Structural Modelling of KCNQ1 and KCNH2 Double Mutant Proteins, Identified in Two Severe Long QT Syndrome Cases, Reveals New Insights into Cardiac Channelopathies.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of the QT interval and T-wave abnormalities, caused, in most cases, by mutations in KCNQ1, KCNH2, and SCN5A. Although the predominant pattern of LQTS inheritance is autosomal dominant, compound heterozygous mutations in genes encoding potassium channels have been reported, often with early disease onset and more severe phenotypes. Since the molecular mechanisms underlying severe phenotypes in carriers of compound heterozygous mutations are unknown, it is possible that these compound mutations lead to synergistic or additive alterations to channel structure and function. In this study, all-atom molecular dynamic simulations of KCNQ1 and hERG channels were carried out, including wild-type and channels with compound mutations found in two patients with severe LQTS phenotypes and limited family history of the disease. Because channels can likely incorporate different subunit combinations from different alleles, there are multiple possible configurations of ion channels in LQTS patients. This analysis allowed us to establish the structural impact of different configurations of mutant channels in the activated/open state. Our data suggest that channels with these mutations show moderate changes in folding energy (in most cases of stabilizing character) and changes in channel mobility and volume, differentiating them from each other and from WT. This would indicate possible alterations in K+ ion flow. Hetero-tetrameric mutant channels showed intermediate structural and volume alterations vis-à-vis homo-tetrameric channels. These findings support the hypothesis that hetero-tetrameric channels in patients with compound heterozygous mutations do not necessarily lead to synergistic structural alterations.

DHH pathogenic variants involved in 46,XY disorders of sex development differentially impact protein self-cleavage and structural conformation.

In humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development.

Exploring the Molecular Aetiology of Preeclampsia by Massive Parallel Sequencing of DNA.

PURPOSE OF REVIEW: This manuscript aims to review (for the first time) studies describing NGS sequencing of preeclampsia (PE) women's DNA. RECENT FINDINGS: Describing markers for the early detection of PE is an essential task because, although associated molecular dysfunction begins early on during pregnancy, the disease's clinical signs usually appear late in pregnancy. Although several biochemical biomarkers have been proposed, their use in clinical environments is still limited, thereby encouraging research into PE's genetic origin. Hundreds of genes involved in numerous implantation- and placentation-related biological processes may be coherent candidates for PE aetiology. Next-generation sequencing (NGS) offers new technical possibilities for PE studying, as it enables large genomic regions to be analysed at affordable cost. This technique has facilitated the description of genes contributing to the molecular origin of a significant amount of monogenic and complex diseases. Regarding PE, NGS of DNA has been used in familial and isolated cases, thereby enabling new genes potentially related to the phenotype to be proposed. For a better understanding of NGS, technical aspects, applications and limitations are presented initially. Thereafter, NGS studies of DNA in familial and non-familial cases are described, including pitfalls and positive findings. The information given here should enable scientists and clinicians to analyse and design new studies permitting the identification of novel clinically useful molecular PE markers.

Functional evidence implicating NOTCH2 missense mutations in primary ovarian insufficiency etiology.

Primary ovarian insufficiency (POI) is a frequently occurring disease affecting women under 40 years old. Recently, we have analyzed unrelated POI women via whole exome sequencing (WES) and identified NOTCH2 mutations underlying possible functional effects. The present study involved reanalyzing of WES assays. We used in the KGN granulosa-like cell model, a synthetic gene reporter construct driving luciferase gene expression to assess the functional effects of five NOTCH2 mutations identified in POI patients. We found that NOTCH2-p.Ser1804Leu, p.Ala2316Val, and p.Pro2359Ala mutations had a functional impact on the protein's transcriptional activity. The results have demonstrated for the first time that NOTCH2 mutations contribute to POI etiology. We therefore recommend sequencing NOTCH2's open reading frame in large panels of POI patients to establish an accurate genotype-phenotype correlation. We cannot rule out the fact that patients affected by Alagille syndrome carrying NOTCH2 mutations may suffer ovarian dysfunction.

The forkhead-box family of transcription factors: key molecular players in colorectal cancer pathogenesis.

Colorectal cancer (CRC) is the third most commonly occurring cancer worldwide and the fourth most frequent cause of death having an oncological origin. It has been found that transcription factors (TF) dysregulation, leading to the significant expression modifications of genes, is a widely distributed phenomenon regarding human malignant neoplasias. These changes are key determinants regarding tumour's behaviour as they contribute to cell differentiation/proliferation, migration and metastasis, as well as resistance to chemotherapeutic agents. The forkhead box (FOX) transcription factor family consists of an evolutionarily conserved group of transcriptional regulators engaged in numerous functions during development and adult life. Their dysfunction has been associated with human diseases. Several FOX gene subgroup transcriptional disturbances, affecting numerous complex molecular cascades, have been linked to a wide range of cancer types highlighting their potential usefulness as molecular biomarkers. At least 14 FOX subgroups have been related to CRC pathogenesis, thereby underlining their role for diagnosis, prognosis and treatment purposes.This manuscript aims to provide, for the first time, a comprehensive review of FOX genes' roles during CRC pathogenesis. The molecular and functional characteristics of most relevant FOX molecules (FOXO, FOXM1, FOXP3) have been described within the context of CRC biology, including their usefulness regarding diagnosis and prognosis. Potential CRC therapeutics (including genome-editing approaches) involving FOX regulation have also been included. Taken together, the information provided here should enable a better understanding of FOX genes' function in CRC pathogenesis for basic science researchers and clinicians.

A homozygous donor splice-site mutation in the meiotic gene MSH4 causes primary ovarian insufficiency.

Premature ovarian insufficiency (POI) is a frequent pathology that affects women under 40 years of age, characterized by an early cessation of menses and high FSH levels. Despite recent progresses in molecular diagnosis, the etiology of POI remains idiopathic in most cases. Whole-exome sequencing of members of a Colombian family affected by POI allowed us to identify a novel homozygous donor splice-site mutation in the meiotic gene MSH4 (MutS Homolog 4). The variant followed a strict mendelian segregation within the family and was absent in a cohort of 135 women over 50 years of age without history of infertility, from the same geographical region as the affected family. Exon trapping experiments showed that the splice-site mutation induced skipping of exon 17. At the protein level, the mutation p.Ile743_Lys785del is predicted to lead to the ablation of the highly conserved Walker B motif of the ATP-binding domain, thus inactivating MSH4. Our study describes the first MSH4 mutation associated with POI and increases the number of meiotic/DNA repair genes formally implicated as being responsible for this condition.

Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia.

BACKGROUND: Human reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women's health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL's origin. METHODS: FOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays. RESULTS: Nine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR. CONCLUSIONS: Our results argue in favour of FOXD1 mutations' central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future.

ATG7 and ATG9A loss-of-function variants trigger autophagy impairment and ovarian failure.

PURPOSE: Primary ovarian insufficiency (POI) is a frequent disorder that affects ~1% of women under 40 years of age. POI, which is characterized by the premature depletion of ovarian follicles and elevated plasma levels of follicle-stimulating hormone (FSH), leads to infertility. Although various etiological factors have been described, including chromosomal abnormalities and gene variants, most cases remain idiopathic. The aim of the present study was to identify and validate functionally new sequence variants in ATG (autophagy-related genes) leading to POI. METHODS: We have reanalyzed, in silico, the exome sequencing data from a previously reported work performed in 69 unrelated POI women. Functional experiments using a classical hallmark of autophagy, the microtubule-associated protein 1 light chain 3ß (LC3), were then used to link these genes to this lysosomal degradation pathway. RESULTS: We venture a functional link between ATG7 and ATG9A variants and POI. We demonstrated that variant ATG7 and ATG9A led to a decrease in autophagosome biosynthesis and consequently to an impairment of autophagy, a key biological process implicated in the preservation of the primordial follicles forming the ovarian reserve. CONCLUSION: Our results unveil that impaired autophagy is a novel pathophysiological mechanism involved in human POI.

Transcriptomic analysis of FUCA1 knock-down in keratinocytes reveals new insights into the pathogenesis of fucosidosis skin lesions.

Fucosidosis is a rare lysosomal storage disease which has been classified into two subtypes, depending on the severity of clinical signs and symptoms. Fucosidosis patients' skin abnormalities include angiokeratoma corporis diffusum, widespread telangiectasia, thick skin, hyperhidrosis and hypohidrosis, acrocyanosis and distal transverse nail bands. It has been described that >50% of fucosidosis patients have angiokeratoma. At molecular level, fucosidosis is caused by lysosomal alpha-L-fucosidase (FUCA1) gene mutations. Obtaining samples for functional studies has been challenging due to the inherent difficulty in finding affected individuals. The effect of FUCA1 dysfunction on gene expression is unknown. The aim of this study was to analyse, in keratinocytes, the transcriptomic effect of FUCA1 knock-down for a better understanding of skin lesions' pathogenesis affecting fucosidosis patients. FUCA1 knock-down (siRNA) was performed in human HaCaT immortalised keratinocytes. Affymetrix arrays and qPCR were used for analysing gene expression. Bioinformatics was used for functional clustering of modified genes. In total, 387 genes showed differential expression between FUCA1 silenced and non-silenced cells (222 up-regulated and 165 down-regulated). Up-regulated genes belonged to two major groups: keratinocyte differentiation/epidermal development (n = 17) and immune response (n = 61). Several transcription factors were up-regulated in FUCA1-siRNA transfected cells. This effect might partly have been produced by abnormal transcription factor expression, that is FOXN1. We thus propose that fucosidosis-related skin lesions (eg angiokeratoma) and those of other diseases (eg psoriasis) might be caused by dysfunctions in common aetiological overlapping molecular cascades.

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing.

STUDY QUESTION: Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? SUMMARY ANSWER: WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. WHAT IS KNOWN ALREADY: POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study performed on 69 women affected by POI. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. LIMITATIONS, REASONS FOR CAUTION: It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WIDER IMPLICATIONS OF THE FINDINGS: WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.

THBD sequence variants potentially related to recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a frequently occurring disease, which is classified as idiopathic in more than 50% of cases. THBD, the endothelial cell receptor for thrombin, has been associated with distinct biological processes and considered a coherent RPL-related candidate gene. In the present study, we have sequenced the complete coding region of THBD in 262 patients affected by RPL. Bioinformatics analysis and screening of controls strongly suggested that the THBD-p.Trp153Gly mutation might be related to RPL aetiology. It could be used, after its validation by functional assays, as a molecular marker for diagnostic/prognostic purposes.

Success stories in genomic medicine from resource-limited countries.

In recent years, the translation of genomic discoveries into mainstream medical practice and public health has gained momentum, facilitated by the advent of new technologies. However, there are often major discrepancies in the pace of implementation of genomic medicine between developed and developing/resource-limited countries. The main reason does not only lie in the limitation of resources but also in the slow pace of adoption of the new findings and the poor understanding of the potential that this new discipline offers to rationalize medical diagnosis and treatment. Here, we present and critically discuss examples from the successful implementation of genomic medicine in resource-limited countries, focusing on pharmacogenomics, genome informatics, and public health genomics, emphasizing in the latter case genomic education, stakeholder analysis, and economics in pharmacogenomics. These examples can be considered as model cases and be readily replicated for the wide implementation of pharmacogenomics and genomic medicine in other resource-limited environments.

Polymorphisms of human placental alkaline phosphatase are associated with in vitro fertilization success and recurrent pregnancy loss.

Fertility is a quantitative, complex character governed by a considerable number of genes. Despite clinical and scientific advances, several cases of human infertility remain unexplained. In the present study, using a positional cloning approach in a mouse model of interspecific recombinant lines, a candidate gene, ALPP, encoding the placental alkaline phosphatase, was identified as being potentially involved in recurrent spontaneous abortion. We then analyzed patients for detecting putative associations between ALPP polymorphisms, in vitro fertilization failures, and miscarriages. ALPP was sequenced in 100 controls and 100 patients affected by recurrent spontaneous abortion, from the same ethnic background. The frequency of several alleles and allelic combinations were different between recurrent spontaneous abortion and control women. One polymorphism induced a coding substitution (Ile89Leu) that was associated with a decreased risk of abortion and in vitro fertilization failure. Thereafter, the population was increased by the analysis of 92 additional controls and 612 additional patients for the coding polymorphism Ile89Leu. We finally show, by functional analysis, that the 89Leu placental alkaline phosphatase has an enhanced alkaline phosphatase activity. This study suggests that ALPP genotyping could be a strong predictor of implantation success.

A high resolution map of mammalian X chromosome fragile regions assessed by large-scale comparative genomics.

Chromosomal evolution involves multiple changes at structural and numerical levels. These changes, which are related to the variation of the gene number and their location, can be tracked by the identification of syntenic blocks (SB). First reports proposed that ~180-280 SB might be shared by mouse and human species. More recently, further studies including additional genomes have identified up to ~1,400 SB during the evolution of eutherian species. A considerable number of studies regarding the X chromosome's structure and evolution have been undertaken because of its extraordinary biological impact on reproductive fitness and speciation. Some have identified evolutionary breakpoint regions and fragile sites at specific locations in the human X chromosome. However, mapping these regions to date has involved using low-to-moderate resolution techniques. Such scenario might be related to underestimating their total number and giving an inaccurate location. The present study included using a combination of bioinformatics methods for identifying, at base-pair level, chromosomal rearrangements occurring during X chromosome evolution in 13 mammalian species. A comparative technique using four different algorithms was used for optimizing the detection of hotspot regions in the human X chromosome. We identified a significant interspecific variation in SB size which was related to genetic information gain regarding the human X chromosome. We found that human hotspot regions were enriched by LINE-1 and Alu transposable elements, which may have led to intraspecific chromosome rearrangement events. New fragile regions located in the human X chromosome have also been postulated. We estimate that the high resolution map of X chromosome fragile sites presented here constitutes useful data concerning future studies on mammalian evolution and human disease.

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF.

Linking genotype to trophoblast phenotype in preeclampsia and HELLP syndrome associated with STOX1 genetic variants.

Preeclampsia is a major hypertensive pregnancy disorder with a 50% heritability. The first identified gene involved in the disease is STOX1, a transcription factor, whose variant Y153H predisposes to the disease. Two rare mutations were also identified in Colombian women affected by the hemolysis, elevated liver enzyme, low platelet syndrome, a complication of preeclampsia (T188N and R364X). Here, we explore the effects of these variants in trophoblast cell models (BeWo) where STOX1 was previously invalidated. We firstly showed that STOX1 knockout alters response to oxidative stress, cell proliferation, and fusion capacity. Then, we showed that mutant versions of STOX1 trigger alterations in gene profiles, growth, fusion, and oxidative stress management. The results also reveal alterations of the STOX interaction with DNA when the mutations affected the DNA-binding domain of STOX1 (Y153H and T188N). We also reveal here that a major contributor of these effects appears to be the E2F3 transcription factor.

Sequence analysis of the ADRA2A coding region in children affected by attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a common neurobehavioral pathology characterized by distinct degrees of inattention, hyperactivity and impulsivity. Although ADHD etiology remains elusive, the ADRA2A candidate gene underlies a particular interest, since it participates in the prefrontal cortex regulation of executive function. Three SNPs located on 5' and 3'UTR regions of the gene have been extensively explored but none of them have been definitely validated as a predisposition or a causative sequence variation. In this study, in order to determine whether ADRA2A non-synonymous sequence variants, resulting in biochemical modifications of the protein, are a common cause of the disease we sequenced the complete ADRA2A coding region in a panel of ADHD children of Colombian origin. We identified the c.1138 C>A (p.Arg380Arg) silent substitution. We conclude that ADRA2A non-synonymous sequence variants do not cause ADHD in our sample population. We cannot formerly discard a potential role of this gene during ADHD pathogenesis since only the coding region was analysed. We hope that these results will encourage further researchers to sequence the promoter and coding regions of ADRA2A in large panels of ADHD patients from distinct ethnical origins.

Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis.

Pachydermoperiostosis, or primary hypertrophic osteoarthropathy (PHO), is an inherited multisystem disorder, whose features closely mimic the reactive osteoarthropathy that commonly accompanies neoplastic and inflammatory pathologies. We previously described deficiency of the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (HPGD) as a cause of this condition, implicating elevated circulating prostaglandin E(2) (PGE(2)) as causative of PHO, and perhaps also as the principal mediator of secondary HO. However, PHO is genetically heterogeneous. Here, we use whole-exome sequencing to identify recessive mutations of the prostaglandin transporter SLCO2A1, in individuals lacking HPGD mutations. We performed exome sequencing of four probands with severe PHO, followed by conventional mutation analysis of SLCO2A1 in nine others. Biallelic SLCO2A1 mutations were identified in 12 of the 13 families. Affected individuals had elevated urinary PGE(2), but unlike HPGD-deficient patients, also excreted considerable quantities of the PGE(2) metabolite, PGE-M. Clinical differences between the two groups were also identified, notably that SLCO2A1-deficient individuals have a high frequency of severe anemia due to myelofibrosis. These findings reinforce the key role of systemic or local prostaglandin excess as the stimulus to HO. They also suggest that the induction or maintenance of hematopoietic stem cells by prostaglandin may depend upon transporter activity.