Reconstruction Using Perforator Propeller Flaps After Malignant Melanoma Resection of the Lower Extremity.

Introduction: Surgical excision with margin of 2 cm is the treatment of choice in malignant cutaneous melanoma when Breslow index is >1 mm. When located on the lower limb, these resections can lead to large defects that require complex reconstruction in order to salvage the limb. The use of propeller-perforator flaps has multiple advantages such as the preservation of adjacent muscles, decrease in morbidity in the donor site, and good aesthetic and functional results. Our objective is to expose our experience and results with the use of propeller-perforator flaps for coverage of this kind of defects. Materials and Methods: Patients with malignant cutaneous melanoma of the lower limb who required reconstruction with a propeller-perforator flap between the years 2015 and 2021 in our plastic surgery department were included in this retrospective research. Demographic, reconstructive, oncologic, and functional data were collected and analyzed. Results: The cohort of 22 patients showed 100% of successful reconstructive outcomes. Only 2 patients experienced distal necrosis of the flap that was resolved with local dressings. Fast recovery and early capacity to walk were achieved in the majority of the patients with an average of 10.1 days until weight-baring walking. The 2-year overall and progression-free survival rates were 86.37% and 81.82%, respectively. Conclusion: The use of propeller perforator flaps for oncological defects coverage in the lower limb location has to be considered as a reliable choice as it takes less surgical time and a faster recovery. It allows limb salvage with an adequate length and functionality, with minimal donor site morbidity and a lower index of complications, contributing to improve patient's quality of life and not delaying other oncological treatments.

Introduction: Une excision chirurgicale aux marges de 2 cm est le traitement de première intention des mélanomes cutanés malins lorsque l'indice de Breslow est supérieur à 1 mm. Situées sur un membre inférieur, ces résections peuvent provoquer de grosses anomalies qui exigent une reconstruction complète pour sauver le membre. Le recours aux lambeaux perforants en hélice comporte de multiples avantages, tels que la préservation des muscles adjacents, la diminution des problèmes médicaux au foyer du donneur ainsi que des résultats esthétiques et fonctionnels satisfaisants. Les auteurs visent à présenter leur expérience et leurs résultats lorsqu'ils recouvrent ce type d'anomalie à l'aide de lambeaux perforants en hélice. Matériel et méthodologie: Les patients atteints d'un mélanome cutané malin d'un membre inférieur qui nécessitaient une reconstruction à l'aide d'un lambeau perforant en hélice entre 2015 et 2021 au département de chirurgie plastique des auteurs ont été inclus dans la présente recherche rétrospective. Les auteurs ont recueilli et analysé les caractéristiques démographiques ainsi que les données reconstructives, oncologiques et fonctionnelles des patients. Résultats: Les résultats reconstructifs ont été satisfaisants dans toute la cohorte de 22 patients. Seuls deux d'entre eux ont vécu une nécrose distale du lambeau, qui a été corrigée par des pansements locaux. La majorité des patients se sont remis rapidement et ont pu recommencer à marcher tôt, soit en moyenne au bout de 10,1 jours avec mise en charge. Le taux de survie global et le taux d'absence de détérioration au bout de deux ans s'élevaient à 86,37 % et à 81,82 %, respectivement. Conclusion: Le recours à des lambeaux perforants en hélice pour couvrir des anomalies oncologiques du membre inférieur doit être considéré comme un choix fiable, car l'intervention chirurgicale et la convalescence sont plus courtes. Cette intervention permet de sauver le membre, dont la longueur et la fonction demeurent appropriées, elle est associée à des problèmes médicaux minimes au foyer du donneur et à un faible indice de complications, elle contribue à améliorer la qualité de vie du patient et elle ne retarde pas les autres traitements oncologiques.

National Policies on Parental Leave and Breastfeeding Breaks: Racial, Ethnic, Gender, and Age Disparities in Access and Implications for Infant and Child Health.

OBJECTIVE: Parental leave and breastfeeding breaks influence the ability to initiate and continue breastfeeding. We investigated how eligibility criteria in the Family and Medical Leave Act (FMLA) and Affordable Care Act (ACA) affect access to unpaid parental leave and breastfeeding breaks and assessed affordability and alternative policy models. METHODS: We used family income data to assess the affordability of unpaid leave by race and ethnicity. We used 2017-2018 US Current Population Survey data to determine the percentage of private sector workers aged 18-44 years who met the minimum hour (1250 hours of work during a 12-month period), tenure (12 months), and firm size (≥50 employees) requirements of FMLA and ACA. We analyzed eligibility by gender, race and ethnicity, and age. We also examined parental leave and breastfeeding break policies in 193 countries. RESULTS: Most Latinx (66.9%), Black (60.2%), and White (55.3%) workers were ineligible and/or unlikely to be able to afford to take unpaid FMLA leave. Of 69 534 workers, more women (16.9%) than men (10.3%) did not meet the minimum hour requirement. Minimum tenure excluded 23.7% of all workers and 42.2% of women aged 18-24 years. Minimum firm size excluded 30.3% of all workers and 37.7% of Latinx workers. Of 27 520 women, 28.8% (including 32.9% of Latina women) were excluded from ACA breastfeeding breaks because of firm size. Nearly all other countries guaranteed mothers paid leave regardless of firm size or minimum hours and guaranteed ≥6 months of paid leave or breastfeeding breaks. CONCLUSIONS: Adopting a comprehensive, inclusive paid parental leave policy and closing gaps in breastfeeding break legislation would remove work-related barriers to breastfeeding; reduce racial, ethnic, and gender inequities; and align US national policies with global norms.

An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.

Drought as a possible contributor to the Visigothic Kingdom crisis and Islamic expansion in the Iberian Peninsula.

The Muslim expansion in the Mediterranean basin was one the most relevant and rapid cultural changes in human history. This expansion reached the Iberian Peninsula with the replacement of the Visigothic Kingdom by the Muslim Umayyad Caliphate and the Muslim Emirate of Córdoba during the 8th century CE. In this study we made a compilation of western Mediterranean pollen records to gain insight about past climate conditions when this expansion took place. The pollen stack results, together with other paleohydrological records, archaeological data and historical sources, indicate that the statistically significant strongest droughts between the mid-5th and mid-10th centuries CE (450-950 CE) occurred at 545-570, 695-725, 755-770 and 900-935 CE, which could have contributed to the instability of the Visigothic and Muslim reigns in the Iberian Peninsula. Our study supports the great sensitivity of the agriculture-based economy and socio-political unrest of Early Medieval kingdoms to climatic variations.

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases.

BACKGROUND: Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches. RESULTS: Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1. CONCLUSIONS: In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.

Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms in selected genes using long-read sequencing.

DNA variants altering the pre-mRNA splicing process represent an underestimated cause of human genetic diseases. Their association with disease traits should be confirmed using functional assays from patient cell lines or alternative models to detect aberrant mRNAs. Long-read sequencing is a suitable technique to identify and quantify mRNA isoforms. Available isoform detection and/or quantification tools are generally designed for the whole transcriptome analysis. However experiments focusing on genes of interest need more precise data fine-tuning and visualization tools.Here we describe VIsoQLR, an interactive analyzer, viewer and editor for the semi-automated identification and quantification of known and novel isoforms using long-read sequencing data. VIsoQLR is tailored to thoroughly analyze mRNA expression in splicing assays of selected genes. Our tool takes sequences aligned to a reference, and for each gene, it defines consensus splice sites and quantifies isoforms. VIsoQLR introduces features to edit the splice sites through dynamic and interactive graphics and tables, allowing accurate manual curation. Known isoforms detected by other methods can also be imported as references for comparison. A benchmark against two other popular transcriptome-based tools shows VIsoQLR accurate performance on both detection and quantification of isoforms. Here, we present VIsoQLR principles and features and its applicability in a case study example using nanopore-based long-read sequencing. VIsoQLR is available at https://github.com/TBLabFJD/VIsoQLR .

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.

Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids.

Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment.

Biallelic intragenic tandem duplication of CPLANE1 in Joubert syndrome: A case report.

Joubert syndrome (JS) is a clinically and genetically heterogeneous genetic disorder. To date, 40 JS-causing genes have been reported and CPLANE1 is one of the most frequently mutated, with biallelic pathogenic missense and truncating variants explaining up to 14% of JS cases. We present a case of JS diagnosed after the identification of a novel biallelic intragenic duplication of exons 20-46 of CPLANE1. The quadruplication was identified by short-read sequencing and copy number variant analysis and confirmed in tandem by long PCR with the breakpoints defined by a nanopore-based long-read sequencing approach. Based on the genetic findings and the clinical presentation of the patient, a brain MRI was ordered, evidencing the molar tooth sign, which confirmed the diagnosis of JS in the patient. This is, to the best of our knowledge, the first report of an intragenic duplication in this gene as the potential molecular mechanism of JS.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

Aggressive Acute Marjolin's Ulcer Arising in a Burn Scar.

Marjolin's ulcer is one of the clinical variants of squamous-cell carcinoma. It is a highly aggressive disease that develops from chronic wounds. Almost 65% of these lesions have been diagnosed on underlying burn scars. Although the mean latency time between the primary lesion and the apparition of the ulcer is around 25 years, some cases with an early debut have been described. Squamous-cell carcinomas arising in chronic wounds are typically aggressive and are related with a poor prognosis due to their late diagnosis. Therefore, it is important to recognize symptoms that indicate malignant degeneration of chronic wounds, allowing the clinician to make an early diagnosis in order not to delay the surgical treatment that is required to improve the global survival of the patient. The time elapsed between our patient's burn and the appearance of Marjolin's ulcer was only 7 months, drawing attention to its fast and aggressive progression.

Extracellular vesicles from Listeria monocytogenes-infected dendritic cells alert the innate immune response.

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against Listeria monocytogenes (Lm) infection and other immune related functions. Here, we show that the protein content of dendritic cells (DCs) and their secreted EVs (DEVs) vary during Lm infection, is enriched in proteins related to antiviral functions compared to non-infected cells and depends on HDAC6 expression. Analyses of the post-translational modifications revealed an alteration of the acetylation and ubiquitination profiles upon Lm infection both in DC lysates and DEVs. Functionally, EVs derived from infected DCs upregulate anti-pathogenic genes (e.g. inflammatory cytokines) in recipient immature DCs, which translated into protection from subsequent infection with vaccinia virus. Interestingly, absence of Listeriolysin O in Lm prevents DEVs from inducing this anti-viral state. In summary, these data underscore a new mechanism of communication between bacteria-infected DC during infection as they alert neighboring, uninfected DCs to promote antiviral responses.

Aggregated Genomic Data as Cohort-Specific Allelic Frequencies can Boost Variants and Genes Prioritization in Non-Solved Cases of Inherited Retinal Dystrophies.

The introduction of NGS in genetic diagnosis has increased the repertoire of variants and genes involved and the amount of genomic information produced. We built an allelic-frequency (AF) database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome sequencing tests available, 1766 with IRD and the rest with diverse genetic diseases. We calculated a subcohort's IRD-specific AF and compared it with suitable pseudocontrols. For non-solved IRD cases, we prioritized variants with a significant increment of frequencies, with eight variants that may help to explain the phenotype, and 10/11 of uncertain significance that were reclassified as probably pathogenic according to ACMG. Moreover, we developed a method to highlight genes with more frequent pathogenic variants in IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. We identified 18 genes for further studies that provided new insights in five cases. This resource can also help one to calculate the carrier frequency in IRD genes. A cohort-specific AF database assists with variants and genes prioritization and operates as an engine that provides a new hypothesis in non-solved cases, augmenting the diagnosis rate.

Presence of rare potential pathogenic variants in subjects under 65 years old with very severe or fatal COVID-19.

Rare variants affecting host defense against pathogens could be involved in COVID-19 severity and may help explain fatal outcomes in young and middle-aged patients. Our aim was to report the presence of rare genetic variants in certain genes, by using whole exome sequencing, in a selected group of COVID-19 patients under 65 years who required intubation or resulting in death (n = 44). To this end, different etiopathogenic mechanisms were explored using gene prioritization-based analysis in which genes involved in immune response, immunodeficiencies or blood coagulation were studied. We detected 44 different variants of interest, in 29 different patients (66%). Some of these variants were previously described as pathogenic and were located in genes mainly involved in immune response. A network analysis, including the 42 genes with candidate variants, showed three main components, consisting of 25 highly interconnected genes related to immune response and two additional networks composed by genes enriched in carbohydrate metabolism and in DNA metabolism and repair processes. In conclusion, we have detected candidate variants that may potentially influence COVID-19 outcome in our cohort of patients. Further studies are needed to confirm the ultimate role of the genetic variants described in the present study on COVID-19 severity.

Past 200 kyr hydroclimate variability in the western Mediterranean and its connection to the African Humid Periods.

The Iberian Peninsula is located at the intersection between the subtropical and temperate climate zones and the paleoclimate records from this region are key to elucidate the varying humidity and changing dominance of atmospheric circulation patterns in the Mediterranean-North African region in the past. Here we present a quantitative hydroclimate reconstruction for the last ca. 200 kyr from southern Iberian Peninsula based on pollen data from the Padul lake sediment record. We use the newly developed Scale-normalized Significant Zero crossing (SnSiZer) method to detect not only the statistically significant precipitation changes but also to estimate the relative magnitude of these oscillations in our reconstruction. We identify six statistically significant main humid phases, termed West Mediterranean Humid Periods (WMHP 1-6). These humid periods correlate with other West/Central Mediterranean paleohydrological records, suggesting that similar climatic factors affected different areas of the Mediterranean. In addition, the WMPHs are roughly coeval with the African Humid Periods (AHPs) during high seasonality, suggesting the same North Atlantic ocean-atmospheric dynamics and orbital forcing as main drivers of both areas. In contrast, during low seasonality periods, the West Mediterranean still appears to be affected by the westerlies and the local Mediterranean rainfall systems with moderate-to-high precipitation, whereas West Africa was characterized by droughts.

An evaluation of pipelines for DNA variant detection can guide a reanalysis protocol to increase the diagnostic ratio of genetic diseases.

Clinical exome (CE) sequencing has become a first-tier diagnostic test for hereditary diseases; however, its diagnostic rate is around 30-50%. In this study, we aimed to increase the diagnostic yield of CE using a custom reanalysis algorithm. Sequencing data were available for three cohorts using two commercial protocols applied as part of the diagnostic process. Using these cohorts, we compared the performance of general and clinically relevant variant calling and the efficacy of an in-house bioinformatic protocol (FJD-pipeline) in detecting causal variants as compared to commercial protocols. On the whole, the FJD-pipeline detected 99.74% of the causal variants identified by the commercial protocol in previously solved cases. In the unsolved cases, FJD-pipeline detects more INDELs and non-exonic variants, and is able to increase the diagnostic yield in 2.5% and 3.2% in the re-analysis of 78 cancer and 62 cardiovascular cases. These results were considered to design a reanalysis, filtering and prioritization algorithm that was tested by reassessing 68 inconclusive cases of monoallelic autosomal recessive retinal dystrophies increasing the diagnosis by 4.4%. In conclusion, a guided NGS reanalysis of unsolved cases increases the diagnostic yield in genetic disorders, making it a useful diagnostic tool in medical genetics.

Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts.

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.