Mutations In Candidate Genes Research Articles

Letter regarding “SCN9A variant in a family of mixed breed dogs with congenital insensitivity to pain”—Navigating the pathogenicity of candidate gene mutations: Spotlight on paralog Nav genes

Letter regarding “<scp><i>SCN9A</i></scp> variant in a family of mixed breed dogs with congenital insensitivity to pain”—Navigating the pathogenicity of candidate gene mutations: Spotlight on paralog Nav genes

Congenital hydrocephalus: new Mendelian mutations and evidence for oligogenic inheritance

BackgroundCongenital hydrocephalus is characterized by ventriculomegaly, defined as a dilatation of cerebral ventricles, and thought to be due to impaired cerebrospinal fluid (CSF) homeostasis. Primary congenital hydrocephalus is a subset of cases with prenatal onset and absence of another primary cause, e.g., brain hemorrhage. Published series report a Mendelian cause in only a minority of cases. In this study, we analyzed exome data of PCH patients in search of novel causal genes and addressed the possibility of an underlying oligogenic mode of inheritance for PCH.Materials and methodsWe sequenced the exome in 28 unrelated probands with PCH, 12 of whom from families with at least two affected siblings and 9 of whom consanguineous, thereby increasing the contribution of genetic causes. Patient exome data were first analyzed for rare (MAF < 0.005) transmitted or de novo variants. Population stratification of unrelated PCH patients and controls was determined by principle component analysis, and outliers identified using Mahalanobis distance 5% as cutoff. Patient and control exome data for genes biologically related to cilia (SYScilia database) were analyzed by mutation burden test.ResultsIn 18% of probands, we identify a causal (pathogenic or likely pathogenic) variant of a known hydrocephalus gene, including genes for postnatal, syndromic hydrocephalus, not previously reported in isolated PCH. In a further 11%, we identify mutations in novel candidate genes. Through mutation burden tests, we demonstrate a significant burden of genetic variants in genes coding for proteins of the primary cilium in PCH patients compared to controls.ConclusionOur study confirms the low contribution of Mendelian mutations in PCH and reports PCH as a phenotypic presentation of some known genes known for syndromic, postnatal hydrocephalus. Furthermore, this study identifies novel Mendelian candidate genes, and provides evidence for oligogenic inheritance implicating primary cilia in PCH.

ERBB2 mutation is associated with sustained tumor cell proliferation after short-term preoperative endocrine therapy in early lobular breast cancer

Invasive lobular breast cancer (ILC) is a special breast cancer (BC) subtype and is mostly hormone receptor (HR)-positive and ERBB2 non-amplified. Endocrine therapy restrains tumor proliferation and is the mainstay of lobular BC treatment. Mutation of ERBB2 has been associated with recurrent ILC. However, it is unknown whether ERBB2 mutation impacts on the otherwise exquisite responsiveness of early ILC to endocrine therapy. We have recently profiled n = 622 HR-positive early BCs from the ADAPT trial for mutations in candidate genes involved in endocrine resistance, including ERBB2. All patients were treated with short-term preoperative endocrine therapy (pET, tamoxifen or aromatase inhibitors) before tumor resection. Tumor proliferation after endocrine therapy (post-pET Ki67 index) was determined prospectively by standardized central pathology assessment supported by computer-assisted image analysis. Sustained or suppressed proliferation were defined as post-pET Ki67 ≥10% or <10%. Here, we report a subgroup analysis pertaining to ILCs in this cohort. ILCs accounted for 179/622 (28.8%) cases. ILCs were enriched in mutations in CDH1 (124/179, 69.3%, P < 0.0001) and ERBB2 (14/179, 7.8%, P < 0.0001), but showed fewer mutations in TP53 (7/179, 3.9%, P = 0.0048) and GATA3 (11/179, 6.1%, P < 0.0001). Considering all BCs irrespective of subtypes, ERBB2 mutation was not associated with proliferation. In ILCs, however, ERBB2 mutations were 3.5-fold more common in cases with sustained post-pET proliferation compared to cases with suppressed post-pET proliferation (10/75, 13.3% versus 4/104, 3.8%, P = 0.0248). Moreover, ERBB2 mutation was associated with high Oncotype DX recurrence scores (P = 0.0087). In summary, our findings support that ERBB2 mutation influences endocrine responsiveness in early lobular BC.

Genetic mutations underlying isoniazid-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan

Tuberculosis, caused by Mycobacterium tuberculosis, is a major public health issue in Pakistan. Isoniazid is a first-line pro-drug that requires activation through an enzyme called catalase peroxidase, but is subject to widespread resistance, driven by mutations in katG and inhA genes and other loci with compensatory effects (e.g., ahpC). Here, we used whole genome sequencing data from 51 M. tuberculosis isolates collected from Khyber Pakhtunkhwa province (years 2016–2019; all isoniazid phenotypically resistant) to investigate the genetic diversity of mutations in isoniazid candidate genes. The most common mutations underlying resistance were katG S315T (37/51), fabG1 -15C>T (13/51; inhA promoter), and inhA −154G>A (7/51). Other less common mutations (n < 5) were also identified in katG (R128Q, V1A, W505*, A109T, D311G) and candidate compensatory genes ahpC (−54C>T, −51G>A) and oxyS (M249T). Using DynaMut2 software, the mutants exhibited various degrees of stability and flexibility on protein structures, with some katG mutations leading to a decrease in KatG protein flexibility. Overall, the characterisation of circulating isoniazid resistant-linked mutations will assist in drug resistant TB management and control activities in a highly endemic area of Pakistan.

Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer.

BackgroundThis is a pilot proof-of-concept study to evaluate the utility of a custom 15-gene circulating tumor DNA (ctDNA) panel as a potential companion molecular next-generation sequencing (NGS) assay for identifying somatic single nucleotide variants and indels in non-small-cell lung cancer (NSCLC) patients. The custom panel covers the hotspot mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET, KIT, PDGFRA, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 genes which serve as biomarkers for guiding treatment decisions in NSCLC patients.MethodThe custom 15-gene ctDNA NGS panel was designed using ArcherDX Assay Designer. A total of 20 ng or 50 ng input ctDNA was used to construct the libraries. The analytical performance was evaluated using reference standards at different allellic frequencies (0.1%, 1%, 5% and parental). The clinical performance was evaluated using plasma samples collected from 10 treatment naïve advanced stage III or IV NSCLC patients who were tested for tissue EGFR mutations. The bioinformatics analysis was performed using the proprietary Archer Analysis Software.ResultsFor the analytical validation, we achieved 100% sensitivity and specificity for the detection of known mutations in the reference standards. The limit of detection was 1% allelic frequency. Clinical validation showed that the clinical sensitivity and specificity of the assay for detecting EGFR mutation were 83.3% and 100% respectively. In addition, the NGS panel also detected other mutations of uncertain significance in 6 out of 10 patients.ConclusionThis preliminary analysis showed that the custom 15-gene ctDNA NGS panel demonstrated good analytical and clinical performances for the EGFR mutation. Further studies incorporating the validation of other candidate gene mutations are warranted.

Exome and Sputum Microbiota as Predictive Markers of Frequent Exacerbations in Chronic Obstructive Pulmonary Disease.

Frequent acute exacerbations are the leading cause of high rates of hospitalization and mortality in chronic obstructive pulmonary disease (COPD). Despite the enormous worldwide medical burden, reliable molecular markers for effective early diagnosis and prognosis of acute exacerbations are still lacking. Both the host genetics and airway microbiome are known to play potential roles in the pathogenesis of frequent exacerbations. Here, we performed whole exome sequencing (WES) and 16S rRNA gene sequencing to explore the interaction between these two factors and their implications in the pathogenesis of frequent exacerbations. We collected peripheral blood (n = 82), sputum samples (n = 59) and clinical data from 50 frequent-exacerbation phenotype (FE) COPD patients and 32 infrequent-exacerbation phenotype (IE) as controls. Based on filtering the deleterious sites, candidate mutated genes shared only in FE patients and did not occur in the IE group were identified. Microbiota analysis revealed significant differences in bacterial diversity and composition between FE and IE groups. We report the underlying pathogenic gene including, AATF, HTT, CEP350, ADAMTS9, TLL2 genes, etc., and explore their possible genotypic-phenotypic correlations with microbiota dysbiosis. Importantly, we observed that AATF gene mutations were significantly negatively correlated with microbial richness and diversity. Our study indicated several deleterious mutations in candidate genes that might be associated with microbial dysbiosis and the increased risk of frequent acute exacerbations in COPD patients. These results provide novel evidence that exomes and related microbiomes may potentially serve as biomarkers for predicting frequent acute exacerbations in COPD patients.

The number of mitochondrial DNA mutations as a genetic feature for hair cycle arrest (alopecia X) in Pomeranian dogs.

Hair cycle arrest (HCA) is a noninflammatory alopecic disease affecting various dog breeds, particularly Pomeranian dogs. This disease is probably a hereditary disorder considering the strong breed predisposition. Despite efforts to identify the pathogenesis of this disease, an underlying specific cause is unknown. To identify candidate gene mutations for HCA in Pomeranian dogs. Four Pomeranian dogs diagnosed with HCA and four unaffected Pomeranian dogs. Whole blood was used for DNA extraction. Whole-genome sequencing (WGS) was performed, and variants were analysed using a Genome Analysis Toolkit (GATK) and SnpEff. All reads were aligned to the reference genome, Dog10K_Boxer_Tasha. Sanger sequencing was performed to define the complex mutations. A total of 113 variants of mitochondrial DNA were found to be effective gene mutations in the eight dogs. The affected dogs showed significantly increased effective mutations (average 57 variants) compared with unaffected dogs (average eight variants; p< 0.05). There was no significant difference in the number of chromosomal DNA mutations between the two groups. We suggest that an increased number of mitochondrial gene mutations are features for HCA in Pomeranian dogs.

Genetic mapping of simply inherited categorical traits, including anthocyanin accumulation profiles and fruit appearance, in eggplant (Solanum melongena).

The appearance quality of the eggplant (Solanum melongena L.) fruit is an important trait that influences its commercial value. It is known that quality traits such as anthocyanin composition and fruit surface pattern are categorical and are inherited simply. However, research examples of gene mapping for the composition (anthocyanin accumulation profile) and the surface pattern in eggplant fruit are limited. To map loci for these traits including the accumulation profiles of two anthocyanins, a widely spreading anthocyanin, delphinidin 3-(p-coumaroyl) rutinoside-5-glucoside (nasunin), and the relatively rare delphinidin 3-glucoside (D3G), we used two F2 intracrossed populations (LWF2 and N28F2). For the LWF2 population, mapping was achieved by reconstructing the linkage map created by Fukuoka et al. [1]. In the case of the N28F2 population, we constructed a linkage map consisting of 13 linkage groups using 238 simple sequence repeats, 75 single-nucleotide polymorphisms. Using the two F2 populations, the nasunin accumulating profile, the striped pattern on the fruit surface, the colors of flowers, fruit, and calyxes, and the D3G accumulating profile were genetically mapped. Furthermore, by utilizing the eggplant reference genome information, mutations in the causative candidate genes for those loci were identified. Overall, the results of this study suggest that inactivation of key enzymes of anthocyanin metabolism and the gene orthologous to the tomato u gene are potential causes of observed variety in eggplant appearance traits.

Post prandial metabolism of lipoproteins in familial chylomicronemia patients treated with lomitapide and tiparvovec

Background and Aims : Familial chylomicronemia syndrome (FCS) is a rare recessive monogenic disease characterized by triglycerides (TG) levels >10 mmol/L. FCS is causally associated with mutations in candidate genes but most patients have mutations in lipoprotein lipase (LPL). Defects in LPL result in reduced clearance of chylomicrons (CM) and development of acute pancreatitis. Treatment of FSC patients is based on combined action of a lipid- and carbohydrate-reduced diet in addition to available hypolipidemic therapies that often fails to achieve a desired TG levels.

Mutations of 1p genes do not consistently abrogate tumor suppressor functions in 1p-intact neuroblastoma

BackgroundDeletion of 1p is associated with poor prognosis in neuroblastoma, however selected 1p-intact patients still experience poor outcomes. Since mutations of 1p genes may mimic the deleterious effects of chromosomal loss, we studied the incidence, spectrum and effects of mutational variants in 1p-intact neuroblastoma.MethodsWe characterized the 1p status of 325 neuroblastoma patients, and correlated the mutational status of 1p tumor suppressors and neuroblastoma candidate genes with survival outcomes among 100 1p-intact cases, then performed functional validation of selected novel variants of 1p36 genes identified from our patient cohort.ResultsAmong patients with adverse disease characteristics, those who additionally had 1p deletion had significantly worse overall survival. Among 100 tumor-normal pairs sequenced, somatic mutations of 1p tumor suppressors KIF1Bβ and CHD5 were most frequent (2%) after ALK and ATRX (8%), and BARD1 (3%). Mutations of neuroblastoma candidate genes were associated with other synchronous mutations and concurrent 11q deletion (P = 0.045). In total, 24 of 38 variants identified were novel and predicted to be deleterious or pathogenic. Functional validation identified novel KIF1Bβ I1355M variant as a gain-of-function mutation with increased expression and tumor suppressive activity, correlating with indolent clinical behavior; another novel variant CHD5 E43Q was a loss-of-function mutation with decreased expression and increased long-term cell viability, corresponding with aggressive disease characteristics.ConclusionsOur study showed that chromosome 1 gene mutations occurred frequently in 1p-intact neuroblastoma, but may not consistently abrogate the function of bonafide 1p tumor suppressors. These findings may augment the evolving model of compounding contributions of 1p gene aberrations toward tumor suppressor inactivation in neuroblastoma.

Focal facial dermal dysplasias type III: Two families with Setleis syndrome in China.

Focal facial dermal dysplasias type III (FFDD III), commonly known as Setleis syndrome (SS; Online Mendelian Inheritance in Man #227260), is a type of focal facial dermal dysplasia, characterized by bitemporal atrophic skin lesion. The homozygous mutations in the TWIST2 gene and copy number variants (CNV) at chromosome 1p36.22p36.21 were reported as the pathogenic mechanism. In this study, we collected DNA samples from a large Chinese family affected by FFDD and found no mutation of TWSIT2. To determine the underlying genetic cause, we performed a multipoint parameter linkage analysis and haplotype analysis of the family 1 and mapped SS to a region Chr1:14.074-20.524cM (rs2401090-rs2294642). Copy number variant was identified by Sanger sequencing, which breakpoints were Chr1:11695972 and Chr1:11829858. The region contains eight genes, including FBXO2, FBXO44, FBXO6, MAD2L2, DRAXIN, AK125437, AGTRAP, and C1orf167. There were no candidate gene mutations of the second family with SS. Our study further reduced the size of CNV resulting in SS (Chr1:11696993-11829858) and focused on eight genes.

Association of gene mutations with clinicopathologic features in patients with external auditory canal squamous cell carcinoma.

External auditory canal squamous cell carcinoma (EACSCC) is a rare form of malignant tumor. Due to the extremely limited understanding of the genomic landscape in EACSCC, the association between gene mutations and clinicopathologic features remains unclear. This study aimed to explore somatic gene mutations associated with the clinicopathological features in patients with EACSCC, and to identify the candidate gene mutations for predicting survival outcome in EACSCC. Twenty-two tissue samples obtained from patients with EACSCC were analyzed for genetic mutations based on targeted next-generation sequencing and genetic expression based on IHC staining to investigate the driver of tumorigenesis and/or the candidates of genes for predicting clinical outcome in EACSCC. Gene alterations were most frequently observed in TP53 (59.1%), followed by CREBBP (9.1%). TP53 mutations showed significant correlation with T classification (P = 0.027) and p53 expression phenotype (P < 0.001). The 5-year overall survival (OS) rates for EACSCC patients with TP53 mutations and wild-type TP53 were 45.0% and 75.0%, respectively. Multivariable analysis using the Cox proportional hazards model demonstrated that TP53 mutations were independent predictors of OS rates for EACSCC patients (P = 0.007). This study has suggested that TP53 mutations have potential for use as a biomarker for identifying individuals at high risk of developing tumors and for predicting survival outcome in EACSCC. IHC staining for p53 might play a useful role as screening tool for detecting TP53 mutations in patients with EACSCC.

Concept of 5P-medicine for prevention of acute cerebral ischemia in women

The objective. To evaluate the effectiveness of the 5P-model for the prevention of acute cerebral ischemia in Dagestan women.Materials and methods. The prospective study included 35 women permanently residing in Dagestan. The mean age was 66.46 ± 10.9 years. Each participant of the study was individually interviewed and examined clinically, neurologically, an additional examination included: complete blood test, biochemical blood test, coagulation test, molecular genetic study for 11 candidate gene mutations significant for the occurrence of ischemic stroke, electrocardiography, neuroimaging.Results. By analyzing the results of the study, individual background conditions that predispose to the occurrence of ischemic stroke were established. The results of the examination were explained to each participant, there were given the recommendations on lifestyle modification, a diet was prescribed and the necessary therapy was selected. In the process of dynamic observation for 4 years the observed group had no episodes of acute cerebrovascular accident.Conclusions. The study confirms the effectiveness of the use of the 5P-model for the prevention of acute cerebral ischemia in women Dagestan and declare a positive experience of its application.

Identification of New Genetic Determinants in Pediatric Patients with Familial Hypercholesterolemia Using a Custom NGS Panel.

The most common form of inherited lipid disorders is familial hypercholesterolemia (FH). It is characterized primarily by high concentrations of the clinical triad of low-density lipoprotein cholesterol, tendon xanthomas and premature CVD. The well-known genetic background are mutations in LDLR, APOB and PCSK9 gene. Causative mutations can be found in 60–80% of definite FH patients and 20–30% of those with possible FH. Their occurrence could be attributed to the activity of minor candidate genes, whose causal mechanism has not been fully discovered. The aim of the conducted study was to identify disease-causing mutations in FH-related and candidate genes in pediatric patients from Poland using next generation sequencing (NGS). An NGS custom panel was designed to cover 21 causative and candidate genes linked to primary dyslipidemia. Recruitment was performed using Simon Broome diagnostic criteria. Targeted next generation sequencing was performed on a MiniSeq sequencer (Illumina, San Diego, CA, USA) using a 2 × 150 bp paired-end read module. Sequencing data analysis revealed pathogenic and possibly pathogenic variants in 33 out of 57 studied children. The affected genes were LDLR, APOB, ABCG5 and LPL. A novel pathogenic 7bp frameshift deletion c.373_379delCAGTTCG in the exon 4 of the LDLR gene was found. Our findings are the first to identify the c.373_379delCAGTTCG mutation in the LDLR gene. Furthermore, the double heterozygous carrier of frameshift insertion c.2416dupG in the LDLR gene and missense variant c.10708C>T in the APOB gene was identified. The c.2416dupG variant was defined as pathogenic, as confirmed by its cosegregation with hypercholesterolemia in the proband’s family. Although the APOB c.10708C>T variant was previously detected in hypercholesterolemic patients, our data seem to demonstrate no clinical impact. Two missense variants in the LPL gene associated with elevated triglyceride plasma level (c.106G>A and c.953A>G) were also identified. The custom NGS panel proved to be an effective research tool for identifying new causative aberrations in a genetically heterogeneous disease as familial hypercholesterolemia (FH). Our findings expand the spectrum of variants associated with the FH loci and will be of value in genetic counseling among patients with the disease.

Mutations in trpγ, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila

Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. Here, we perform a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, we used the fruit fly, Drosophila melanogaster, to show that null mutations in transient receptor gamma (trpγ; the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John’s wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, our results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes.

Identification of Prognostic Markers in Argentinian Acute Lymphoblastic Leukemia Pediatric Patients Through Transcriptome Analysis: Differential Gene Expression, Mutational Status and Immune Microenvironment

PURPOSE Acute lymphoblastic leukemia (ALL) is the most incident pediatric cancer. Although considerable progress has been made on treatment efficacy and survival rates, 15%-30% of patients relapse and/or die. We aimed to identify molecular profiles and microenvironment makeup in leukemic bone marrow samples that could help to better predict disease outcome. METHODS We performed RNA-seq on bone marrow samples from pediatric ALL patients at diagnosis (n = 37). Patients were recruited under a multi-center clinical protocol in Argentina (median follow-up: 31 months). We analyzed differential gene expression, gene set variations, mutations in candidate genes, and fusion genes among clinico-pathological features. The abundance of immune-cell populations was inferred by digital cytometry (MIXTURE) and a “cytolytic score” based on the expression of five genes specific to cytotoxic cells. RESULTS We detected: (1) 37 differentially expressed genes (DEG) between poor vs good responders to prednisone; (2) 71 DEG between high vs standard-risk groups; (3) 13 DEG between patients who died/relapsed vs those who did not; and (4) 35 DEG between patients that developed severe therapy-related acute toxicity vs those who did not (|log2FC|&gt; 1; P.adj &lt; .05). We observed that 15%-30% of the DEG corresponded to lncRNAs. We found six differentially expressed pathways relevant to cancer and leukocyte biology among risk groups ( P &lt; .01). We identified 17 mutations and three fusion genes in 44% of patients; the presence of these mutations shortened the relapse-free survival (Cox- P-val = .006). High cytolytic score was associated with activated CD8+T cells, immune cell trafficking, and bone marrow niche signaling genesets, suggesting potential candidates for immunotherapies. Higher CD8+T-cell/NK at diagnosis was marginally associated with worse event-free survival (hazard-ratio = 5.39; Cox- P-val = .08). CONCLUSION Transcriptomic sequencing allowed the analysis and integration of multiple molecular features that might improve pediatric ALL outcome prediction.

Genomic selection and genetic architecture of agronomic traits during modern rapeseed breeding.

Rapeseed (Brassica napus L.) is an important oil-producing crop for the world. Its adaptation, yield and quality have been considerably improved in recent decades, but the genomic basis underlying successful breeding selection remains unclear. Hence, we conducted a comprehensive genomic assessment of rapeseed in the breeding process based on the whole-genome resequencing of 418 diverse rapeseed accessions. We unraveled the genomic basis for the selection of adaptation and agronomic traits. Genome-wide association studies identified 628 associated loci-related causative candidate genes for 56 agronomically important traits, including plant architecture and yield traits. Furthermore, we uncovered nonsynonymous mutations in plausible candidate genes for agronomic traits with significant differences in allele frequency distributions across the improvement process, including the ribosome recycling factor (BnRRF) gene for seed weight. This study provides insights into the genomic basis for improving rapeseed varieties and a valuable genomic resource for genome-assisted rapeseed breeding.

Cathepsin F genetic mutation is associated with familial papillary thyroid cancer

BackgroundThyroid cancer is one of the most common cancers in the world. Genetic factors are important in the occurrence and development of thyroid cancer, and genetic diagnosis has become an important basis for the prognosis of benign and malignant nodules. We identify a family of six siblings with inherited thyroid cancer susceptibility. All six members of this generation have been definitely diagnosed with papillary thyroid carcinoma. This work aims at confirming the relevant causative genes for thyroid cancer in this pedigree. MethodsWe extract DNA from the peripheral blood of six individuals and perform whole genome sequencing. Sanger sequencing and immunohistochemistry further testify the cathepsin F (CTSF) mutation and expression. ResultsWe identify 57 single nucleotide variations (SNVs) out of at least 4 affected family members via certain filter criteria. The CTSF gene found in five of the six family members is here considered the most promising candidate gene mutation for familial thyroid cancer. Besides, our research also proves several known genes including CTSB, TEKT4, ESR1, MSH6, DIRC3, GNAS, and BANCR that act as probable oncogenic drivers in this family. The Sanger sequencing identifies the existence and veracity of CTSF somatic mutations. The CTSF immunohistochemistry of thyroid cancer tissue specimens displays that higher CTSF expression in mutated patients than that in wild-type patient as well as pericarcinomatous tissue. ConclusionsWe conclude that the evaluation of CTSF gene mutations of patients in thyroid cancer families may be predictive and valuable for the familial heredity of thyroid cancer.

The genetic factors contributing to the risk of cleft lip-cleft palate and their clinical utility.

Cleft lip and cleft palate (CL/P) are among the most common congenital malformations in neonates and have syndromic or nonsyndromic forms. Nonsyndromic forms of malformation are being reported to be associated with chromosomal DNA modification by teratogenic exposure and to complex genetic contributions of multiple genes. Syndromic forms are shown to be related to chromosomal aberrations or monogenic diseases. There is a growing body of data illustrating the association of several genes with risk of developing this malformation, including genetic defects in T-box transcription factor-22 (TBX22), interferon regulatory factor-6 (IRF6), and poliovirus receptor-like-1 (PVRL1), responsible for X-linked cleft palate, cleft lip/palate-ectodermal dysplasia syndrome, and Van der Woude and popliteal pterygium syndromes, respectively. Genetic variants in MTR, PCYT1A, ASS1, SLC 25A13, GSTM1, GSTT1, SUMO1 BHMT1, and BHMT2 are being reported to be linked with CL/P risk. The etiology of nonsyndromic CLP is still remained to be unknown, although mutations in candidate genes have been found. Here, we provide an overview about the potential variants to be associated with CL/P for identification of the relative risk of CLP with respect to the basis of genetic background and environmental factors (e.g., dietary factors, alcohol use).

Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction.

Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.