83T (first intron) of the Apo A-1 gene may increase the risk of developing MI.

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An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold. [ABSTRACT FROM AUTHOR]

Background: Genetic mutations of IKZF1 have been frequently delineated in B‐lineage acute leukaemia (B‐ALL) but rarely elucidated in acute myeloid leukaemia (AML). IKZF1 mutations confer a poor prognosis in AML, and hotspot mutations of IKZF1, N159Y and N159S tend to occur in B‐ALL and AML respectively. However, the pathogenesis of IKZF1 N159S in AML and IKZF1 lineage susceptibility are largely unknown. Methods: The genetic and clinical characteristics of IKZF1‐mutated AML patients were evaluated. Multi‐omics analysis and functional assays were performed in vitro using IKZF1 mutations knock‐in AML cell lines. Results: 23 (4.84%) small sequence variants of IKZF1 were identified in 475 newly diagnosed AML (non‐M3) patients. Based on RNA sequencing, three classes of IKZF1‐related AML were defined, including 9 patients (39.13%) with IKZF1 N159S mutations, 10 (43.47%) with CEBPA mutations and 4 others (17.39%). IKZF1 N159S may define a unique subgroup with higher HOXA/B expression and native B‐cell immune fractions. Gene expression data of multiple knock‐in cell lines indicate that the lymphocyte differentiation‐related MME and CD44 kept high expression in IKZF1 N159Y but were downregulated in N159S. CUT&TAG sequencing showed that IKZF1 N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis of IKZF1 N159S may depend on the deregulation of several cofactors, such as oncogenic MYC and CPNE7 targets. Conclusions: Collectively, we dissected the molecular spectrum and clinical features of IKZF1‐related AML, which may promote an in‐depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML. [ABSTRACT FROM AUTHOR]

Objectives: Allele counts of sequence variants obtained by whole genome sequencing (WGS) often play a central role in interpreting the results of genetic and genomic research. However, such variant counts are not readily available for individuals in the Danish population. Here, we present a dataset with allele counts for sequence variants (single nucleotide variants (SNVs) and indels) identified from WGS of 8,671 (5,418 females) individuals from the Danish population. The data resource is based on WGS data from three independent research projects aimed at assessing genetic risk factors for cardiovascular, psychiatric, and headache disorders. To enable the sharing of information on sequence variation in Danish individuals, we created summarized statistics on allele counts from anonymized data and made them available through the European Genome-phenome Archive (EGA, https://identifiers.org/ega.dataset:EGAD00001009756) and in a dedicated browser, DanMAC5 (available at www.danmac5.dk). The summary level data and the DanMAC5 browser provide insight into the allelic spectrum of sequence variants segregating in the Danish population, which is important in variant interpretation. Data description: Three WGS datasets with an average coverage of 30x were processed independently using the same quality control pipeline. Subsequently, we summarized, filtered, and merged allele counts to create a high-quality summary level dataset of sequence variants. [ABSTRACT FROM AUTHOR]

• c.919A>G genotypes AG/GG are associated with higher CPR versus genotype AA • c.919A>G genotypes AG/GG are associated with higher LBR versus genotype AA • c.2039A>G genotype GG is associated with lower CLBR versus genotype AA Is there an association between FSHR sequence variants and reproductive outcomes following IVF in predicted normoresponders? Multicentre prospective cohort study conducted from November 2016 to June 2019 in Vietnam, Belgium and Spain including patients aged <38 years, and undergoing IVF with a predicted normal response with fixed-dose 150 IU rFSH in an antagonist protocol. Genotyping was performed for three FSHR (c.919A>G, c.2039A>G, c.-29G>A) and one FSHB sequence variants (c.-211G>T). Clinical pregnancy rate (CPR), live birth rate (LBR) and miscarriage rate in the first embryo transfer and cumulative live birth rate (CLBR) were compared between the different genotypes. A total of 351 patients underwent at least one embryo transfer. Genetic model analysis that adjusted for patient age, body mass index, ethnicity, type of embryo transfer, embryo stage and number of top-quality embryos transferred revealed a higher CPR for homozygous patients for the variant allele G of c.919A>G when compared to patients with genotype AA (60.3% versus 46.3%, adjusted odds ratio [ORadj] 1.96, 95% confidence interval [CI] 1.09–3.53). Also, c.919A>G genotypes AG and GG presented a higher CPR and LBR when compared with genotype AA (59.1% versus 46.3%, ORadj 1.80, 95% CI 1.08–3.00, and 51.3% versus 39.0%, ORadj 1.69, 95% CI 1.01–2.80, respectively). Cox regression models revealed a statistically significantly lower CLBR for c.2039A>G genotype GG in the codominant model (hazard ratio [HR] 0.66, 95% CI 0.43–0.99). These results demonstrate a previously unreported association between variant c.919A>G genotype GG and higher CPR and LBR in infertile patients and reinforce a potential role for genetic background in predicting the reproductive prognosis following IVF. [ABSTRACT FROM AUTHOR]

Detecting and identifying viral infections in perennial plants, such as grapevines, can be challenging. Therefore, the aim of this study was to perform a real-time RT-PCR (RT-qPCR) high-resolution melting (HRM) curve analysis to detect and differentiate Brazilian variants of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine fanleaf virus (GFLV) in 74 and 10 infected plants, respectively, maintained in a collection block of grapevines. A single amplification curve was generated for each sample by RT-qPCR. Considering the amplified region of genomes of these two viruses, it was possible to identify and distinguish different variants of GLRaV-3 and of GFLV, which showed significantly different melting temperature (Tm) values between themselves, reflecting differences in the nucleotide sequences of the respective amplicons, and allowing discriminating variants and assess the viral diversity in grapevine accessions. The HRM analysis was validated by sequencing and nucleotide comparisons among Brazilian isolates of GLRaV-3 and GFLV.

Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow. [ABSTRACT FROM AUTHOR]

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene has come to prominence due to its reported function in the clearance of low‐density lipoprotein cholesterol. The vervet monkey (Chlorocebus aethiops) was utilized to study the genetics of PCSK9 gene. Method: Sixteen vervet monkeys were selected to screen for possible PCSK9 polymorphisms and to determine gene expression. Results: Four PCSK9 sequence variants (T112T, R148S, H177N and G635G) were identified and three of these variants (H177N, R148S, and G635G) were categorized as loss of function mutations. A decline in gene expression levels was also observed in animals harboring these three variants. Although the selected variants might have affected the level of gene expression in the selected animals, individual variation was also noticed in some of these individuals with the G635G variant. Conclusion: Based on the findings obtained from this study, it is suggestive that the activity of PCSK9 was hindered. [ABSTRACT FROM AUTHOR]

Abstract Background Genetic mutations of IKZF1 have been frequently delineated in B‐lineage acute leukaemia (B‐ALL) but rarely elucidated in acute myeloid leukaemia (AML). IKZF1 mutations confer a poor prognosis in AML, and hotspot mutations of IKZF1, N159Y and N159S tend to occur in B‐ALL and AML respectively. However, the pathogenesis of IKZF1 N159S in AML and IKZF1 lineage susceptibility are largely unknown. Methods The genetic and clinical characteristics of IKZF1‐mutated AML patients were evaluated. Multi‐omics analysis and functional assays were performed in vitro using IKZF1 mutations knock‐in AML cell lines. Results 23 (4.84%) small sequence variants of IKZF1 were identified in 475 newly diagnosed AML (non‐M3) patients. Based on RNA sequencing, three classes of IKZF1‐related AML were defined, including 9 patients (39.13%) with IKZF1 N159S mutations, 10 (43.47%) with CEBPA mutations and 4 others (17.39%). IKZF1 N159S may define a unique subgroup with higher HOXA/B expression and native B‐cell immune fractions. Gene expression data of multiple knock‐in cell lines indicate that the lymphocyte differentiation‐related MME and CD44 kept high expression in IKZF1 N159Y but were downregulated in N159S. CUT&TAG sequencing showed that IKZF1 N159S reshaped the binding profiles of IKZF1. Integration analysis suggested that the pathogenesis of IKZF1 N159S may depend on the deregulation of several cofactors, such as oncogenic MYC and CPNE7 targets. Conclusions Collectively, we dissected the molecular spectrum and clinical features of IKZF1‐related AML, which may promote an in‐depth understanding of the pathogenesis, lineage susceptibility and clinical research of AML.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre‐mRNA splicing via disruption/creation of splicing motifs such as 5′/3′ splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high‐throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/ [ABSTRACT FROM AUTHOR]

In the present study, we further analyzed the data obtained in our previous study, where we investigated the cell-free DNA (cfDNA) of 34 progressive prostate cancer patients via targeted sequencing. Here, we studied the occurrence and prognostic impact of sequence variants according to their clinical pathological significance (CPS) or their functional impact (FI) in 23 DNA damage repair (DDR) genes with a focus on the ATM serine/threonine kinase gene (ATM). All patients had at least one DDR gene with a CPS or FI variant. Kaplan-Meier analysis indicated that the group with a higher number of CPS variants in DDR genes had a shorter time to treatment change (TTC) compared to the group with a lower number of CPS variants (p = 0.038). Analysis of each DDR gene revealed that CPS variants in the ATM gene and FI variants in the nibrin (NBN) gene showed a shorter TTC (p = 0.034 and p = 0.042). In addition, patients with CPS variants in the ATM gene had shorter overall survival (OS; p = 0.022) and disease-specific survival (DSS; p = 0.010) than patients without these variants. Interestingly, patients with CPS variants in seven DDR genes possessed a better OS (p = 0.008) and DSS (p = 0.009), and patients with FI variants in four DDR genes showed a better OS (p = 0.007) and DSS (p = 0.008). Together, these findings demonstrated that the analysis of cfDNA for gene variants in DDR genes provides prognostic information that may be helpful for future temporal and targeted treatment decisions for advanced PCa patients. [ABSTRACT FROM AUTHOR]

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare, severe subtype of epidermolysis bullosa (EB), characterized by blisters and miliary rashes of the skin. Dystrophic EB (DEB) includes variants inherited both in an autosomal-dominant or autosomal-recessive manner. Recessive dystrophic EB (RDEB) is divided into many subtypes and prevails as a result of biallelic genetic mutations in COL7A1 gene encoding type VII collagen, a major stabilizing molecule of the dermo-epidermal junction. The blister formation is mainly due to the variable structural and functional impairment of anchoring fibrils in VII collagen (COLVII), responsible for the adhesion of the epidermis to the dermis. Method: Three Pakistani families (A, B and C) affected with congenital dystrophic epidermolysis bullosa were recruited in the present study. The whole-exome sequencing (WES) approach was utilized for the detection of the pathogenic sequence variants in probands. The segregation of these variants in other participants was confirmed by Sanger sequencing. Results: This study identified a novel missense variant c.7034G>A, p. Gly2345Asp in exon 91, a novel Frameshift mutation c.385del (p. His129MetfsTer18) in a homozygous form in exon no 3, and a previously known nonsense variation (c.1573 C>T; p. Arg525Ter) in exon 12 of COL7A1 gene in families A, B, and C, respectively, as causative mutations responsible for dystrophic epidermolysis bullosa in these families. Conclusion: Our study validates the involvement of the COL7A1 gene in the etiology of dystrophic epidermolysis bullosa. It further expands the COL7A1 gene mutation database and provides an additional scientific basis for diagnosis, genetic counseling, and prognosis purposes for EB patients. [ABSTRACT FROM AUTHOR]

Abstract Background Reference-guided read alignment and variant genotyping are prone to reference allele bias, particularly for samples that are greatly divergent from the reference genome. A Hereford-based assembly is the widely accepted bovine reference genome. Haplotype-resolved genomes that exceed the current bovine reference genome in quality and continuity have been assembled for different breeds of cattle. Using whole genome sequencing data of 161 Brown Swiss cattle, we compared the accuracy of read mapping and sequence variant genotyping as well as downstream genomic analyses between the bovine reference genome (ARS-UCD1.2) and a highly continuous Angus-based assembly (UOA_Angus_1). Results Read mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. We discovered 22,744,517 and 22,559,675 high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. The concordance between sequence- and array-called genotypes was high and the number of variants deviating from Hardy-Weinberg proportions was low at segregating sites for both assemblies. More artefactual INDELs were genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. Using the 161 sequenced Brown Swiss cattle as a reference panel, we imputed sequence variant genotypes into a mapping cohort of 30,499 cattle that had microarray-derived genotypes using a two-step imputation approach. The accuracy of imputation (Beagle R2) was very high (0.87) for both assemblies. Genome-wide association studies between imputed sequence variant genotypes and six dairy traits as well as stature produced almost identical results from both assemblies. Conclusions The ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. Although differences in read mapping and genotyping accuracy between both assemblies are negligible, the choice of the reference genome has a large impact on detecting signatures of selection that already reached fixation using the composite likelihood ratio test. We developed a workflow that can be adapted and reused to compare the impact of reference genomes on genome analyses in various breeds, populations and species.

Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow.

Avian influenza A virus subtype H9N2 has been in circulation since last two decades in poultry flocks of Pakistan. It is causing immense economic losses to the farmers. Avian influenza H9N2 virus were isolated from infected birds of different poultry farms in the Province of Punjab, Pakistan. Segment eight gene of the virus was amplified using RT-PCR and sequenced to analyze mutations in this viral segment. Phylogenetic tree analysis showed sequences from 2015 to 2017 form a single evolving clade. Valdar residues conservation scores by multiple sequence alignment showed the C terminal region of NEP protein is conserved while C terminal region of effector domain (ED) of NSI protein exhibit mutations. These mutations are enhancing the total hydrophobicity of the molecules. Hydrophobicity was calculated by using Kyte and Doolittle method. High hydrophobicity of NSI protein is also posing a potential for H9N2 virus to adapt in host, which might be contributing to the increase in pathogenicity of the circulating H9N2 virus. [ABSTRACT FROM AUTHOR]

The SARS‐CoV‐2 spike (S) protein facilitates viral infection, and has been the focus of many structure determination efforts. Its flexible loop regions are known to be involved in protein binding and may adopt multiple conformations. This article identifies the S protein loops and studies their conformational variability based on the available Protein Data Bank structures. While most loops had essentially one stable conformation, 17 of 44 loop regions were observed to be structurally variable with multiple substantively distinct conformations based on a cluster analysis. Loop modeling methods were then applied to the S protein loop targets, and the prediction accuracies discussed in relation to the characteristics of the conformational clusters identified. Loops with multiple conformations were found to be challenging to model based on a single structural template. [ABSTRACT FROM AUTHOR]

In our study, four single nucleotide polymorphisms (SNPs) were identified in exon 2 of cofilin-1 (CFL1) gene in 488 Chinese Qinchuan (QC) cattle, which included two missense mutations T 2084G and G 2107C, two synonymous mutations T 2052C and T 2169C. Further, we evaluated haplotype frequency and linkage disequilibrium (LD) coefficient of four SNPs. At SNP T 2052C, G 2107C and T 2169C, the QC cattle population belonged to intermediate genetic diversity (0.25 < PIC-value < 0.5), whereas SNP T-2084G belonged to low polymorphism (PIC-value < 0.25). Haplotype analysis showed that 6 different haplotypes (frequency > 0.03). LD analysis showed that SNP G 2107C and T 2169C, SNP G 2107C and T 2084G were high LD, respectively (r2 > 0.33). Association analysis indicated that SNP T 2052C was significantly associated with body length, chest breadth, chest depth and body mass in the QC population (p < 0.01 or p < 0.05). SNP G 2107C was significantly associated with rump length (p < 0.05). SNP T 2169C was significantly associated with chest breadth and chest depth (p <.01 or p <.05). The results of our study suggest that the CFL1 gene may be a strong candidate gene that affects growth traits in the QC cattle breeding program. [ABSTRACT FROM AUTHOR]

Abstract Background Usher syndrome (USH) is the most prevalent cause of the human genetic deafness and blindness. USH type II (USH2) is the most common form of USH, and USH2A is the major pathogenic gene for USH2. For expanding the spectrum of USH2A mutations and further revealing the role of USH2A in USH2, we performed the USH2A gene variant screening in Chinese patients with USH2. Methods Genomic DNA was extracted from peripheral blood of unrelated Chinese USH2 patients, we designed specific primers for amplifying the coding region (exons 2–72) of the USH2A gene. Sanger sequencing was used to study alleles. Silico prediction tools were used to predict the pathogenicity of the variants identified in these patients. Results Five heterozygous pathogenic variants were detected in four patients. Two patients were found to have two-mutations and two patients only have one. Two novel variants c.4217C > A (p.Ser1406X) and c.11780A > G (p.Asp3927Gly)) were predicted deleterious by computer prediction algorithms. In addition, three reported mutations (c.8559-2A > G, c.8232G > C and c.11389 + 3A > T) were also found in this study. Conclusions We identified five heterozygous pathogenic variants in the USH2A gene in Chinese patients diagnosed with Usher syndrome type 2, two of which were not reported. It expands the spectrum of USH2A variants in USH.

Abstract Background APOC3 is important in lipid transport and metabolism with limited studies reporting genetic sequence variations in specific ethnic groups. The present study aimed to analyze the full APOC3 sequence among Kuwaiti Arabs and test the association of selected variants with lipid levels and BMI. Methods Variants were identified by Sanger sequencing the entire APOC3 gene in 100 Kuwaiti Arabs. Variants and their genotypes were fully characterized and used to construct haplotype blocks. Four variants (rs5128, rs2854117, rs2070668, KUAPOC3N3 g.5196 A > G) were selected for testing association with serum lipid levels and BMI in a cohort (n = 733). Results APOC3 sequence (4.3 kb) of a Kuwaiti Arab was deposited in Genbank (accession number KJ437193). Forty-two variants including 3 novels were identified including an “A” insertion at genomic positions 116,700,599–116,700,600 (promoter region) and two substitutions in intron 1 at genomic positions 116,700,819 and 116,701,159. Only three variants, (rs5128, rs2854117, and rs2070668) were analyzed for association of which rs5128 showed a trend for association with increased BMI, TG and VLDL levels that was further investigated using multivariate analysis. A significant association of rs5128 with BMI (p G for population stratification of Arabs.

Abstract Background Oxytocin receptor (OXTR) gene variants have been shown to affect the prevalence of preterm birth, mode of delivery and oxytocin (OXT) requirements for labor induction and augmentation. We hypothesized that this might be associated with different myometrium responses to oxytocin. Our aim was to investigate the influence of a selection of eight OXTR gene single nucleotide variants on oxytocin-induced stimulation of human myometrium contractility in vitro. Methods Human myometrium biopsies were collected during elective cesarean sections at term, if patients had given informed consent. Myometrial strips were submerged under tension in an organ bath and allowed to contract; the remaining material was stored at − 80 °C for further determination of relevant genetics and mRNA level. The area under the curve (AUC) of all contractions taking place in the absence of OXT and of those occurring upon OXT addition (for 30 min each) was measured. OXT stimulation, defined as the ratio between AUC measurements after OXT addition and those in the absence of OXT was calculated for each strip. TaqMan™ Assays were used to detect the allele distribution of the eight OXTR variants and to determine the relative amounts of OXTR-mRNA in the samples. For each variant, oxytocin stimulation of contractility was compared between samples homozygous for the reference allele (reference group) and samples with at least one variant allele (variant group) by linear regression. Results Sixty samples were included in the present study. For rs1042778, rs11706648, rs4686301, rs53576, rs237895, and rs237902, OXT stimulation was similar in the reference and in the variant groups. However, the values of OXT stimulation differed significantly between the reference and the variant groups for rs4686302 (3.1 vs. 4.1 times; p = 0.022) and rs237888 (3.2 vs. 5.5 times; p = 0.001). No significant differences between the levels of OXTR-mRNA in the various reference and corresponding variant groups were detected. Conclusions Patients with variant alleles of rs237888 and/or rs4686302 may be more sensitive to oxytocin stimulation, explaining why these sequence variants have been associated with lower cesarean section prevalence and premature birth, respectively.

In the present study, we further analyzed the data obtained in our previous study, where we investigated the cell-free DNA (cfDNA) of 34 progressive prostate cancer patients via targeted sequencing. Here, we studied the occurrence and prognostic impact of sequence variants according to their clinical pathological significance (CPS) or their functional impact (FI) in 23 DNA damage repair (DDR) genes with a focus on the ATM serine/threonine kinase gene (ATM). All patients had at least one DDR gene with a CPS or FI variant. Kaplan-Meier analysis indicated that the group with a higher number of CPS variants in DDR genes had a shorter time to treatment change (TTC) compared to the group with a lower number of CPS variants (p = 0.038). Analysis of each DDR gene revealed that CPS variants in the ATM gene and FI variants in the nibrin (NBN) gene showed a shorter TTC (p = 0.034 and p = 0.042). In addition, patients with CPS variants in the ATM gene had shorter overall survival (OS; p = 0.022) and disease-specific survival (DSS; p = 0.010) than patients without these variants. Interestingly, patients with CPS variants in seven DDR genes possessed a better OS (p = 0.008) and DSS (p = 0.009), and patients with FI variants in four DDR genes showed a better OS (p = 0.007) and DSS (p = 0.008). Together, these findings demonstrated that the analysis of cfDNA for gene variants in DDR genes provides prognostic information that may be helpful for future temporal and targeted treatment decisions for advanced PCa patients.

Objective: To evaluate comprehensively, using chromosomal microarray analysis (CMA) and exome sequencing (ES), the prevalence of chromosomal abnormalities and sequence variants in unselected fetuses with congenital heart defect (CHD) and to evaluate the potential diagnostic yields of CMA and ES for different CHD subgroups.Methods: This was a study of 360 unselected singleton fetuses with CHD detected by echocardiography, referred to our department for genetic testing between February 2018 and December 2019. We performed CMA, as a routine test for aneuploidy and copy number variations (CNV), and then, in cases without aneuploidy or pathogenic CNV on CMA, we performed ES.Results: Overall, positive genetic diagnoses were made in 84 (23.3%) fetuses: chromosomal abnormalities were detected by CMA in 60 (16.7%) and sequence variants were detected by ES in a further 24 (6.7%) cases. The detection rate of pathogenic and likely pathogenic genetic variants in fetuses with non-isolated CHD (32/83, 38.6%) was significantly higher than that in fetuses with isolated CHD (52/277, 18.8%) (P < 0.001), this difference being due mainly to the difference in frequency of aneuploidy between the two groups. The prevalence of a genetic defect was highest in fetuses with an atrioventricular septal defect (36.8%), ventricular septal defect with or without atrial septal defect (28.4%), conotruncal defect (22.2%) or right ventricular outflow tract obstruction (20.0%). We also identified two novel missense mutations (c.2447G>C, p.Arg816Pro; c.1171C>T, p.Arg391Cys) and a new phenotype caused by variants in PLD1.Conclusions: Chromosomal abnormalities were identified in 16.7% and sequence variants in a further 6.7% of fetuses with CHD. ES should be offered to all pregnant women with a CHD fetus without chromosomal abnormality or pathogenic CNV identified by CMA, regardless of whether the CHD is isolated. © 2020 International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Background: Reference-guided read alignment and variant genotyping are prone to reference allele bias, particularly for samples that are greatly divergent from the reference genome. A Hereford-based assembly is the widely accepted bovine reference genome. Haplotype-resolved genomes that exceed the current bovine reference genome in quality and continuity have been assembled for different breeds of cattle. Using whole genome sequencing data of 161 Brown Swiss cattle, we compared the accuracy of read mapping and sequence variant genotyping as well as downstream genomic analyses between the bovine reference genome (ARS-UCD1.2) and a highly continuous Angus-based assembly (UOA_Angus_1). Results: Read mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. We discovered 22,744,517 and 22,559,675 high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. The concordance between sequence- and array-called genotypes was high and the number of variants deviating from Hardy-Weinberg proportions was low at segregating sites for both assemblies. More artefactual INDELs were genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. Using the 161 sequenced Brown Swiss cattle as a reference panel, we imputed sequence variant genotypes into a mapping cohort of 30,499 cattle that had microarray-derived genotypes using a two-step imputation approach. The accuracy of imputation (Beagle R2) was very high (0.87) for both assemblies. Genome-wide association studies between imputed sequence variant genotypes and six dairy traits as well as stature produced almost identical results from both assemblies. Conclusions: The ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. Although differences in read mapping and genotyping accuracy between both assemblies are negligible, the choice of the reference genome has a large impact on detecting signatures of selection that already reached fixation using the composite likelihood ratio test. We developed a workflow that can be adapted and reused to compare the impact of reference genomes on genome analyses in various breeds, populations and species. [ABSTRACT FROM AUTHOR]

Introduction of microbial contaminations in the dairy value chain starts at the farm level and the initial microbial composition may severely affect the production of high-quality dairy products. Therefore, understanding the farm-to-farm variation and longitudinal shifts in the composition of the bulk tank milk microbiota is fundamental to increase the quality and reduce the spoilage and waste of milk and dairy products. In this study, we performed a double experiment to study long- and short-term longitudinal shifts in microbial composition using 16S rRNA gene amplicon sequencing. We analyzed milk from 37 farms, that had also been investigated two years earlier, to understand the stability and overall microbial changes over a longer time span. In addition, we sampled bulk tank milk from five farms every 1–2 weeks for up to 7 months to observe short-term changes in microbial composition. We demonstrated that a persistent and farm-specific microbiota is found in bulk tank milk and that changes in composition within the same farm are mostly driven by bacterial genera associated with mastitis (e.g., Staphylococcus and Streptococcus). On a long-term, we detected that major shift in milk microbiota were not correlated with farm settings, such as milking system, number of cows and quality of the milk but other factors, such as weather and feeding, may have had a greater impact on the main shifts in composition of the bulk tank milk microbiota. Our results provide new information regarding the ecology of raw milk microbiota at the farm level.

Introduction of microbial contaminations in the dairy value chain starts at the farm level and the initial microbial composition may severely affect the production of high-quality dairy products. Therefore, understanding the farm-to-farm variation and longitudinal shifts in the composition of the bulk tank milk microbiota is fundamental to increase the quality and reduce the spoilage and waste of milk and dairy products. In this study, we performed a double experiment to study long- and short-term longitudinal shifts in microbial composition using 16S rRNA gene amplicon sequencing. We analyzed milk from 37 farms, that had also been investigated two years earlier, to understand the stability and overall microbial changes over a longer time span. In addition, we sampled bulk tank milk from five farms every 1–2 weeks for up to 7 months to observe short-term changes in microbial composition. We demonstrated that a persistent and farm-specific microbiota is found in bulk tank milk and that changes in composition within the same farm are mostly driven by bacterial genera associated with mastitis (e.g., Staphylococcus and Streptococcus). On a long-term, we detected that major shift in milk microbiota were not correlated with farm settings, such as milking system, number of cows and quality of the milk but other factors, such as weather and feeding, may have had a greater impact on the main shifts in composition of the bulk tank milk microbiota. Our results provide new information regarding the ecology of raw milk microbiota at the farm level. [ABSTRACT FROM AUTHOR]

Inherited retinal dystrophy (IRD) is a heterogenous blinding eye disease and affects more than 200,000 Americans and millions worldwide. By far, 270 protein-coding genes have been identified to cause IRD when defective. However, only one microRNA (miRNA), miR-204, has been reported to be responsible for IRD when a point-mutation occurs in its seed sequence. Previously, we identified that a conserved, polycistronic, paralogous miRNA cluster, the miR-183/96/182 cluster, is highly specifically expressed in all photoreceptors and other sensory organs; inactivation of this cluster in mice resulted in syndromic IRD with multi-sensory defects. We hypothesized that mutations in the miR-183/96/182 cluster in human cause IRD. To test this hypothesis, we perform mutation screening in the pre-miR-183, -96, -182 in >1000 peripheral blood DNA samples of patients with various forms of IRD. We identified six sequence variants, three in pre-miR-182 and three in pre-miR-96. These variants are in the pre-miRNA-182 or -96, but not in the mature miRNAs, and are unlikely to be the cause of the IRD in these patients. In spite of this, the nature and location of these sequence variants in the pre-miRNAs suggest that some may have impact on the biogenesis and maturation of miR-182 or miR-96 and potential roles in the susceptibility to diseases. Although reporting on negative results so far, our study established a system for mutation screening in the miR-183/96/182 cluster in human for a continued effort to unravel and provides deeper insight into the potential roles of miR-183/96/182 cluster in human diseases.

Rice blast, caused by Magnaporthe oryzae, is a destructive disease. A total of 23 rice genotypes, were screened using a set of 51 simple sequence repeats SSR/ InDel markers and 51 single nucleotide polymorphic (SNP) markers to evaluate the sequence variation. Six newly identified Malaysian isolates of M. oryzae were used for blast screening. The resistant genotypes, consisting of Pongsu Seribu 2, O. rufipogon accessions, Tetep and IR64 had a resistant disease score of 2/3 showing minimal symptoms of blast. In contrast, Mahsuri and MR264 were the most susceptible and had a disease score of 5. In total, 11 sequence variations and two putative haplotype blocks, were observed in the SNP data representing Pi54 and Pita. The SNPs for Pita and Pi54 revealed the presence of resistant alleles that generated reproducible and aggressive calls based on the Sequenom data, but when screened with SSR and INDel markers showed poor correspondence to phenotype effects. Therefore, SNPs for Pi54 and Pita were found to be the most reliable for marker-assisted selection. The blast-resistant alleles identified in this study indicate that they are involved in the defence response against diverse isolates and play a role in putative broad spectrum resistance. [ABSTRACT FROM AUTHOR]

Skeletal remains encountered frequently in forensic applications are a challenging specimen, since their DNA is usually degraded due to harsh conditions, limiting the utilization of skeletal DNA. Forensic scientists have tried various methods to extract DNA from skeletal remains of low quantity and poor quality or improve detecting technology for more information from compromised DNA. Compared with traditional capillary electrophoresis (CE), massively parallel sequencing (MPS) is more sensitive to shorter fragments, able to detect allele sequences for variations from core motif or flanking regions, and able to detect more markers with a higher discrimination power. In this study, short tandem repeats (STR) and single nucleotide polymorphisms (SNP) from 35 human skeletons were genotyped by MPS platform, and CE method was also used to perform STR genotyping. The results indicated that the detection rates reached 100.00% in 16 of 35 samples with MPS method, while the same 100.00% was reached in only 9 samples with CE. The success rates of MPS were also higher than that of CE method in shared 21 loci (excluding Y-indel, DYS391, and SE33), especially in loci detected by MPS method only. Besides, all SNPs (124 and 90 SNPs in males and females) were detected in 18 samples of 35 samples by MPS method. Some intra-allelic sequence variants were observed in eight loci (D21S11, D8S1179, D5S2800, D3S1358, vWA, D2S1338, D1S1656, D12S391) using MPS technology. Interestingly, there is a sample showing genotyping disagreement in FGA locus. The clone sequencing verified that a "T" deletion discovered in flanking sequence of FGA led to wrong genotyping on Ampliseq Converge. Our results indicated that MPS could be adopted in qualified labs as a supplementary when the DNA of skeletal remains are hard to identify. [ABSTRACT FROM AUTHOR]

Abstract Background Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by psychiatric and neurological features. Most reported cases are caused by 22q13.3 deletions, leading to SHANK3 haploinsufficiency, but also usually encompassing many other genes. While the number of point mutations identified in SHANK3 has increased in recent years due to large-scale sequencing studies, systematic studies describing the phenotype of individuals harboring such mutations are lacking. Methods We provide detailed clinical and genetic data on 17 individuals carrying mutations in SHANK3. We also review 60 previously reported patients with pathogenic or likely pathogenic SHANK3 variants, often lacking detailed phenotypic information. Results SHANK3 mutations in our cohort and in previously reported cases were distributed throughout the protein; the majority were truncating and all were compatible with de novo inheritance. Despite substantial allelic heterogeneity, four variants were recurrent (p.Leu1142Valfs*153, p.Ala1227Glyfs*69, p.Arg1255Leufs*25, and c.2265+1G>A), suggesting that these are hotspots for de novo mutations. All individuals studied had intellectual disability, and autism spectrum disorder was prevalent (73%). Severe speech deficits were common, but in contrast to individuals with 22q13.3 deletions, the majority developed single words, including 41% with at least phrase speech. Other common findings were consistent with reports among individuals with 22q13.3 deletions, including hypotonia, motor skill deficits, regression, seizures, brain abnormalities, mild dysmorphic features, and feeding and gastrointestinal problems. Conclusions Haploinsufficiency of SHANK3 resulting from point mutations is sufficient to cause a broad range of features associated with PMS. Our findings expand the molecular and phenotypic spectrum of PMS caused by SHANK3 point mutations and suggest that, in general, speech impairment and motor deficits are more severe in the case of deletions. In contrast, renal abnormalities associated with 22q13.3 deletions do not appear to be related to the loss of SHANK3.

Prostate cancer (PCa) is the second most common malignant cancer and is a major cause of morbidity and mortality among men worldwide. There is still an urgent need for biomarkers applicable for diagnosis, prognosis, therapy prediction, or therapy monitoring in PCa. Liquid biopsies, including cell-free DNA (cfDNA) and circulating tumor cells (CTCs), are a valuable source for studying such biomarkers and are minimally invasive. In our study, we investigated the cfDNA of 34 progressive PCa patients, via targeted sequencing, for sequence variants and for the occurrence of CTCs, with a focus on androgen receptor splice variant 7 (AR-V7)-positive CTCs. The cfDNA content was associated with overall survival (OS; p = 0.014), disease-specific survival (DSS; p = 0.004), and time to treatment change (TTC; p = 0.001). Moreover, when considering all sequence variants grouped by their functional impact and allele frequency, a significant association with TTC (p = 0.017) was observed. When investigating only pathogenic or likely pathogenic gene variants, variants of the BRCA1 gene (p = 0.029) and the AR ligand-binding domain (p = 0.050) were associated with a shorter TTC. Likewise, the presence of CTCs was associated with a shorter TTC (p = 0.031). The presence of AR-V7-positive CTCs was associated with TTC (p < 0.001) in Kaplan–Meier analysis. Interestingly, all patients with AR-V7-positive CTCs also carried TP53 point mutations. Altogether, analysis of cfDNA and CTCs can provide complementary information that may support temporal and targeted treatment decisions and may elucidate the optimal choice within the variety of therapy options for advanced PCa patients.

Genes are unique in functional role and differ in their sensitivities to genetic defects, but with difficulties in pathogenicity prediction. This study attempted to improve the performance of existing in silico algorithms and find a common solution based on individualization strategy. We initiated the individualization with the epilepsy-related SCN1A variants by sub-regional stratification. SCN1A missense variants related to epilepsy were retrieved from mutation databases, and benign missense variants were collected from ExAC database. Predictions were performed by using 10 traditional tools with stepwise optimizations. Model predictive ability was evaluated using the five-fold cross-validations on variants of SCN1A, SCN2A, and KCNQ2. Additional validation was performed in SCN1A variants of damage-confirmed/familial epilepsy. The performance of commonly used predictors was less satisfactory for SCN1A with accuracy less than 80% and varied dramatically by functional domains of Nav1.1. Multistep individualized optimizations, including cutoff resetting, domain-based stratification, and combination of predicting algorithms, significantly increased predictive performance. Similar improvements were obtained for variants in SCN2A and KCNQ2. The predictive performance of the recently developed ensemble tools, such as Mendelian clinically applicable pathogenicity, combined annotation-dependent depletion and Eigen, was also improved dramatically by application of the strategy with molecular sub-regional stratification. The prediction scores of SCN1A variants showed linear correlations with the degree of functional defects and the severity of clinical phenotypes. This study highlights the need of individualized optimization with molecular sub-regional stratification for each gene in practice. [ABSTRACT FROM AUTHOR]

Background: Usher syndrome (USH) is the most prevalent cause of the human genetic deafness and blindness. USH type II (USH2) is the most common form of USH, and USH2A is the major pathogenic gene for USH2. For expanding the spectrum of USH2A mutations and further revealing the role of USH2A in USH2, we performed the USH2A gene variant screening in Chinese patients with USH2.Methods: Genomic DNA was extracted from peripheral blood of unrelated Chinese USH2 patients, we designed specific primers for amplifying the coding region (exons 2-72) of the USH2A gene. Sanger sequencing was used to study alleles. Silico prediction tools were used to predict the pathogenicity of the variants identified in these patients.Results: Five heterozygous pathogenic variants were detected in four patients. Two patients were found to have two-mutations and two patients only have one. Two novel variants c.4217C > A (p.Ser1406X) and c.11780A > G (p.Asp3927Gly)) were predicted deleterious by computer prediction algorithms. In addition, three reported mutations (c.8559-2A > G, c.8232G > C and c.11389 + 3A > T) were also found in this study.Conclusions: We identified five heterozygous pathogenic variants in the USH2A gene in Chinese patients diagnosed with Usher syndrome type 2, two of which were not reported. It expands the spectrum of USH2A variants in USH. [ABSTRACT FROM AUTHOR]

Background: The prevalence of Parkinson's disease (PD) is increasing in sub-Saharan Africa, but little is known about the genetics of PD in these populations. Due to their unique ancestry and diversity, sub-Saharan African populations have the potential to reveal novel insights into the pathobiology of PD. In this study, we aimed to characterise the genetic variation in known and novel PD genes in a group of Black South African and Nigerian patients. Methods: We recruited 33 Black South African and 14 Nigerian PD patients, and screened them for sequence variants in 751 genes using an Ion AmpliSeq™ Neurological Research panel. We used bcftools to filter variants and annovar software for the annotation. Rare variants were prioritised using MetaLR and MetaSVM prediction scores. The effect of a variant on ATP13A2's protein structure was investigated by molecular modelling. Results: We identified 14,655 rare variants with a minor allele frequency ≤ 0.01, which included 2448 missense variants. Notably, no common pathogenic mutations were identified in these patients. Also, none of the known PD-associated mutations were found highlighting the need for more studies in African populations. Altogether, 54 rare variants in 42 genes were considered deleterious and were prioritized, based on MetaLR and MetaSVM scores, for follow-up studies. Protein modelling showed that the S1004R variant in ATP13A2 possibly alters the conformation of the protein. Conclusions: We identified several rare variants predicted to be deleterious in sub-Saharan Africa PD patients; however, further studies are required to determine the biological effects of these variants and their possible role in PD. Studies such as these are important to elucidate the genetic aetiology of this disorder in patients of African ancestry. [ABSTRACT FROM AUTHOR]

Massive parallel sequencing technologies are facilitating the faster identification of sequence variants with the consequent capability of untangling the molecular bases of many human genetic syndromes. However, it is not always easy to understand the impact of novel variants, especially for missense changes, which can lead to a spectrum of phenotypes. This study presents a custom‐designed multistep methodology to evaluate the impact of novel variants aggregated in the genome aggregation database for the HBB, HBA2, and HBA1 genes, by testing and improving its performance with a dataset of previously described alterations affecting those same genes. This approach scored high sensitivity and specificity values and showed an overall better performance than sequence‐derived predictors, highlighting the importance of protein conformation and interaction specific analyses in curating variant databases. This study also describes the strengths and limitations of these structural studies and allows identifying residues in the globin chains more prone to tolerate substitutions. [ABSTRACT FROM AUTHOR]

Abstract Background Late-onset Pompe disease is a rare genetic neuromuscular disorder caused by a primary deficiency of α-glucosidase and the associated accumulation of glycogen in lysosomal vacuoles. The deficiency of α-glucosidase can often be detected using an inexpensive and readily accessible dried blood spot test when Pompe disease is suspected. Like several neuromuscular disorders, Pompe disease typically presents with progressive weakness of limb-girdle muscles and respiratory insufficiency. Due to the phenotypic heterogeneity of these disorders, however, it is often difficult for clinicians to reach a diagnosis for patients with Pompe disease. Six hundred and six patients from a European population were recruited onto our study. Inclusion criteria stipulated that index cases must present with limb-girdle weakness or elevated serum creatine kinase activity. Whole exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. A panel of 169 candidate genes for limb-girdle weakness was analysed for disease-causing variants. Results A total of 35 variants within GAA were detected. Ten distinct variants in eight unrelated index cases (and four siblings not sequenced in our study) were considered disease-causing, with the patients presenting with heterogeneous phenotypes. The eight unrelated individuals were compound heterozygotes for two variants. Six patients carried the intronic splice site c.-13 T > G transversion and two of the six patients also carried the exonic p.Glu176ArgfsTer45 frameshift. Four of the ten variants were novel in their association with Pompe disease. Conclusions Here, we highlight the advantage of using whole exome sequencing as a tool for detecting, diagnosing and treating patients with rare, clinically variable genetic disorders.

Background: APOC3 is important in lipid transport and metabolism with limited studies reporting genetic sequence variations in specific ethnic groups. The present study aimed to analyze the full APOC3 sequence among Kuwaiti Arabs and test the association of selected variants with lipid levels and BMI. Methods: Variants were identified by Sanger sequencing the entire APOC3 gene in 100 Kuwaiti Arabs. Variants and their genotypes were fully characterized and used to construct haplotype blocks. Four variants (rs5128, rs2854117, rs2070668, KUAPOC3N3 g.5196 A > G) were selected for testing association with serum lipid levels and BMI in a cohort (n = 733). Results: APOC3 sequence (4.3 kb) of a Kuwaiti Arab was deposited in Genbank (accession number KJ437193). Forty-two variants including 3 novels were identified including an "A" insertion at genomic positions 116,700,599–116,700,600 (promoter region) and two substitutions in intron 1 at genomic positions 116,700,819 and 116,701,159. Only three variants, (rs5128, rs2854117, and rs2070668) were analyzed for association of which rs5128 showed a trend for association with increased BMI, TG and VLDL levels that was further investigated using multivariate analysis. A significant association of rs5128 with BMI (p < 0.05) was observed following a dominant genetic model with increased risk by an OR of 4.022 (CI: 1.13–14.30). Conclusion: The present study is the first to report sequence analysis of APOC3 in an Arab ethnic group. This study supports the inclusion of rs5128 as a marker for assessing genetic risk to dyslipidemia and obesity and the inclusion of the novel variant g.5196 A > G for population stratification of Arabs. [ABSTRACT FROM AUTHOR]

Background: Oxytocin receptor (OXTR) gene variants have been shown to affect the prevalence of preterm birth, mode of delivery and oxytocin (OXT) requirements for labor induction and augmentation. We hypothesized that this might be associated with different myometrium responses to oxytocin. Our aim was to investigate the influence of a selection of eight OXTR gene single nucleotide variants on oxytocin-induced stimulation of human myometrium contractility in vitro. Methods: Human myometrium biopsies were collected during elective cesarean sections at term, if patients had given informed consent. Myometrial strips were submerged under tension in an organ bath and allowed to contract; the remaining material was stored at − 80 °C for further determination of relevant genetics and mRNA level. The area under the curve (AUC) of all contractions taking place in the absence of OXT and of those occurring upon OXT addition (for 30 min each) was measured. OXT stimulation, defined as the ratio between AUC measurements after OXT addition and those in the absence of OXT was calculated for each strip. TaqMan™ Assays were used to detect the allele distribution of the eight OXTR variants and to determine the relative amounts of OXTR-mRNA in the samples. For each variant, oxytocin stimulation of contractility was compared between samples homozygous for the reference allele (reference group) and samples with at least one variant allele (variant group) by linear regression. Results: Sixty samples were included in the present study. For rs1042778, rs11706648, rs4686301, rs53576, rs237895, and rs237902, OXT stimulation was similar in the reference and in the variant groups. However, the values of OXT stimulation differed significantly between the reference and the variant groups for rs4686302 (3.1 vs. 4.1 times; p = 0.022) and rs237888 (3.2 vs. 5.5 times; p = 0.001). No significant differences between the levels of OXTR-mRNA in the various reference and corresponding variant groups were detected. Conclusions: Patients with variant alleles of rs237888 and/or rs4686302 may be more sensitive to oxytocin stimulation, explaining why these sequence variants have been associated with lower cesarean section prevalence and premature birth, respectively. [ABSTRACT FROM AUTHOR]

Abstract Background Sequence variants in the NOS1AP gene have repeatedly been reported to influence QTc, albeit with moderate effect sizes. In the long QT syndrome (LQTS), this may contribute to the substantial QTc variance seen among carriers of identical pathogenic sequence variants. Here we assess three non-coding NOS1AP sequence variants, chosen for their previously reported strong association with QTc in normal and LQTS populations, for association with QTc in two Swedish LQT1 founder populations. Methods This study included 312 individuals (58% females) from two LQT1 founder populations, whereof 227 genotype positive segregating either Y111C (n = 148) or R518* (n = 79) pathogenic sequence variants in the KCNQ1 gene, and 85 genotype negatives. All were genotyped for NOS1AP sequence variants rs12143842, rs16847548 and rs4657139, and tested for association with QTc length (effect size presented as mean difference between derived and wildtype, in ms), using a pedigree-based measured genotype association analysis. Mean QTc was obtained by repeated manual measurement (preferably in lead II) by one observer using coded 50 mm/s standard 12-lead ECGs. Results A substantial variance in mean QTc was seen in genotype positives 476 ± 36 ms (Y111C 483 ± 34 ms; R518* 462 ± 34 ms) and genotype negatives 433 ± 24 ms. Female sex was significantly associated with QTc prolongation in all genotype groups (p