Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development.

BACKGROUND: Forty-six ,XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic. METHODS: To determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed. RESULTS: Cytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the SRY gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a sub-group of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients. CONCLUSION: Rare or novel pathogenic variants were identified in the AR, SRD5A2, ZNRF3, SOX8, SOX9 and HHAT genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.

QUANTITATIVE ANALYSIS OF CHORIOCAPILLARIS ALTERATIONS IN SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY IN DIABETIC PATIENTS.

PURPOSE: To evaluate quantitative alterations of the choriocapillaris in swept-source optical coherence tomography angiography in diabetic patients. METHODS: We included normal patients and diabetic patients with and without diabetic retinopathy (DR), excluding patients with macular edema. Angiograms in 3 × 3 mm were acquired with Plexelite 9000 swept-source optical coherence tomography angiography. Choroidal flow voids were analyzed after removal of projection artifacts. The main evaluation was the correlation between choroidal flow voids area (FVA-CC) and DR stage. RESULTS: A total of 120 eyes of 72 patients were analyzed. There were 17 eyes from healthy subjects, 30 eyes without DR, 22 eyes with minimal nonproliferative DR, 30 eyes with moderate nonproliferative DR, 16 eyes with severe nonproliferative DR, and 5 eyes with proliferative DR (PDR). The percentage of FVA-CC for each group was, respectively, 10.9 ± 3.4%, 14.6 ± 4.8%, 17.6 ± 3.5%, 20.7 ± 5.9%, 19.9 ± 2.9%, and 26.6 ± 4.4%. FVA-CC and DR stage significantly correlated (P < 0.0001). FVA-CC was significantly increased in diabetic patients without DR compared with healthy subjects (P = 0.008). CONCLUSION: Diabetes is associated with quantifiable choriocapillaris alterations in swept-source optical coherence tomography angiography. These alterations precede clinical signs of DR and are correlated with DR stage.

Neuronal migration genes and a familial translocation t (3;17): candidate genes implicated in the phenotype.

BACKGROUND: While Miller-Dieker syndrome critical region deletions are well known delineated anomalies, submicroscopic duplications in this region have recently emerged as a new distinctive syndrome. So far, only few cases have been described overlapping 17p13.3 duplications. METHODS: In this study, we report on clinical and cytogenetic characterization of two new cases involving 17p13.3 and 3p26 chromosomal regions in two sisters with familial history of lissencephaly. Fluorescent In Situ Hybridization and array Comparative Genomic Hybridization were performed. RESULTS: A deletion including the critical region of the Miller-Dieker syndrome of at least 2,9 Mb and a duplication of at least 3,6 Mb on the short arm of chromosome 3 were highlighted in one case. The opposite rearrangements, 17p13.3 duplication and 3p deletion, were observed in the second case. This double chromosomal aberration is the result of an adjacent 1:1 meiotic segregation of a maternal reciprocal translocation t(3,17)(p26.2;p13.3). CONCLUSIONS: 17p13.3 and 3p26 deletions have a clear range of phenotypic features while duplications still have an uncertain clinical significance. However, we could suggest that regardless of the type of the rearrangement, the gene dosage and interactions of CNTN4, CNTN6 and CHL1 in the 3p26 and PAFAH1B1, YWHAE in 17p13.3 could result in different clinical spectrums.

A subset of genomic alterations detected in rolandic epilepsies contains candidate or known epilepsy genes including GRIN2A and PRRT2.

OBJECTIVES: Rolandic epilepsies (REs) represent the most frequent epilepsy in childhood. Patients may experience cognitive, speech, language, reading, and behavioral issues. The genetic origin of REs has long been debated. The participation of rare copy number variations (CNVs) in the pathophysiology of various human epilepsies has been increasingly recognized. However, no systematic search for microdeletions or microduplications has been reported in RE so far. METHODS: Array comparative genomic hybridization (aCGH) and quantitative polymerase chain reaction (qPCR) were used to analyze the genomic status of a series of 47 unrelated RE patients who displayed various types of electroclinical manifestations. RESULTS: Thirty rare CNVs were detected in 21 RE patients. Two CNVs were de novo, 12 were inherited, and 16 were of unknown inheritance. Each CNV was unique to one given patient, except for a 16p11.2 duplication found in two patients. The CNVs of highest interest comprised or disrupted strong candidate or confirmed genes for epileptic and other neurodevelopmental disorders, including BRWD3, GRIN2A, KCNC3, PRKCE, PRRT2, SHANK1, and TSPAN7. SIGNIFICANCE: Patients with REs showed rare microdeletions and microduplications with high frequency and heterogeneity. Whereas only a subset of all genomic alterations found here may actually participate in the phenotype, the novel de novo events as well as several inherited CNVs contain or disrupt genes, some of which are likely to influence the emergence, the presentation, or the comorbidity of RE. The future screening of cohorts of larger size will help in detecting more de novo or recurrent events and in appreciating the possible enrichment of specific CNVs in patients with RE.

Investigation on the effect of several parameters involved in the biodegradation of polyethylene (PE) and low-density polyethylene (LDPE) under various seawater environments.

This work investigates the biodegradation of polyethylene (PE) and low-density polyethylene (LDPE) and the leaching of their harmful additives. Micro/macro-plastics of both types were subjected to different laboratory-controlled conditions for 3 months. Gas Chromatography-Mass Spectroscopy (GC-MS) results revealed that leachate concentrations ranged from 0.40 ± 0.07 µg/L to 96.36 ± 0.11 µg/L. It was concluded that the additives' leaching process was promoted by light. However, light was not the only factor examined; microorganisms, pH, salinity, aeration/mixing and temperature influenced the biodegradation process, too. GC-MS results showed a prodigious impact on the biodegradation process when Pseudomonas aeruginosa was added to the artificial seawater compared to plastics exposed to light/air only. Scanning Electron Microscopy (SEM) micrographs demonstrated a significant alteration in the plastics' morphologies. Similarly, Fourier-Transform Infrared Spectroscopy (FTIR) spectra showed obvious changes in plastics characteristic peaks, especially microplastics. Furthermore, it was shown that PE was more susceptible to degradation/biodegradation than LDPE. Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) findings showed that some toxic metals were present in water samples after experiments, with concentrations above the permissible limits. For instance, bio-augmentation/bio-stimulation experiments showed that the concentrations of Pb, Sr, and Zn were 0.59 mg/L, 70.09 mg/L, and 0.17 mg/L, respectively; values above the permissible limits. It is crucial to emphasise that plastics must be meticulously engineered to avoid environmental and human impacts, originated from their degradation by-products. Furthermore, a holistic approach engaging stakeholders, researchers, policymakers, industries and consumers, is essential to effectively tackle the global challenge of marine plastic pollution.

Interstitial 12p13.1 deletion involving GRIN2B in three patients with intellectual disability.

We report on three patients presenting moderate intellectual disability, delayed language acquisition, and mild facial dysmorphia. Array-CGH studies revealed overlapping interstitial 12p13.1 microdeletions encompassing all or part of GRIN2B. GRIN2B encodes the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor. This receptor is a heteromeric glutamate-activated ion channel, present throughout the central nervous system. It plays a critical role in corticogenesis, neuronal migration, and synaptogenesis during brain development. GRIN2B alterations, including mutation and gene disruption by apparently balanced chromosomal rearrangements, have been described in patients with intellectual disability and autism spectrum disorder. We report here on the first cases of GRIN2B deletion, enlarging the spectrum of GRIN2B abnormalities. Our findings confirm the involvement of this gene in neurodevelopmental disorders. © 2013 Wiley Periodicals, Inc.

Effect of temperature and sunlight on the leachability potential of BPA and phthalates from plastic litter under marine conditions.

This study investigates the leaching potential of several additives embedded in six different plastic types when exposed to extreme simulated marine conditions for 140 days. The findings achieved herein contribute to a better understanding of the impact of macro- and microplastics leaching harmful compounds (bisphenol A (BPA) and phthalates) in the marine environment when exposed to harsh climatic conditions. Leachability experiments showed that bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and BPA were detected in seawater (SW) samples. Furthermore, while analysing 100 mL of SW per each sample, the total leachate concentrations of the identified compounds ranged from 5 µg/L to 123 µg/L, after 140 days of exposing a total of 120 plastic samples (96 samples micro- and 24 macro-plastics) to SW conditions It was observed that the leaching of DEHP was promoted by wave abrasion, high temperature and sunlight, while the leaching of DBP was favoured by wave abrasion. Findings showed that polypropylene (PP) was the most attributable plastic type in the leaching of DBP with an average concentration of 5.3 µg/L, whereas high-density polyethylene (HDPE) was the most responsible plastic-type for the leaching of DEHP, with an average concentration of 123 µg/L. Our results suggest that most of the phthalates and BPA will, ultimately, leach out to the SW environment after a longer period.

Insights into the degradation mechanism of PET and PP under marine conditions using FTIR.

Plastics possess diverse functional properties that have made them extremely desirable. However, due to poor waste management practices, large quantities eventually end up in the oceans where their degradation begins. Hence, it is imperative to understand and further investigate the dynamics of this process. Currently, most relevant studies have been carried out under benign and/or controlled weather conditions. This study investigates the natural degradation of polypropylene (PP) and polyethylene terephthalate (PET) in more extreme environments. Simulated and real marine conditions, both in the laboratory (indoors) and outdoors were applied for a duration of 140 days and results were assessed using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) with energy dispersive X-ray analysis. SEM micrographs revealed variations in the morphologies of both plastic types. Degradation signs were shown in both plastic types, under all conditions. Findings indicated that microplastics (MPs) degraded faster than macroplastics, with PP MPs having higher weight loss (49%) than PET MPs (1%) when exposed to outdoor marine conditions. Additionally, the degradation rates of MPs-PP were higher than MPs-PET for outdoor and indoor treatments, with 1.07 × 10-6 g/d and 4.41 × 10-7 g/d, respectively. FTIR combined with PCA was efficient in determining the most degraded plastic types.

X-linked recessive ichthyosis in 8 Tunisian patients: awareness of misdiagnosis due to the technical trap of the STS pseudogene.

INTRODUCTION: X-linked recessive ichthyosis (XLI) is a genodermatosis, caused by a deficiency of the steroid sulphatase enzyme encoded by the STS gene (OMIM # 300,747). Adopted XLI molecular diagnosis approaches differ from one laboratory to another depending on available technical facilities. Our work aims to figure out a sound diagnostic strategy for XLI. PATIENTS AND METHODS: We collected 8 patients with XLI, all males, from 3 unrelated Tunisian families from central Tunisia. Genetic diagnosis was conducted through a large panel of genetic techniques including: Sanger Sequencing, haplotype analysis of STR markers, MLPA analysis, FISH and array CGH. RESULTS: Direct Sanger sequencing of the STS gene showed the same deletion of 13 base pairs within the exon 4 in all patients resulting in a premature stop codon. However, all patients' mothers were not carriers of this variant and no common haplotype flanking STS gene was shared between affected patients. Sequence alignment with reference human genome revealed an unprocessed pseudogene of the STS gene located on the Y chromosome, on which the 13 bp deletion was actually located. STS MLPA analysis identified a deletion of the entire STS gene on X chromosome for all affected patients. This deletion was confirmed by FISH and delineated by array CGH. CONCLUSION: All our patients shared a deletion of the entire STS gene revealed by MLPA, confirmed by FISH and improved by array CGH. Geneticists must be aware of the presence of pseudogenes that can lead to XLI genetic misdiagnosis.

Clinical and molecular characterization of 1q43q44 deletion and corpus callosum malformations: 2 new cases and literature review.

BACKGROUND: Corpus callosum malformations (CCM) represent one of the most common congenital cerebral malformations with a prevalence of around one for 4000 births. There have been at least 230 reports in the literature concerning 1q43q44 deletions of varying sizes discovered using chromosomal microarrays. This disorder is distinguished by global developmental delay, seizures, hypotonia, corpus callosum defects, and significant craniofacial dysmorphism. In this study, we present a molecular cytogenetic analysis of 2 Tunisian patients with corpus callosum malformations. Patient 1 was a boy of 3 years old who presented psychomotor retardation, microcephaly, behavioral problems, interventricular septal defect, moderate pulmonary stenosis, hypospadias, and total CCA associated with delayed encephalic myelination. Patient 2 was a boy of 9 months. He presented a facial dysmorphia, a psychomotor retardation, an axial hypotonia, a quadri pyramidal syndrome, a micropenis, and HCC associated with decreased volume of the periventricular white matter. Both the array comparative genomic hybridization and fluorescence in situ hybridization techniques were used. RESULTS: Array CGH analysis reveals that patient 1 had the greater deletion size (11,7 Mb) at 1q43. The same region harbors a 2,7 Mb deletion in patient 2. Here, we notice that the larger the deletion, the more genes are likely to be involved, and the more severe the phenotype is likely to be. In both patients, the commonly deleted region includes six genes: PLD5, AKT3, ZNF238, HNRNPU, SDCCAG8 and CEP170. Based on the role of the ZNF238 gene in neuronal proliferation, migration, and cortex development, we hypothesized that the common deletion of ZNF238 in both patients seems to be the most responsible for corpus callosum malformations. Its absence may directly cause CCM. In addition, due to their high expression in the brain, PLD5 and FMN2 could modulate in the CCM phenotype. CONCLUSION: Our findings support and improve the complex genotype-phenotype correlations previously reported in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of several genes related to this pathology.

Retinal Vascularization Analysis on Optical Coherence Tomography Angiography before and after Intraretinal or Subretinal Fluid Resorption in Exudative Age-Related Macular Degeneration: A Pilot Study.

The aim was to analyze the variations in macular vascularization on optical coherence tomography angiography (OCTA) according to the presence of intraretinal fluid (IRF) induced by exudative age-related macular degeneration (AMD). We included exudative AMD patients with IRF and/or subretinal fluid (SRF) and age-matched control eyes. All patients underwent a macular 6 × 6 mm swept-source OCTA. The mean perfusion density (MPD) and mean vascular density (MVD) were calculated in the superficial (SCP) and the deep (DCP) capillary plexus at two timepoints: during an episode of exudation (T0) and after its total resorption (T1). A total of 22 eyes in the IRF ± SRF group, 11 eyes in the SRF group and 11 eyes in the healthy group were analyzed. At T0, the IRF ± SRF group showed significantly lower MPD and MVD than healthy eyes in the SCP (p < 0.001) and DCP (p < 0.001). At T1, MPD and MVD significantly increased from T0 in the SCP (p = 0.027 and p = 0.0093) and DCP (p = 0.013 and p = 0.046) but remained statistically lower than in the healthy eyes. For the SRF group, only the DCP showed significantly lower MPD (p = 0.012) and MVD (p = 0.046) in comparison to the healthy eyes at T0. The present study shows that retinal vascular changes do occur in the case of exudative AMD.

Clinical and molecular findings in nine new cases of tetrasomy 18p syndrome: FISH and array CGH characterization.

BACKGROUND: Small Supernumerary Marker Chromosomes (sSMC) are rare chromosomal abnormalities, which have abnormal banding arrangement and take many shapes. Several disorders have been correlated with sSMC presence. The aim of this study is to characterize the sSMC derived from chromosome 18 by Fluorescence in situ hybridization (FISH) and Array Comparative Genomic Hybridization (aCGH). RESULTS: Nine children with dysmorphic features have been investigated. They have these features in common: a triangular face, low-set ears, a large mouth with a thin upper lip, and a horizontal palpebral fissure. Epicanthus and strabismus were present in two patients. In addition, we have noticed microcephaly and mental and/or developmental delay with low birth weight. However, two patients had standard birth weight; one patient had hypospadias; two had skin problems; and three showed different congenital heart defects. One patient had corpus callosum hypoplasia. Systematic karyotype analysis revealed a de novo supernumerary chromosome. Array CGH showed a gain in copy number on the short arm of chromosome 18 in the nine cases. In one case, the sSMC seemed to be in mosaic. The breakpoints of the marker were identified using aCGH and FISH. Thus, the sSMC led to 18p tetrasomy with approximately 14 Mb lengths, between 364344 and 14763575 based on the human genome version 18. CONCLUSIONS: These results have been completed by FISH in order to ascertain the shape of the sSMC. Our results confirm the uniqueness and particularity of the iso18p syndrome on the phenotypic as well as on the genetic level.

New MCM8 mutation associated with premature ovarian insufficiency and chromosomal instability in a highly consanguineous Tunisian family.

OBJECTIVE: To identify the gene(s) involved in the etiology of premature ovarian insufficiency in a highly consanguineous Tunisian family. DESIGN: Genetic analysis of a large consanguineous family with several affected siblings. SETTING: University hospital-based cytogenetics and molecular genetics laboratories. PATIENT(S): A highly consanguineous Tunisian family with several affected siblings born to healthy second-degree cousins. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Targeted exome sequencing was performed by next-generation sequencing for affected family members. Mutations were validated by Sanger sequencing. Functional experiments were performed to explore the deleterious effects of the identified mutation. DNA damage was induced by increasing mitomycin C (MMC) concentrations on cultured peripheral lymphocytes. RESULT(S): Analysis of the next-generation sequencing data revealed a new homozygous missense mutation in the minichromosome maintenance 8 gene (MCM8).This homozygous mutation (c. 482A>C; p.His161Pro) was predicted to be deleterious and segregated with the disease in the family. MCM8 participates in homologous recombination during meiosis and DNA double-stranded break repair by dimerizing with MCM9. Mcm8 knock out results in an early block in follicle development and small gonads. Given this, we tested the chromosomal breakage repair capacity of homozygous and heterozygous MCM8 p.His161Pro mutation on cultured peripheral lymphocytes exposed to increasing MMC concentrations. We found that chromosomal breakage after MMC exposure was significantly higher in cells from homozygously affected individuals than in those from a healthy control. CONCLUSION(S): Our findings provide additional support to the view that MCM8 mutations are involved in the primary ovarian insufficiency phenotype.

Novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene associated with 46,XY primary amenorrhea.

OBJECTIVE: To determine the genetic cause of 46,XY primary amenorrhea in three 46,XY girls. DESIGN: Whole exome sequencing. SETTING: University cytogenetics center. PATIENT(S): Three patients with unexplained 46,XY primary amenorrhea were included in the study. INTERVENTION(S): Potentially pathogenic variants were confirmed by Sanger sequencing, and familial segregation was determined where parents' DNA was available. MAIN OUTCOME MEASURE(S): Exome sequencing was performed in the three patients, and the data were analyzed for potentially pathogenic mutations. The functional consequences of mutations were predicted. RESULT(S): Three novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene were identified:c.1573 C→T, p.Gln525Ter, c.1435 C→T p.Arg479Ter, and c.508 C→T, p.Gln170Ter. CONCLUSION(S): Inactivating mutations of the LHCGR gene may be a more common cause of 46,XY primary amenorrhea than previously considered.

Molecular characterization of a cohort of 73 patients with infantile spasms syndrome.

Infantile Spasms syndrome (ISs) is a characterized by epileptic spasms occurring in clusters with an onset in the first year of life. West syndrome represents a subset of ISs that associates spasms in clusters, a hypsarrhythmia EEG pattern and a developmental arrest or regression. Aetiology of ISs is widely heterogeneous including many genetic causes. Many patients, however, remain without etiological diagnosis, which is critical for prognostic purpose and genetic counselling. In the present study, we performed genetic screening of 73 patients with different types of ISs by array-CGH and molecular analysis of 5 genes: CDKL5, STXBP1, KCNQ2, and GRIN2A, whose mutations cause different types of epileptic encephalopathies, including ISs, as well as MAGI2, which was suggested to be related to a subset of ISs. In total, we found a disease-causing mutation or CNV (Copy Number Variation) in 15% of the patients. These included 6 point mutations found in CDKL5 (n = 3) and STXBP1 (n = 3), 3 microdeletions (10 Mb in 2q24.3, 3.2 Mb in 5q14.3 including the region upstream to MEF2C, and 256 kb in 9q34 disrupting EHMT1), and 2 microduplications (671 kb in 2q24.3 encompassing SCN2A, and 11.93 Mb in Xq28). In addition, we discuss 3 CNVs as potential risk factors, including one 16p12.1 deletion, one intronic deletion of the NEDD4 gene, and one intronic deletion of CALN1 gene. The present findings highlight the efficacy of combined cytogenetic and targeted mutation screening to improve the diagnostic yield in patient with ISs.

Comparison of two next-generation sequencing kits for diagnosis of epileptic disorders with a user-friendly tool for displaying gene coverage, DeCovA.

In recent years, molecular genetics has been playing an increasing role in the diagnostic process of monogenic epilepsies. Knowing the genetic basis of one patient's epilepsy provides accurate genetic counseling and may guide therapeutic options. Genetic diagnosis of epilepsy syndromes has long been based on Sanger sequencing and search for large rearrangements using MLPA or DNA arrays (array-CGH or SNP-array). Recently, next-generation sequencing (NGS) was demonstrated to be a powerful approach to overcome the wide clinical and genetic heterogeneity of epileptic disorders. Coverage is critical for assessing the quality and accuracy of results from NGS. However, it is often a difficult parameter to display in practice. The aim of the study was to compare two library-building methods (Haloplex, Agilent and SeqCap EZ, Roche) for a targeted panel of 41 genes causing monogenic epileptic disorders. We included 24 patients, 20 of whom had known disease-causing mutations. For each patient both libraries were built in parallel and sequenced on an Ion Torrent Personal Genome Machine (PGM). To compare coverage and depth, we developed a simple homemade tool, named DeCovA (Depth and Coverage Analysis). DeCovA displays the sequencing depth of each base and the coverage of target genes for each genomic position. The fraction of each gene covered at different thresholds could be easily estimated. None of the two methods used, namely NextGene and Ion Reporter, were able to identify all the known mutations/CNVs displayed by the 20 patients. Variant detection rate was globally similar for the two techniques and DeCovA showed that failure to detect a mutation was mainly related to insufficient coverage.