A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

46,XX disorder of sex development associated with skin abnormalities due to homozygous R-Spondin 1 loss of function mutation.

A child, who was reared as male, presented in his early childhood to the endocrine clinic with penoscrotal hypospadias which was noticed at birth. On examination, he had both gonads in the scrotal sacs with complete scrotal fusion, rugosities and chordee with a single opening. He had increased palmoplantar skin desquamation. As an initial part of the workup, karyotyping was done, which was 46,XX. To rule out the most common cause of 46,XX disorder of sex development (DSD) in phenotypical males (SRY - Sex Determining Region Y gene - translocation), fluorescent in situ hybridisation for SRY was done, which was negative. Whole exome sequencing revealed a homozygous loss of function mutation in the R-Spondin1 gene. Here we report a rare case of 46,XX DSD with loss of function mutation in the R-Spondin1 gene associated with skin abnormalities.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Loss of function mutations at the tomato SSI2 locus impair plant growth and development by altering the fatty acid desaturation pathway.

The stearoyl-ACP desaturase (SACPD) is a key enzyme in the regulation of saturated to unsaturated fatty acid ratio, playing a crucial role in regulating membrane stability and fluidity, as well as photosynthesis efficiency, which makes it an important research focus in crop species. This study reports the characterization and molecular cloning of pale dwarf (pad), a new tomato (Solanum lycopersicum L.) T-DNA recessive mutant, which exhibits a dwarf and chlorotic phenotype. Functional studies of the T-DNA tagged gene were conducted, including phylogenetic analysis, expression and metabolomic analyses, and generation of CRISPR/Cas9 knockout lines. The cloning of T-DNA flanking genomic sequences and a co-segregation analysis found the pad phenotype was caused by a T-DNA insertion disrupting the tomato homologue of the Arabidopsis SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY 2 (SlSSI2), encoding a plastid localized isoform of SACPD. The phenotype of CRISPR/Cas9 SlSSI2 knockout lines confirmed that the morphological abnormalities in pad plants were due to SlSSI2 loss of function. Functional, metabolomic and expression analyses proved that SlSSI2 disruption causes deficiencies in 18:1 fatty acid desaturation and leads to diminished jasmonic acid (JA) content and increased salicylic acid (SA) levels. Overall, these results proved that SSI2 plays a crucial role in the regulation of polyunsaturated fatty acid profiles in tomato, and revealed that SlSSI2 loss of function results in an inhibited JA-responsive signalling pathway and a constitutively activated SA-mediated defence signalling response. This study lays the foundation for further research on tomato SACPDs and their role in plant performance and fitness.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.

Mapping and Functional Dissection of the Rumpless Trait in Piao Chicken Identifies a Causal Loss of Function Mutation in the Novel Gene Rum.

Rumpless chickens exhibit an abnormality in their tail development. The genetics and biology of this trait has been studied for decades to illustrate a broad variation in both the types of inheritance and the severity in the developmental defects of the tail. In this study, we created a backcross pedigree by intercrossing Piao (rumpless) with Xianju (normal) to investigate the genetic mechanisms and molecular basis of the rumpless trait in Piao chicken. Through genome-wide association and linkage analyses, the candidate region was fine-mapped to 798.5 kb (chromosome 2: 86.9 to 87.7 Mb). Whole-genome sequencing analyses identified a single variant, a 4.2 kb deletion, which was completely associated with the rumpless phenotype. Explorations of the expression data identified a novel causative gene, Rum, that produced a long, intronless transcript across the deletion. The expression of Rum is embryo-specific, and it regulates the expression of MSGN1, a key factor in regulating T-box transcription factors required for mesoderm formation and differentiation. These results provide genetic and molecular experimental evidence for a novel mechanism regulating tail development in chicken and report the likely causal mutation for the tail abnormity in the Piao chicken. The novel regulatory gene, Rum, will, due to its role in fundamental embryo development, be of interest for further explorations of a potential role in tail and skeletal development also in other vertebrates.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Novel frameshift variants expand the map of the genetic defects in IRF2BP2.

Background: At present, the knowledge about disease-causing mutations in IRF2BP2 is very limited because only a few patients affected by this condition have been reported. As previous studies have described, the haploinsufficiency of this interferon transcriptional corepressors leads to the development of CVID. Very recently, a more accurate phenotype produced by truncating variants in this gene has been defined, manifesting CVID with gastrointestinal inflammatory symptoms and autoimmune manifestations. Methods: We analyzed 5 index cases with suspected primary immunodeficiency by high throughput sequencing. They were submitted for a genetic test with a panel of genes associated with immune system diseases, including IRF2BP2. The screening of SNVs, indels and CNVs fulfilling the criteria with very low allelic frequency and high protein impact, revealed five novel variants in IRF2BP2. In addition, we isolated both wild-type and mutated allele of the cDNA from one of the families. Results: In this study, we report five novel loss-of-function (LoF) mutations in IRF2BP2 that likely cause primary immunodeficiency, with CVID as more frequent phenotype, variable expression of inflammatory gastrointestinal features, and one patient with predisposition of viral infection. All identified variants were frameshift changes, and one of them was a large deletion located on chromosome 1q42, which includes the whole sequence of IRF2BP2, among other genes. Both de novo and dominant modes of inheritance were observed in the families here presented, as well as incomplete penetrance. Conclusions: We describe novel variants in a delimited low-complex region, which may be considered a hotspot in IRF2BP2. Moreover, this is the first time that a large CNV in IRF2BP2 has been reported to cause CVID. The distinct mechanisms than LoF in IRF2BP2 could cause different phenotype compared with the mainly described. Further investigations are necessary to comprehend the regulatory mechanisms of IRF2BP2, which could be under variable expression of the disease.

Genotyping, sequencing and analysis of 140,000 adults from Mexico City.

The Mexico City Prospective Study is a prospective cohort of more than 150,000 adults recruited two decades ago from the urban districts of Coyoacán and Iztapalapa in Mexico City1. Here we generated genotype and exome-sequencing data for all individuals and whole-genome sequencing data for 9,950 selected individuals. We describe high levels of relatedness and substantial heterogeneity in ancestry composition across individuals. Most sequenced individuals had admixed Indigenous American, European and African ancestry, with extensive admixture from Indigenous populations in central, southern and southeastern Mexico. Indigenous Mexican segments of the genome had lower levels of coding variation but an excess of homozygous loss-of-function variants compared with segments of African and European origin. We estimated ancestry-specific allele frequencies at 142 million genomic variants, with an effective sample size of 91,856 for Indigenous Mexican ancestry at exome variants, all available through a public browser. Using whole-genome sequencing, we developed an imputation reference panel that outperforms existing panels at common variants in individuals with high proportions of central, southern and southeastern Indigenous Mexican ancestry. Our work illustrates the value of genetic studies in diverse populations and provides foundational imputation and allele frequency resources for future genetic studies in Mexico and in the United States, where the Hispanic/Latino population is predominantly of Mexican descent.

The fading guardian: clinical relevance of TP53 null mutation in high-grade serous ovarian cancers.

Background: we evaluated the concordance between immunohistochemical p53 staining and TP53 mutations in a series of HGSOC. Moreover, we searched for prognostic differences between p53 overexpression and null expression groups. Methods: patients affected by HGSOC were included. For each case p53 immunohistochemical staining and molecular assay (Sanger sequencing) were performed. Kaplan-Meier survival analyses were undertaken to determine whether the type of TP53 mutation, or p53 staining pattern influenced overall survival (OS) and progression free survival (PFS). Results: 34 HGSOC were considered. All cases with a null immunohistochemical p53 expression (n=16) showed TP53 mutations (n=9 nonsense, n=4 in-frame deletion, n=2 splice, n=1 in-frame insertion). 16 out of 18 cases with p53 overexpression showed TP53 missense mutation. Follow up data were available for 33 out of 34 cases (median follow up time 15 month). We observed a significant reduction of OS in p53 null group [HR = 3.64, 95% CI 1.01-13.16]. Conclusion: immunohistochemical assay is a reliable surrogate for TP53 mutations in most cases. Despite the small cohort and the limited median follow up, we can infer that HGSOC harboring p53 null mutations are a more aggressive subgroup.

Single-cell brain organoid screening identifies developmental defects in autism.

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

YAP silencing by RB1 mutation is essential for small-cell lung cancer metastasis.

Small cell lung cancer (SCLC) is highly lethal due to its prevalent metastasis. Most SCLCs have inactivating mutations in TP53 and RB1. We find that loss of YAP expression is key for SCLC cells to acquire rapid ameboid migration and high metastatic potential. YAP functions through its target genes CCN1/CCN2 to inhibit SCLC ameboid migration. RB1 mutation contributes to YAP transcriptional silencing via E2F7, which recruits the RCOR co-repressor complex to YAP promoter. We discover that benzamide family HDAC inhibitors stimulate YAP expression by inhibiting the RCOR-HDAC complex, thereby suppressing SCLC metastasis and improving survival in a mouse model. Our study unveils the molecular and cellular basis underlying SCLC's high metastatic potential, the previously unrecognized role of YAP in suppressing ameboid migration and tumor metastasis, and the mechanism of YAP transcription regulation involving E2F7, RCOR, and Sin3 HDAC. This study reveals a therapeutic potential of benzamides for SCLC treatment.

A novel loss of function mutation in the PHD domain of the RAG2 gene, affecting zinc-binding affinity.

BACKGROUND: Severe combined immune deficiencies (SCIDs) are genetically heterogeneous disorders that lead to the absence or malfunction of adaptive immune cells, including T- and B-cells. Pathogenic variants in the RAG2 gene are associated with this disease. METHODS: A couple with consanguineous marriage from the Iranian-Azeri-Turkish ethnic group was referred to the genetic lab. Two children of this family died due to SCID disease with symptoms of skin granulomas, lack of developed T- and B-cells, and intact NK cells. To infer their genotypes, DNA samples obtained from the parents were subjected to whole-exome sequencing (WES). RESULTS: WES data analysis revealed that both parents were carriers of a pathogenic variant, NC_000011.10 (NM_000536.4):c.1268G > C, in the RAG2 gene. This variant was absent in our cohort of 400 healthy individuals from the same ethnic group. To gain insight into the consequence of the variant on the protein function, further analysis was performed by applying bioinformatics tools. This study revealed that the replacement of cysteine with serine at the zinc-binding domain diminished the domain's affinity to zinc ion, resulting in the loss of the mutant protein's ability to bind to the recombination signal sequence (RSS). The formation of the RAG2-RSS complex is vital for T- and B-cell development. CONCLUSION: The identification of a novel pathogenic variant, c.1268G > C, revealed that this variant in the zinc-binding domain diminished the affinity of the zinc ion to the mutant protein and consequently led to the loss of its ability to bind to the RSS.

An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis.

Transient receptor potential cation channel-6 (TRPC6) gene mutations cause familial focal segmental glomerulosclerosis (FSGS), which is inherited as an autosomal dominant disease. In patients with TRPC6-related FSGS, all mutations map to the N- or C-terminal TRPC6 protein domains. Thus far, the majority of TRPC6 mutations are missense resulting in increased or decreased calcium influx; however, the fundamental molecular mechanisms causing cell injury and kidney pathology are unclear. We report a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite a largely truncated TRPC6 protein. We studied the molecular effects of V691Kfs* TRPC6 mutant using the tridimensional cryo-EM structure of the tetrameric TRPC6 protein. The results indicated that V691 is localized at the pore-forming transmembrane region affecting the ion conduction pathway, and predicted that V691Kfs* causes closure of the ion-conducting pathway leading to channel inactivation. We assessed the impact of V691Kfs* and two previously reported TRPC6 disease mutants (P112Q and G757D) on calcium influx in cells. Our data show that the V691Kfs* fully inactivated the TRCP6 channel-specific calcium influx consistent with a complete loss-of-function phenotype. Furthermore, the V691Kfs* truncation exerted a dominant negative effect on the full-length TRPC6 proteins. In conclusion, the V691Kfs* non-functional truncated TRPC6 is not sufficient to cause FSGS. Our data corroborate recently characterized TRPC6 loss-of-function and gain-of-function mutants suggesting that one defective TRPC6 gene copy is not sufficient to cause FSGS. We underscore the importance of increased rather than reduced calcium influx through TRPC6 for podocyte cell death.

The PARN, TOE1, and USB1 RNA deadenylases and their roles in non-coding RNA regulation.

The levels of non-coding RNAs (ncRNAs) are regulated by transcription, RNA processing, and RNA degradation pathways. One mechanism for the degradation of ncRNAs involves the addition of oligo(A) tails by non-canonical poly(A) polymerases, which then recruit processive sequence-independent 3' to 5' exonucleases for RNA degradation. This pathway of decay is also regulated by three 3' to 5' exoribonucleases, USB1, PARN, and TOE1, which remove oligo(A) tails and thereby can protect ncRNAs from decay in a manner analogous to the deubiquitination of proteins. Loss-of-function mutations in these genes lead to premature degradation of some ncRNAs and lead to specific human diseases such as Poikiloderma with Neutropenia (PN) for USB1, Dyskeratosis Congenita (DC) for PARN and Pontocerebellar Hypoplasia type 7 (PCH7) for TOE1. Herein, we review the biochemical properties of USB1, PARN, and TOE1, how they modulate ncRNA levels, and their roles in human diseases.

Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95.

Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor's transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow.