Balanced chromosomal rearrangements implicate YIPF5 and SPATC1L in non-obstructive oligoasthenozoospermia and oligozoospermia and of a derivative chromosome 22 in recurrent miscarriage.

Naturally occurring balanced, unbalanced, and complex chromosomal rearrangements have been reported to cause pathogenic genomic or genetic variants leading to infertility and recurrent miscarriage. Therefore, balanced chromosomal rearrangements were used as genomic signposts for identification of candidate genes or genomic loci associated with male infertility due to defects of spermatogenesis, or with recurrent miscarriage. In three male probands, structural chromosomal variants and copy number variants were identified at nucleotide resolution by long-insert genome sequencing approaches and Sanger sequencing. The pathogenic potential of these and affected candidate genes was assessed based on convergent genomic and genotype-phenotype correlation data. Identification of balanced chromosomal rearrangement breakpoints and interpretation in the context of their genomic background of structural and copy number variants led us to conclude that the infertility due to oligoasthenozoospermia and oligozoospermia is most likely associated with a position effect on YIPF5 and SPATC1L, respectively. In a third proband with intellectual disability and recurrent miscarriage, disruption of CAMK2B causing autosomal dominant, intellectual developmental disorder 54 and increased meiotic segregation during gametogenesis of a der(22) are responsible for the reported phenotype. Our data further support the existence of loci at 5q23 and 21q22.3 for these spermatogenesis defects and highlight the importance of the naturally occurring balanced chromosomal rearrangements for assessment of the pathogenic mechanisms. Furthermore, we show comorbidities due to the same balanced chromosomal rearrangement caused by different pathogenic mechanisms.

Corrigendum: SVInterpreter: A Comprehensive Topologically Associated Domain Based Clinical Outcome Prediction Tool for Balanced and Unbalanced Structural Variants.

[This corrects the article DOI: 10.3389/fgene.2021.757170.].

A Novel Frameshift CHD4 Variant Leading to Sifrim-Hitz-Weiss Syndrome in a Proband with a Subclinical Familial t(17;19) and a Large dup(2)(q14.3q21.1).

The genetic complexity of neurodevelopmental disorders (NDD), combined with a heterogeneous clinical presentation, makes accurate assessment of their molecular bases and pathogenic mechanisms challenging. Our purpose is to reveal the pathogenic variant underlying a complex NDD through identification of the "full" spectrum of structural genomic and genetic variants. Therefore, clinical phenotyping and identification of variants by genome and exome sequencing, together with comprehensive assessment of these and affected candidate genes, were carried out. A maternally-inherited familial translocation [t(17;19)(p13.1;p13.3)mat] disrupting the GSG1 like 2 gene (GSG1L2), a 3.2 Mb dup(2)(q14.3q21.1) encompassing the autosomal dominant OMIM phenotype-associated PROC and HS6ST1 gene, and a novel frameshift c.4442del, p.(Gly1481Valfs*21) variant within exon 30 of the Chromodomain helicase DNA binding protein 4 (CHD4) have been identified. Considering the pathogenic potential of each variant and the proband's phenotype, we conclude that this case basically fits the Sifrim-Hitz-Weiss syndrome or CHD4-associated neurodevelopmental phenotype. Finally, our data highlight the need for identification of the "full" spectrum of structural genomic and genetic variants and of reverse comparative phenotyping, including unrelated patients with variants in same genes, for improved genomic healthcare of patients with NDD.

SVInterpreter: A Comprehensive Topologically Associated Domain-Based Clinical Outcome Prediction Tool for Balanced and Unbalanced Structural Variants.

With the advent of genomic sequencing, a number of balanced and unbalanced structural variants (SVs) can be detected per individual. Mainly due to incompleteness and the scattered nature of the available annotation data of the human genome, manual interpretation of the SV's clinical significance is laborious and cumbersome. Since bioinformatic tools developed for this task are limited, a comprehensive tool to assist clinical outcome prediction of SVs is warranted. Herein, we present SVInterpreter, a free Web application, which analyzes both balanced and unbalanced SVs using topologically associated domains (TADs) as genome units. Among others, gene-associated data (as function and dosage sensitivity), phenotype similarity scores, and copy number variants (CNVs) scoring metrics are retrieved for an informed SV interpretation. For evaluation, we retrospectively applied SVInterpreter to 97 balanced (translocations and inversions) and 125 unbalanced (deletions, duplications, and insertions) previously published SVs, and 145 SVs identified from 20 clinical samples. Our results showed the ability of SVInterpreter to support the evaluation of SVs by (1) confirming more than half of the predictions of the original studies, (2) decreasing 40% of the variants of uncertain significance, and (3) indicating several potential position effect events. To our knowledge, SVInterpreter is the most comprehensive TAD-based tool to identify the possible disease-causing candidate genes and to assist prediction of the clinical outcome of SVs. SVInterpreter is available at http://dgrctools-insa.min-saude.pt/cgi-bin/SVInterpreter.py.

Comprehensive clinically oriented workflow for nucleotide level resolution and interpretation in prenatal diagnosis of de novo apparently balanced chromosomal translocations in their genomic landscape.

We present a comprehensive clinically oriented workflow for large-insert genome sequencing (liGS)-based nucleotide level resolution and interpretation of de novo (dn) apparently balanced chromosomal abnormalities (BCA) in prenatal diagnosis (PND). Retrospective or concomitant with conventional PND and liGS, molecular and newly developed clinically inspired bioinformatic tools (TAD-GConTool and CNV-ConTool) are applied to analyze and assess the functional and phenotypic outcome of dn structural variants (dnSVs). Retrospective analysis of four phenotype-associated dnSVs identified during conventional PND precisely reveal the genomic elements disrupted by the translocation breakpoints. Identification of autosomal dominant disease due to the disruption of ANKS1B and WDR26 by t(12;17)(q23.1;q21.33)dn and t(1;3)(q24.11;p25.3)dn breakpoints, respectively, substantiated the proposed workflow. We then applied this workflow to two ongoing prenatal cases with apparently balanced dnBCAs: 46,XX,t(16;17)(q24;q21.3)dn referred for increased risk on combined first trimester screening and 46,XY,t(2;19)(p13;q13.1)dn referred due to a previous trisomy 21 pregnancy. Translocation breakpoints in the t(16;17) involve ANKRD11 and WNT3 and disruption of ANKRD11 resulted in KBG syndrome confirmed in postnatal follow-up. Breakpoints in the t(2;19) are within ATP6V1B1 and the 3' UTR of CEP89, and are not interpreted to cause disease. Genotype-phenotype correlation confirms the causative role of WDR26 in the Skraban-Deardorff and 1q41q42 microdeletion phenocopy syndromes, and that disruption of ANKS1B causes ANKS1B haploinsufficiency syndrome. In sum, we show that an liGS-based approach can be realized in PND care providing additional information concerning clinical outcomes of dnBCAs in patients with such rearrangements.

Identification of OAF and PVRL1 as candidate genes for an ocular anomaly characterized by Peters anomaly type 2 and ectopia lentis.

Keratolenticular dysgenesis (KLD) and ectopia lentis are congenital eye defects. The aim of this study is the identification of molecular genetic alterations responsible for those ocular anomalies with neurologic impairment in an individual with a de novo balanced chromosome translocation t(11;18)(q23.3;q11.2)dn. Disruption of OAF, the human orthologue of the Drosophila oaf, by the 11q23.3 breakpoint results in reduced expression of this transcriptional regulator. Furthermore, four most likely nonfunctional chimeric transcripts comprising up to OAF exon 3, derived from the der(11) allele, have also been identified. This locus has been implicated by publicly available genome-wide association data in corneal disease and corneal topography. The expression of the poliovirus receptor-related 1(PVRL1) or nectin cell adhesion molecule 1 (NECTIN1), a paralogue of nectin cell adhesion molecule 3 (PVRL3) associated with congenital ocular defects, situated 500 kb upstream from 11q23.3 breakpoint, is increased. The 18q11.2 breakpoint is localized between cutaneous T-cell lymphoma-associated antigen 1(CTAGE1) and retinoblastoma binding protein 8 (RBBP8) genes. Genomic imbalance that could contribute to the observed phenotype was excluded. Analysis of gene expression datasets throughout normal murine ocular lens embryogenesis suggests that OAF expression is significantly enriched in the lens from early stages of development through adulthood, whereas PVRL1 is lens-enriched until E12.5 and then down-regulated. This contrasts with the observation that the proposita's lymphoblastoid cell lines exhibit low OAF and high PVRL1 expression as compared to control, which offers further support that the alterations described above are most likely responsible for the clinical phenotype. Finally, gene interaction topology data for PVRL1 also agree with our proposal that disruption of OAF by the translocation breakpoint and misregulation of PVRL1 due to a position effect contribute to the observed ocular and neurological phenotype.

Structure_threader: An improved method for automation and parallelization of programs structure, fastStructure and MavericK on multicore CPU systems.

Structure_threader is a program to parallelize multiple runs of genetic clustering software that does not make use of multithreading technology (structure, fastStructure and MavericK) on multicore computers. Our approach was benchmarked across multiple systems and displayed great speed improvements relative to the single-threaded implementation, scaling very close to linearly with the number of physical cores used. Structure_threader was compared to previous software written for the same task-ParallelStructure and StrAuto and was proven to be the faster (up to 25% faster) wrapper under all tested scenarios. Furthermore, Structure_threader can perform several automatic and convenient operations, assisting the user in assessing the most biologically likely value of 'K' via implementations such as the "Evanno," or "Thermodynamic Integration" tests and automatically draw the "meanQ" plots (static or interactive) for each value of K (or even combined plots). Structure_threader is written in python 3 and licensed under the GPLv3. It can be downloaded free of charge at https://github.com/StuntsPT/Structure_threader.

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification.

KEY MESSAGE: RNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment. The molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

Shared and divergent pathways for flower abscission are triggered by gibberellic acid and carbon starvation in seedless Vitis vinifera L.

BACKGROUND: Abscission is a highly coordinated developmental process by which plants control vegetative and reproductive organs load. Aiming at get new insights on flower abscission regulation, changes in the global transcriptome, metabolome and physiology were analyzed in 'Thompson Seedless' grapevine (Vitis vinifera L.) inflorescences, using gibberellic acid (GAc) spraying and shading as abscission stimuli, applied at bloom. RESULTS: Natural flower drop rates increased from 63.1% in non-treated vines to 83% and 99% in response to GAc and shade treatments, respectively. Both treatments had a broad effect on inflorescences metabolism. Specific impacts from shade included photosynthesis inhibition, associated nutritional stress, carbon/nitrogen imbalance and cell division repression, whereas GAc spraying induced energetic metabolism simultaneously with induction of nucleotide biosynthesis and carbon metabolism, therefore, disclosing alternative mechanisms to regulate abscission. Regarding secondary metabolism, changes in flavonoid metabolism were the most represented metabolic pathways in the samples collected following GAc treatment while phenylpropanoid and stilbenoid related pathways were predominantly affected in the inflorescences by the shade treatment. However, both GAc and shade treated inflorescences revealed also shared pathways, that involved the regulation of putrescine catabolism, the repression of gibberellin biosynthesis, the induction of auxin biosynthesis and the activation of ethylene signaling pathways and antioxidant mechanisms, although often the quantitative changes occurred on specific transcripts and metabolites of the pathways. CONCLUSIONS: Globally, the results suggest that chemical and environmental cues induced contrasting effects on inflorescence metabolism, triggering flower abscission by different mechanisms and pinpointing the participation of novel abscission regulators. Grapevine showed to be considered a valid model to study molecular pathways of flower abscission competence acquisition, noticeably responding to independent stimuli.

Molecular candidates for early-stage flower-to-fruit transition in stenospermocarpic table grape (Vitis vinifera L.) inflorescences ascribed by differential transcriptome and metabolome profiles.

Flower-to-fruit transition depends of nutrient availability and regulation at the molecular level by sugar and hormone signalling crosstalk. However, in most species, the identities of fruit initiation regulators and their targets are largely unknown. To ascertain the main pathways involved in stenospermocarpic table grape fruit set, comprehensive transcriptional and metabolomic analyses were conducted specifically targeting the early phase of this developmental stage in 'Thompson Seedless'. The high-throughput analyses performed disclosed the involvement of 496 differentially expressed genes and 28 differently accumulated metabolites in the sampled inflorescences. Our data show broad transcriptome reprogramming of molecule transporters, globally down-regulating gene expression, and suggest that regulation of sugar- and hormone-mediated pathways determines the downstream activation of berry development. The most affected gene was the SWEET14 sugar transporter. Hormone-related transcription changes were observed associated with increased indole-3-acetic acid, stimulation of ethylene and gibberellin metabolisms and cytokinin degradation, and regulation of MADS-box and AP2-like ethylene-responsive transcription factor expression. Secondary metabolism, the most representative biological process at transcriptome level, was predominantly repressed. The results add to the knowledge of molecular events occurring in grapevine inflorescence fruit set and provide a list of candidates, paving the way for genetic manipulation aimed at model research and plant breeding.