Microarray Comparative Genomic Hybridization Research Articles

First report on chromosomal abnormalities in Eastern Morocco: Identification of a new case of a de novo partial trisomy 13q using single-nucleotide polymorphism array

BackgroundChromosomal abnormalities are the main cause of birth defects, intellectual disability, and miscarriages. They contribute to significant human morbidity and infant mortality. Here we report for the first time the chromosomal abnormalities encountered in the population of Eastern Morocco. Furthermore, we describe a new case of a de novo partial trisomy 13q combined with a terminal deletion in an 11-day-old girl. MethodsFrom November 2015 to March 2022, 195 patients from the BRO Biobank who were clinically suspected of having chromosomal abnormalities were referred to the cytogenetics laboratory of the Genetics Unit of the Faculty of Medicine and Pharmacy of Oujda for cytogenetic study. Karyotyping analysis was performed on peripheral blood samples using standard R banding techniques. To identify single-nucleotide polymorphism (SNP) and copy number variants (CNVs), Illumina SNP array was used. ResultsAmong 195 studied cases, 32 (16.4 %) had abnormal karyotypes, of which 12 cases had numerical aberrations while 20 cases had structural aberrations. The most common numerical aberrations were Turner syndrome and Down syndrome followed by Edward, Patau, and Klinefelter syndromes. For structural aberrations, translocations were the most common, followed by derivative chromosomes, inversions, deletions, and an addition on chromosome 13 identified in an 11-day-old girl. To further characterize this addition, SNP array was carried out and revealed a 58.8-Mb duplication in region 13q14.3q34 associated with a 1-Mb deletion in region 13q34. Follow-up parental chromosomes analysis showed normal karyotypes for the parents, confirming that this partial trisomy 13q was de novo. Comparison of the phenotype associated with this novel duplication on chromosome 13q with those previously reported confirmed the considerable variability in the phenotype of the patients with partial trisomy 13q. ConclusionThis study provided the first report on chromosomal abnormalities in Eastern Morocco and it enriched the phenotype spectrum of partial trisomy 13q and further confirmed the genotype–phenotype correlations. Furthermore, these findings justify the need to set up microarray comparative genomic hybridization techniques in Morocco for better genetic diagnosis.

Comparative genomic hybridisation and transcriptome microarray analysis in triple negative breast cancer: An INDIAN study.

Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancers, defined as lack of expression of estrogen receptor, progesterone receptor and Her-2 neu receptors. TNBC has two subtypes basal like and non-basal like, the former characterised by aggressive biology with limited therapeutic options. This study explored molecular markers involved in pathogenesis of TNBC and investigated novel potential diagnostic and therapeutic targets by CGH array and transcriptome array. aCGH analysis in TNBC demonstrated genes amplified were 3888, number of pathway hits was 1554 and major pathways amplified was found to be WNT signalling pathway and Cadherin signalling pathway. Among all metastatic sites and remission, activation of WNT signalling pathway is commonly observed. TNBC exhibited 1486 copy number variations (CNVR) which is approximately 250 times higher than controls. More than 20 CNVR was observed in all chromosomes and more than 80 CNVR was observed in Chr 1 to Chr 4, Chr 7, Chr 11 and Chr X. Common CNVR associated with amplified regions in Chr 22, Chr 14, Chr 8 and Chr 2 was observed in TNBC and CNVR associated with Chr 22q11.22–23, Chr 6p21.32–33, Chr11q12.2, Chr14q32.22–23, Chr 8p11.22–23, was observed in metastatic disease. In transcriptome array analysis a total of 11,359 differentially expressed genes with fold change 2.0 were in observed in TNBC comprise of 7639 upregulated genes and 3720 downregulated genes. Further, with fold change 10, 1526 upregulated genes and 839 down regulated genes were identified. Panther pathway analysis identified the main pathways of upregulated genes were Wnt signalling pathway, Integrin signalling pathway and Cadherin signalling pathway. The main pathways of down regulated genes were Inflammation mediated by chemokine and cytokine signalling pathways. PPI network shows that COL12A1, COL6A3, FN1, MMP3, WNT5A were key upregulated genes and ITGB7, PTPRC, ITGA4, LCK and CD247 were key down regulated genes. Cytoscape analysis followed by multiple list comparator tool identified top 5 significant hub genes were FN1, MMP3, COLL11A1, COL12A1 and COL3A1. The significant pathway genes obtained by CGH array and transcriptome array when compared, exhibited 5 common genes COL4A1, FN1, COL6A3, COL5A2 and PCDH7. These genes were not overexpressed in Controls and therefore involved in pathogenesis of TNBC. Expressions of these genes were validated by studying protein expression by immunohistochemistry. FN1 and COL6A3 protein over expression predicted worse DFS in TNBC and can be considered as therapeutic targets at diagnosis to reduce the disease metastases. These findings provide new insights into the pathogenesis of TNBC and guide for selection of targets related to diagnosis, prognosis and prediction of treatment in TNBC.

Variants in FREM1 and trisomy 18 identified in a neonatal progeria patient.

Neonatal progeroid disorders are rare disorders with clinical features including low body mass index, proptosis, aged and dysmorphic facial features at the time of birth, prominent veins, sparse scalp hairs, and severe growth retardation. Very few cases have been identified with an unknown genetic cause. Here, we report clinical and genetic findings of a proband with hallmark features of neonatal progeria. Microarray comparative genomic hybridization, whole exome sequencing (WES) and Sanger sequencing were performed using standard methods. Array combined genome hybridization data revealed trisomy 18 in the proband (II-1), and WES data identified novel compound heterozygous variants (c.247C > T; p.H83Y and c.14769868InsA) in the FREM1 gene. We report a novel complex case of neonatal progeria with atrial septal defects, trisomy 18 without typical features of Edward syndrome. The phenotype of the patient was more consistent with neonatal progeria, thus we speculate it to be caused by the FREM1 variants.

Abstract F1-2: Clonal evolution of DCIS to invasion

Abstract Clonal evolution of DCIS to invasion Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive breast cancer (IBC). If not treated, at least 3 out of 4 women with DCIS will not develop IBC1-3. This implies many women with non-progressive, low-risk DCIS are likely to carry the burden of overtreatment. To solve this DCIS dilemma, two fundamental questions need to be answered. The first question is, how the subsequent IBC is related to the initial DCIS lesion. The second question is how to distinguish high- from low-risk DCIS at the time of diagnosis. This is essential to take well-informed DCIS management decisions, i.e., surgery, followed by radiotherapy in case of breast conserving treatment with or without subsequent endocrine treatment, or test whether active surveillance for low-risk DCIS is safe. How is the subsequent IBC related to the initial DCIS? The high genomic concordance in DNA aberrations between DCIS and IBC suggest that most driver mutations and CNA events are acquired at the earliest stages of DCIS initiation. It has therefore been assumed that most solid tumours arise from a single cell and that the probability of two independent tumours arising from the same tissue is low4-6. However, lineage tracing and genomic studies strongly suggest both direct and independent clonal lineages during the initiation of DCIS and evolution to IBC. In these processes, mammary stem cells have been implicated in DCIS initiation. Role of mammary stem cells in DCIS initiation Lineage tracing mouse model experiments have shown the fate of individual cells and lineages that acquire mutations before a tumour is established7-9. This is also relevant for DCIS initiation10,11, as different pools of MaSCs drive the growth and development of the ductal network and are considered the cell of origin for breast cancers9,10. The ductal trees remain quiescent until puberty, during which extension, branching and termination of terminal end buds (TEBs) leads to its expansion throughout the fat pad7,12,13. Any oncogenic mutation that occurs in a fetal MaSC will spread throughout the ductal network to a large part of the ductal tree, leading to sick lobes9. By contrast, oncogenic mutations acquired by a single MaSC during puberty spread to a smaller number of offspring located in small clusters in a part of the ductal network8,14. Direct lineage models for DCIS progression Direct lineage models postulate that DCIS has a single cell of origin that acquires mutations and progresses to IBC15-18. This is also supported by the high genomic concordance of CNAs and mutations in synchronous DCIS–IBC regions6,15,17,19-21 and the results of a recent large longitudinal study that profiled pure DCIS and recurrent IBC using multiple sequencing techniques, which estimated direct clonal lineages in approximately ~80% of patients18. Two distinct direct lineage models have been proposed: the evolutionary bottleneck model and the multiclonal invasion model. In the evolutionary bottleneckmodel, a single clone (or a limited number of clones) with an invasive genotype is selected and breaks through the basement membrane to migrate into surrounding tissues15,16,22, while other clones are unable to escape the ducts21-28. The multiclonal invasion model posits that most or all subclones can escape the basement membrane, establishing invasive disease6,16,17,20. The multiclonal model has not been studied widely in pure DCIS and recurrent IBC samples. Independent lineage model for DCIS progression DCIS lesions and IBCs can arise from different initiating cells in the same breast independently5,20,29-32. An analysis of sequential DCIS–IBC pairs in a unique, large-scale, in-depth study of 95 matched pure DCIS and recurrent IBC showed that ~20% of the IBC recurrences were indeed clonally unrelated to the primary DCIS18, as is also supported by some mathematical model studies33. The potential role of a field effect IBC can develop in the same breast as an initial DCIS even after treatment, which could be explained by the presence of a field effect34-37. Alternatively, the sick lobe hypothesis proposes that a single lobe harbours first-hit mutations, acquired in utero or during early mammary development37-42. This could also explain the restriction of IBC to the ipsilateral side of the breast39,43,44. Germline mutations may also explain the emergence of independent lineages in DCIS and IBC patients, lowering the threshold for cancer development32,43-46. Convergent evolution model of DCIS progression A third model for the emergence of IBC from DCIS is convergent evolution, in which the same mutations and CNA are selected and expanded during tumour growth such that environmental factors fuel competition between distinct clones and push them towards a similar genotype. Ultimately, two independent clonal lineages from different ancestral cells then happen to share multiple genomic aberrations or driver mutations across regions47-49. Although independent lineages are considered uncommon (~20%) in ipsilateral recurrences, they occur at much higher frequencies in contralateral recurrences (>80%), in which single-nucleotide polymorphism and comparative genomic hybridization microarrays show few (or no) genomic alterations shared in tumours from the contralateral breast cancer18,50,51. How to distinguish high- from low-risk DCIS at the time of diagnosis? The genomic and transcriptomic profile present at the time of DCIS diagnosis may contain crucial information on the risk of progression of DCIS to IBC. Thus far, it has been unclear whether prognostic gene expression markers can be used to separate indolent DCIS from potentially progressive DCIS. To this end, microarrays and RNA-seq have been applied for the comparison of bulk RNA from microdissected DCIS and IBC tissue. In synchronous DCIS–IBC, a limited number of transcriptional differences have been found and the few events discovered often varied extensively across different tumours52-56. Although these differences were strong, the added value of these studies is uncertain as they are often confounded by small sample size, lack of matched receptor status data, and low sample purity. Despite these limitations, these studies have implicated the epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) remodelling pathways as potentially relevant for the progression of DCIS to IBC55-62. We studied two large DCIS cohorts: the Sloane cohort, a prospective breast screening cohort from the UK (median follow-up of 12.5 years), and a Dutch population-based cohort (NKI, median follow-up of 13 years). FFPE tissue specimens from patients with pure primary DCIS after breast-conserving surgery (BCS) +/- RT that did develop a subsequent ipsilateral event (DCIS or invasive) were considered as cases, whereas patients that did not develop any form of recurrence up to the last follow-up or death were considered as controls. We performed copy number analysis (CNA) and RNAseq analysis on 229 cases (149 IBC recurrences and 80 DCIS recurrences) and 344 controls. We classified DCIS into the PAM50 subtypes using RNAseq data which revealed an enrichment of luminal A phenotype in DCIS that did not recur (P = 0.01, Fisher Exact test). No single copy number aberration was more common in cases compared to controls. RNAseq data did not reveal any genes significantly over/under expressed in cases versus controls after false discovery rate (FDR) correction. However, by limiting the analysis to samples that had not had RT and excluding pure DCIS recurrences we developed a penalized Cox model from RNAseq data. The model was trained on weighted samples (to correct for the biased sampling of the case control dataset) from the NKI series with double loop cross validation. Using this predicted hazard ratio, the samples were split into high, medium and low risk quantiles, with a recurrence risk of 20%, 9% and 2.5%, respectively at 5 years (p<0.001, Wald test). The NKI-trained predictor was independently validated in the Sloane No RT cohort (p = 0.02, Wald test). GSEA analysis revealed proliferation hallmarks enriched in the recurrence predictor (FDR = 0.058). The NKI-RNAseq predictor was more predictive of invasive recurrence than PAM50, clinical features (Grade, Her2 and ER) and the 12-gene Oncotype DCIS score (p < 0.001, permutation test using the Wald statistic) in both the NKI and Sloane series. In the methylation analysis, 50 controls were compared with 35 cases. We could identify Variably Methylation Regions (VMRs) and Differentially Methylated Regions (DMRs) between cases and controls. Interestingly, VMRs were enriched in cell adhesion pathways Conclusion The recently acquired knowledge described above on how often the subsequent IBC is directly related to the initial DCIS and on molecular markers predicting the risk of DCIS progression is essential for accurate DCIS risk assessment. This is essential to aid accurate clinical decision making to personalize DCIS management in the near future.

Genetic data protection as an indispensable element of genomic medicine development.

The immense progress in molecular biology observed in the last decades has led to a fundamental change in our understanding of the etiology of human diseases. Whole genome analyses, both DNA sequencing and microarray comparative genomic hybridization, allowed for identification of previously unknown diseases and syndromes. Therefore, in difficult‑to‑diagnose cases, clinical diagnosis is being replaced by molecular diagnosis (molecular dysmorphology, genomic medicine). For both scientific development of human genetics and clinical characteristics of rare genetic diseases, the construction and sharing of internationally available large databases has become crucial. However, genetic data have to be considered on the individual level too; therefore, they have to be treated as sensitive personal information. The context of ethical and legal risks involved in genetic testing has been long analyzed, although recognition of personal data protection issues is a more recent topic. The respective legal acts and protective measures should take into account several different aspects. The present paper explores major benefits and risks associated with international sharing of vast databases of genetic material, and presents legal provisions applied in the European Union, the United States, and China. The latter part is based on the respective acts themselves, as well as on analyses and commentaries by other scholars.

Multiplex Proteomic Evaluation in Inborn Errors with Deregulated IgE Response.

(1) Background: Atopic dermatitis constitutes one of the most common inflammatory skin manifestations of the pediatric population. The onset of many inborn errors occurs early in life with an AD-like picture associated with a deregulated IgE response. The availability of proteomic tests for the simultaneous evaluation of hundreds of molecules allows for more precise diagnosis in these cases. (2) Methods: Comparative genomic hybridization microarray (Array-CGH) analysis and specific IgE evaluation by using allergenic microarray (ISAC) and microarray (ALEX2) systems were performed. (3) Results: Proteomic investigations that use multiplex methods have proven to be extremely useful to diagnose the sensitization profile in inborn errors with deregulated IgE synthesis. Four patients with rare diseases, such as recessive X-linked ichthyosis (RXLI, OMIM 308100), Comel-Netherton syndrome (NS, OMIM256500), monosomy 1p36 syndrome (OMIM: 607872), and a microduplication of Xp11.4 associated with extremely high levels of IgE: 7.710 kU/L, 5.300 kU/L, 1.826 kU/L, and 10.430 kU/L, respectively, were evaluated by micro- and macroarray multiplex methods. Polyreactivity to both environmental and food allergens was observed in all cases, including the first described case of association of X-chromosome microduplication and HIE. (4) Conclusions: Extensive use of proteomic diagnostics should be included among the procedures to be implemented in inborn errors with hyper-IgE.

Real-world clinical and molecular management of 50 prospective patients with microphthalmia, anophthalmia and/or ocular coloboma

Background/aimsMicrophthalmia, anophthalmia and coloboma (MAC) are clinically and genetically heterogenous rare developmental eye conditions, which contribute to a significant proportion of childhood blindness worldwide. Clear understanding of MAC aetiology and...

Long-term outcome of consecutive case series of congenital isolated agenesis of corpus callosum.

To describe the long-term outcome of children with prenatally diagnosed isolated complete agenesis of the corpus callosum (cACC). In this single-center case series, we reviewed retrospectively the charts of fetuses referred to our fetal therapy unit from January 2004 to July 2020 for a suspected anomaly of the corpus callosum (CC). Cases with prenatally diagnosed isolated cACC were included. Fetal karyotype and comparative genomic hybridization microarray of amniotic fluid, in addition to fetal magnetic resonance imaging, were offered to all pregnant women with a diagnosis of fetal CC malformation. The surviving children were enrolled in the neurodevelopmental follow-up program at our institution, which included postnatal magnetic resonance imaging, serial neurological examinations and neurodevelopmental evaluations with standardized tests according to age. Families living in remote areas or far from our institution were offered a structured ad-hoc phone interview. A total of 128 pregnancies with fetal CC malformation were identified (mean gestational age at diagnosis, 24.5 (range, 21-34) weeks), of which 53 cases were diagnosed prenatally with apparently isolated cACC. Of these, 12 cases underwent termination of pregnancy, one resulted in intrauterine demise at 24 weeks of gestation and 13 cases were lost to follow-up. Of the remaining 27 children, one was excluded due to an associated chromosomal anomaly (8p21.3q11.21 mosaic duplication) diagnosed after birth, which could have been detected prenatally if the parents had consented to amniocentesis. In the 26 children included in the analysis, neurodevelopmental follow-up was available for a median of 3 (range, 1-16) years. Three (11.5%) infants had severe neurodevelopmental impairment, two of which were diagnosed postnatally with a genetic syndrome (Mowat-Wilson syndrome and Vici syndrome) that would not have been diagnosed prenatally. Seven (26.9%) children had mild neurodevelopmental impairment and 16 (61.5%) had normal neurodevelopmental outcome. The Full-Scale Intelligence Quotients of the three children with severe neurodevelopmental impairment were 50, 64 and 63, respectively, while that of the remaining children was in the normal range (median, 101; range, 89-119). In 88% of the children with cACC included in this study, neurodevelopment was not severely impaired. However, long-term follow-up is recommended in all cases of congenital isolated cACC to recognize subtle neurodevelopmental disorders as early as possible. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Bi-allelic mutation of CTNNB1 causes a severe form of syndromic microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia

BackgroundInherited vitreoretinopathies arise as a consequence of congenital retinal vascularisation abnormalities. They represent a phenotypically and genetically heterogeneous group of disorders that can have a major impact on vision. Several genes encoding proteins and effectors of the canonical Wnt/β-catenin pathway have been associated and precise diagnosis, although difficult, is essential for proper clinical management including syndrome specific management where appropriate. This work aimed to investigate the molecular basis of disease in a single proband born to consanguineous parents, who presented with microphthalmia, persistent foetal vasculature, posterior lens vacuoles, vitreoretinal dysplasia, microcephaly, hypotelorism and global developmental delay, and was registered severely visually impaired by 5 months of age.MethodsExtensive genomic pre-screening, including microarray comparative genomic hybridisation and sequencing of a 114 gene panel associated with cataract and congenital ophthalmic disorders was conducted by an accredited clinical laboratory. Whole exome sequencing (WES) was undertaken on a research basis and in vitro TOPflash transcriptional reporter assay was utilised to assess the impact of the putative causal variant.ResultsIn the proband, WES revealed a novel, likely pathogenic homozygous mutation in the cadherin-associated protein beta-1 gene (CTNNB1), c.884C>G; p.(Ala295Gly), which encodes a co-effector molecule of the Wnt/β-catenin pathway. The proband’s parents were shown to be heterozygous carriers but ophthalmic examination did not detect any abnormalities. Functional assessment of the missense variant demonstrated significant reduction of β-catenin activity.ConclusionsThis is the first report of a biallelic disease-causing variation in CTNNB1. We conclude that this biallelic, transcriptional inactivating mutation of CTNNB1 causes a severe, syndromic form of microphthalmia, persistent foetal vasculature and vitreoretinal dysplasia that results in serious visual loss in infancy.

The extended concurrent genes signature for disease-free survival in breast cancer

Previously we had identified concurrent genes, which highlighted the interplay between copy number variation (CNV) and differential gene expression (GE) for Han Chinese breast cancers. The merit of the approach is to discovery biomarkers not identifiable by conventional GE only data, for which phenotype-correlation or gene variability is the criteria of gene selection. Thirty-one comparative genomic hybridization (CGH) and 83GE microarrays were performed, with 29 breast cancers assayed from both platforms. Potential targets were revealed by Genomic Identification of Significant Targets in Cancer (GISTIC) from CGH arrays. Concurrent genes and genes with significant GISTIC scores were used to derive the extended concurrent genes signature, which was consensus from leading edge analysis across all studies and a supervised partial least square (PLS) regression predictive model of disease-free survival was constructed. There were 1584 concurrent genes from 29 samples with both CGH and GE microarrays. Enriched concurrent genes sets for disease-free survival were identified independently from 83GE arrays and another one with Han Chinese origin as well as three studies of Western origin. For five studies with disease-free survival follow up, prognostic discrepancy was observed between predicted high-risk and low-risk group patients. We concluded that through parallel analyses of CGH and GE microarrays, the proposed extended concurrent gene expression signature can identify biomarkers with prognostic values.

Prenatal Diagnosis by Array Comparative Genomic Hybridization in Fetuses with Cardiac Abnormalities.

Congenital heart defects (CHDs) appear in 8–10 out of 1000 live born newborns and are one of the most common causes of deaths. In fetuses, the congenital heart defects are found even 3–5 times more often. Currently, microarray comparative genomic hybridization (array CGH) is recommended by worldwide scientific organizations as a first-line test in the prenatal diagnosis of fetuses with sonographic abnormalities, especially cardiac defects. We present the results of the application of array CGH in 484 cases with prenatally diagnosed congenital heart diseases by fetal ultrasound scanning (256 isolated CHD and 228 CHD coexisting with other malformations). We identified pathogenic aberrations and likely pathogenic genetic loci for CHD in 165 fetuses and 9 copy number variants (CNVs) of unknown clinical significance. Prenatal array-CGH is a useful method allowing the identification of all unbalanced aberrations (number and structure) with a much higher resolution than the currently applied traditional assessment techniques karyotype. Due to this ability, we identified the etiology of heart defects in 37% of cases.

X-linked sideroblastic anaemia in a female fetus: a case report and a literature review

BackgroundX-linked sideroblastic anaemia (XLSA) is commonly due to mutations in the ALAS2 gene and predominantly affects hemizygous males. Heterozygous female carriers of the ALAS2 gene mutation are often asymptomatic or only mildly anaemic. XLSA is usually characterized by microcytic erythrocytes (reduced mean corpuscular volume (MCV)) and hypochromia, along with increased red cell distribution width. However, in females with XLSA the characteristic laboratory findings can be dimorphic and present with macrocytic (elevated MCV) in addition to microcytic red cells.Case presentationWe report a case of fetal anaemia, presenting in the early third trimester of pregnancy, in a female fetus. Ultrasound findings at 29 weeks were of cardiomegaly, prominent umbilical veins, a small rim of ascites, and mean cerebral artery peak systolic velocity (PSV) value above 1.5 Multiples of the Median (MoM). She underwent non-invasive prenatal testing that determined the rhesus genotype of the fetus to be rhesus B negative. No red blood cell antibodies were reported. Other investigations to determine the underlying cause of fetal anaemia included microarray comparative genomic hybridization, serology to exclude congenital infection and a peripheral blood film and fetal bilirubin to detect haemolysis. The maternal grandmother had a history of sideroblastic anaemia diagnosed at the age of 17 years. The mother had mild macrocytic anaemia with haemoglobin of 10.4 g/dl and MCV of 104 fl. The fetal anaemia was successfully treated with two in utero transfusions (IUTs), and delivery occurred via caesarean section at 37 weeks of gestation. The red cell gene sequencing in both the mother and fetus were heterozygous for an ALAS2 mutation causing in utero manifestations of XLSA. The haemoglobin on discharge to the local hospital at five days of age was 19.1 g/dl. Subsequently, the infant became anaemic, requiring regular 3–4 monthly blood transfusions and demonstrating overall normal development. Her anaemia was unresponsive to pyridoxine.ConclusionsThis is one of four cases reporting multiple female members presenting with discordant clinical features of XLSA from being entirely asymptomatic to hydropic in utero. Our report is novel in that there are no previous cases in the literature of anaemia in a female fetus heterozygous for ALAS2 mutation.

A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes

To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on invitro maturation of human oocytes. Randomized study. Fertility center in an academic hospital. Not applicable. Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 μM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. Invitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. The percentage of oocytes undergoing maturation invitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. cFEE significantly increased the percentage of oocytes that matured invitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. cFEE improves human oocyte maturation invitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for invitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

Profile of sulfur oxidizing bacteria in full-scale Biotrickling filter to remove H2S in biogas from in cassava starch industry

The success of Next generation sequencing (NGS) technology over the past decades has led to a better understanding of complex microbial communities in such environment. Here, we employed NGS to monitor sulfur oxidizing bacteria (SOB) community from cassava starch industry (BPT), which can oxidize H2S into sulfate or elemental sulfur under aerobic conditions. Microbial sludge was collected from two sampling points comprising starting sludge (BPT1) and recirculating sludge (BPT2) of the biotrickling filter. gDNA was extracted from BPT1 and BPT2 samples and removed other contaminations by using the GenepHarM Gel/PCR Kit (Geneaid). DNA concentrations and DNA purity were analyzed by using gel electrophoresis and NanoDrop™ 1000 Spectrophotometer, respectively. Analysis of SOB species using NGS technique was based on the hypervariable regions V4 on the 16S rRNA gene. The comparative genomic hybridization (CGH) microarray probes for detection and identification of SOB species-strain that high expression of soxAXBYZ and fccAB genes in the cassava starch industry. For the recirculating sludge (BPT2), the bacterial communities were dominated by Sulfurimonas (13.7%) followed by Synechococcus (9.4%), Hydrogenophaga (6.5%), Methanosaeta (2.4%), and Acidithiobacillus (2.1%), the same dominant genera were also found in the starting sludge (BPT1), but with slightly different abundancy. The heatmap revealed that Acidithiobacillus caldus species (33.9%–49.9%) could play a vital role in eliminating H2S in biogas from the cassava starch industry. The CGH microarray indicated that Thiothrix, Paracoccus, Sulfurimonas, Hydrogenophaga, Magnetospirillum, Rhodoplanes, Syntrophomonas, Pseudomonas, Sulfuricurvum, and Arcobacter were dominant genus, that has a high expression of sulfur oxidation genes in biotrickling filter.

[Advances in identification methods of alien genomic components in plants].

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.

Two Cases of Ocular Manifestations in Patients with Microdeletion of the Chromosome 7 Long Arm

Purpose: We report ocular manifestations in two patients with 7q microdeletion. Case summary: (Case 1) A 62-day-old male infant was admitted to the ophthalmology outpatient department for ocular examination after being diagnosed with microdeletion of chromosome seven (7q36.2q36.3 deletion) in DNA microarray comparative genomic hybridization (DNA microarray CGH) and fluorescence in situ hybridization (FISH) tests. Fundus examination showed optic disc hypoplasia in both eyes and retinopathy of prematurity, accompanied by retinal hemorrhage in his right eye. Around the age of 24 months, the patient was diagnosed with intermittent exotropia with anisometropia and was prescribed spectacles. (Case 2) A 3-year-old male infant was referred to the ophthalmology clinic to evaluate poor fixation, which was found during rehabilitation therapy for cerebral palsy and developmental delay. Fundus examination showed an increased cup/disc ratio bilaterally. A flash visual evoked potential test indicated a decrease in amplitude in his right eye. Intermittent exotropia of forty prism diopters was observed. DNA microarray CGH and FISH tests performed at another hospital revealed microdeletion of chromosome seven (7q35 microdeletion) and CNTNAP2 gene loss. Conclusions: When genetic anomalies associated with ocular development are identified, it is necessary to detect the ophthalmic abnormalities early and provide the appropriate treatment to allow for the development of normal visual function.

Comprehensive Genetic Analysis of Non-syndromic Autism Spectrum Disorder in Clinical Settings.

Although genetic factors are involved in the etiology of autism spectrum disorder (ASD), the significance of genetic analysis in clinical settings is unclear. Forty-nine subjects diagnosed with non-syndromic ASD were analyzed by microarray comparative genomic hybridization (CGH) analysis, whole-exome sequencing (WES) analysis, and panel sequencing analysis for 52 common causative genes of ASD to detect inherited rare variants. Genetic analysis by microarray CGH and WES analyses showed conclusive results in about 10% of patients, however, many inherited variants detected by panel sequencing analysis were difficult to interpret and apply in clinical practice in the majority of patients. Further improvement of interpretation of many variants detected would be necessary for combined genetic tests to be used in clinical settings.

Lissencephaly in an epilepsy cohort: Molecular, radiological and clinical aspects.

Lissencephaly is a rare malformation of cortical development due to abnormal transmantle migration resulting in absent or reduced gyration. The lissencephaly spectrum consists of agyria, pachygyria and subcortical band heterotopia. In this study we compared genetic aetiology, neuroradiology, clinical phenotype and response to antiepileptic drugs in patients with epilepsy and lissencephaly spectrum malformations. The study group consisted of 20 patients - 13 males and 7 females, aged 18 months to 21yearsat the time of data collection. Genetic testing was performed by oligonucleotide array comparative genomic hybridization (microarray), multiplex ligation-dependent probe amplification (MLPA), targeted gene panels and whole exome/genome sequencing. All neuroradiological investigations were re-evaluated and the malformations were classified by the same neuroradiologist. Clinical features and response to anti-epileptic drugs (AEDs) were evaluated by retrospective review of medical records. In eleven patients (55%) mutations in PAFAH1B1 (LIS1) or variable microdeletions of 17p13.3 including the PAFAH1B1 gene were detected. Four patients (20%) had tubulin encoding gene mutations (TUBA1A, TUBG1 and TUBGCP6). Mutations in DCX, DYNC1H1, ADGRG1 and WDR62 were identified in single patients. In one patient, a possibly pathogenic intragenic deletion in TRIO was detected. A clear radiologic distinction could be made between tubulinopathies and PAFAH1B1 related lissencephaly. The majority of the patients had therapy resistant epilepsy and epileptic spasms was the most prominent seizure type. The best therapeutic response to seizure control in our cohort was obtained by the ketogenic diet, vigabatrin, clobazam, phenobarbital and valproate. The most common genetic aetiologies in our cohort of 20 individuals with epilepsy and lissencephaly spectrum were intragenic deletions or single nucleotide mutations in PAFAH1B1 or larger deletions in 17p13.3, encompassing PAFAH1B1, followed by mutations in tubulin encoding genes. Radiological findings could reliably predict molecular results only in agyria with a posterior to anterior gradient. Radiological and molecular findings did not correlate consistently with severity of clinical outcome or therapeutic response.

Intellectual disability and dysmorphisms in a child with 14q32.31-q32.33 deletion: a case report

INTRODUCTION: Intellectual disability and global developmental delay are frequent causes of genetic consultation requested by pediatricians. Around 30% of severe intellectual disability and 70% of mild intellectual disability do not present a defined diagnosis. OBJECTIVES: Chromosome microarray comparative genomic hybridization test is now the standard diagnosis for patients presenting unexplained global developmental delay or intellectual disability, autism spectrum disorders, and multiple congenital anomalies due to the tests sensibility, increasing the chance for a diagnosis and elucidating size and region affected. CASE REPORT: This paper reports a girl with intellectual disability, prominent hypotonia, postnatal growth retardation and several dysmorphisms with a small subtelomeric 14q terminal deletion whose diagnosis was sought during six years and only became possible through the chromosome microarray test.

The Association of Scoliosis and NSD1 Gene Deletion in Sotos Syndrome Patients.

A retrospective comparative study. The aim of this study was to examine the NSD1 abnormalities in patients diagnosed with Sotos syndrome and its correlation with the presence, severity, and progression of associated scoliosis. Scoliosis has been reported in approximately 30% of patients diagnosed with Sotos syndrome, a genetic disorder characterized by a distinctive facial appearance, learning disability, and overgrowth. Sotos syndrome is mainly attributed to NSD1 haploinsufficiency, but with ethnical differences in genetic profile: NSD1 microdeletions are frequently identified in Japanese Sotos patients whereas intragenic mutations are more frequently found in non-Japanese patients. Although possible genotype-phenotype correlations have been proposed, the genotype of Sotos syndrome patients suffering from scoliosis has not been examined. The medical records and spinal radiographs of 63 consecutive Sotos syndrome patients at a single center were reviewed. Fluorescent in situ hybridization or microarray comparative genomic hybridization and DNA sequencing or multiplex ligation-dependent probe amplification were performed to detect 5q35 microdeletion involving the NSD1 gene and intragenic mutations of the NSD1 gene, respectively. The phenotypes of all cases and radiological assessments for the presence and progression of scoliosis were studied. NSD1 abnormalities were identified in 55 patients (87%): microdeletion in 34 patients (54%) and intragenic mutation in 22 patients (33%). Scoliosis was observed in 26 patients (41%), with a significantly higher ratio of microdeletions than mutations. The 10 patients with progressive scoliosis all had NSD1 microdeletions. Scoliosis was a common phenotypical trait in children with Sotos syndrome and its presence as well as progression were higher in cases with NSD1 microdeletions. Although all Sotos syndrome patients should be monitored for scoliosis, clinicians should be made aware that patients with NSD1 microdeletions have a higher probability of scoliosis development and progression that may require early intervention.Level of Evidence: 3.