Differential expression of chromosome 14 open reading frame 180 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding chromosome 14 open reading frame 180, C14orf180, when comparing primary tumors of the breast to the tissue of origin, the normal breast. C14orf180 was also differentially expressed in the brain metastases of patients with metastatic breast cancer. C14orf180 mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of C14orf180 in primary tumors of the breast was correlated with overall survival in patients with HER2+ subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. C14orf180 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Differential expression of C14orf132 in cancer of the vulva.

In these brief notes we document work using published microarray data (1, 2) to pioneer integrative transcriptome analysis comparing vulvar carcinoma to its tissue of origin, the vulva. We report the differential expression of chromosome 14 open reading frame 132, encoded by C14orf132, in cancer of the vulva. C14orf132 may be of pertinence to understanding transformation and disease progression in vulvar cancer (3).

LRFN5 locus structure is influenced by the individual’s sex and associated with autism

BackgroundLRFN5 is a brain-specific gene needed for synaptic development and plasticity. It is the only gene in a large 5.4 Mb topologically associating domain (TAD) on chromosome 14, which we term the LRFN5 locus. This locus is highly conserved, but has extensive copy number variation. MethodsLocus structure was studied by chromatin immunoprecipitation (chIP-onchip) in fibroblasts from individuals with autism and controls, supplemented with a capture-HiC determination of TAD structures in a family trio. LRFN5 expression was studied in foetal brain cell cultures. In addition, locus interaction was studied in four large and independent cohorts by measuring deviations from Hardy-Weinberg equilibrium of a common deletion polymorphism. ResultsWe found that locus structural changes are associated with developmental delay (DD) and autism spectrum disorders (ASD). In a large family, ASD in males segregated with a chromosome 14 haplotype carrying a 172 kb deletion upstream of LRFN5 . In a fibroblast capture-HiC study on an ASD-patient-parent trio, the ASDsusceptible haplotype (in the mother and her autistic son) had a TAD pattern different from both the father and a female control. When the trimethylated histone-3-lysine-9 chromatin (H3K9me3) profiles in fibroblasts from control males (n=6) and females (n=7) were compared, a male-female difference was observed around the LRFN5 gene itself (p<0.01). Intriguingly, in three cohorts of individuals with DD (n=8757), the number of heterozygotes of a common deletion polymorphism upstream of LRFN5 was 20-26% lower than expected from Hardy-Weinberg equilibrium. This indicates early allelic interaction, and the genomic conversions from heterozygosity to wildtype or deletion homozygosity were of equal magnitudes. In a control group of medical students (n=1416), such conversions were three times more common than in the DD-patient cohorts (p=0.00001). Hypothetically, such allelic interaction is needed to establish monoallelic expression, which we found in foetal brain cell cultures. LimitationsThe male-female difference in H3K9me3 profiles was based on fibroblast data from a small number of individuals, and the monoallelic expression data on a single experiment. ConclusionsTaken together, allelic interaction, monoallelic expression and sexdependent differences make the LRFN5 locus attractive for exploring the genetic basis of synaptic memory and high-functioning male autism.

Frequent Aneuploidy in Primary Human T Cells Following CRISPR-Cas9 cleavage

Multiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy. In particular, the first U.S. clinical trial using CRISPR-Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients. Here, we used the same guide RNA sequences and applied single-cell RNA sequencing (scRNAseq) to more than 7000 primary human T cells, transfected with CRISPR-Cas9. Four days post-transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells, and a chromosome 14 gain in up to 1.4% of the cells. We further identified truncations of chromosome 7, harboring the TCRβ locus, in 9.9% of the cells. Loss of heterozygosity (LOH) was further validated using fluorescence in situ hybridization (FISH) and the temporal dynamics of cleavage and incomplete repair were monitored using digital droplet PCR (ddPCR). Aneuploidy was found among all T cell subsets and was associated with transcriptional signatures of reduced proliferation and metabolism as well as with induced p53 activation and cell death. We conclude that aneuploidy and chromosomal truncations are frequent outcomes of CRISPR-Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.

Higher CNV frequencies in Chromsome-14 of girls with Turner syndrome phenotype – A chromosomal microarray study

Context Precise genotype-phenotype correlations in Turner syndrome (TS) have not yet been deciphered. The chromosomal basis of the clinical TS-phenotype in the absence of X chromosome aberrations on the conventional karyotyping remains more and less unexplored. Objective To elucidate the high resolution chromosomal picture and analyze the genotype -phenotype associations in girls with clinical phenotype of TS by chromosomal microarray (CMA). Design and Patients: Cross sectional observational study conducted between October 2018 to January 2020 on 47 girls presenting the clinical TS-phenotype and fulfilling the criteria for chromosomal analysis. Setting Out Patient Department at Department of Endocrinology and the Molecular research lab at tertiary care teaching institution. Results The CNV (copy number variation) polymorphs were more frequent on autosomes than X chromosomes and they were detected in 89.3%, 61.7 %, and 92.8 % of patients respectively on chromosome 14 or X or both. Total 445 and 64 CNV polymorphs were discovered on chromosome X and 14 respectively. The latter exhibiting either gain at 14q32.33 or loss at 14q11.2 or both. Karyotype was available for 27 patients; 55.6% cases displayed X chromosome abnormalities while 44.4% cases had a normal karyotype. Functional interactomes of the genes those were present in Chromosome 14 CNVs and those known to be associated with TS showed an overlap of 67% and enriched various development-related cellular pathways underlying TS-phenotype. Conclusions On high resolution karyotype analysis, clinical phenotype of TS could be found associated with CNV defects in autosomes (specifically Chr. 14,) or X chromosome or both. The syndrome of chromosome 14 CNVs defects with and without X-chromosomal defects clinically mimics TS and shares a common genomic network deserves further investigations.

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1hu2787 Model of Fragile X Syndrome

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1hu2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the “leading edge” differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher’s assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1hu2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

Genome-wide admixture mapping of eGFR and CKD identify European and African ancestry-of-origin loci in U.S. Hispanics/Latinos

Background Admixture mapping is a powerful approach for gene mapping of complex traits that leverages the diverse genetic ancestry in populations with recent admixture such as U.S. Hispanics/Latinos (HL), who have increased risk of chronic kidney disease (CKD). Methods Genome-wide admixture mapping was performed for CKD and estimated glomerular filtration rate (eGFR) in a sample of 12601 participants from the Hispanic Community Health Study/Study of Latinos, with validation in a sample of 8191 African Americans from the Women's Health Initiative (WHI). Results Three novel ancestry-of-origin loci were identified on chromosomes 2, 14 and 15 for CKD and eGFR. The chromosome 2 locus (2p16.3) consisted of two European ancestry regions encompassing the FSHR and NRXN1 genes, with European ancestry at this locus associated with increased risk for CKD. The chromosome 14 locus (14q32.2) located within the DLK1-DIO3 imprinted domain was driven by European ancestry, and was associated with lower eGFR. The chromosome 15 locus (15q13.3-14) included intronic variants of RYR3 and was within an African-specific genomic region that was associated with higher eGFR. These findings were compared to the conventional genome-wide association study that failed to identify significant associations in these regions. We validated the chromosome 14 and 15 loci for eGFR in the WHI African Americans. Conclusions This study provides evidence of shared ancestry-specific genomic regions influencing eGFR in HL and African Americans, and illustrates the potential for leveraging genetic ancestry in recently admixed populations for novel discovery of kidney trait loci.

Copy Number Variations Are More Frequent on Chromosome 14 as Compared to X Chromosome in Suspected Turner Syndrome Girls - A Chromosomal Microarray Analysis

Introduction: Turner syndrome(TS) is defined by complete/partial monosomy of X chromosome in association with classic clinical manifestations. Conventional karyotyping is the gold standard test for diagnosis of TS. However it is labour intensive and inaccurate for detecting mosaicism, marker chromosomes and sub-microscopic deletions/duplications. TS is characterized by heterogeneous phenotypes despite identical karyotypes and precise genotype-phenotype correlations have not yet been deciphered. Presence of TS specific features in absence of X chromosome abnormality, evokes the hypothesis of possible autosomal involvement. Here, we report detailed Chromosomal microarray (CMA) analysis of 47 girls with clinically suspected TS, using Affymetrix CytoScan 750K array. Materials and Methods: The clinical diagnosis of TS was based on recommendations by clinical practice guidelines from 2016 Cincinnati International TS meeting. Peripheral venous sample was collected in EDTA tubes and DNA was extracted using Qiagen-DNAeasy Blood and Tissue kit (Cat No. 69504). DNA samples were then hybridized to the Affymetrix CytoScan 750K array as per manufacturer’s instructions. The data obtained was analysed using Chromosomal Analysis suite software and public genomic databases- ISCA, OMIM, DGV, DECIPHER. For bioinformatic analysis, all the genes (172) implicated in TS were retrieved from DisGeNET database. A TS-interactome of 4033 genes was then constructed from these genes and their first-degree neighbours from complete human interactome. Thereafter compilation was done based on CMA results and a protein-protein interaction network of 316 nodes was constructed. Results: Mean age of study cohort was 15.8 ± 3.64 years with short stature being the most common presenting phenotype (91.4%). CMA analysis detected copy number variations (CNVs) on chromosome 14 in 42 (89.3%) of 47 cases while X chromosome CNVs were present in only 28 (59.5%) cases, with all patients clinically qualifying as TS. Total 445 CNVs were discovered on X chromosome and 64 CNVs were found on Chromosome 14 exhibiting either CNV gain at 14q32.33 or CNV loss at 14q11.2 or both. The 30 cell karyotype was available for 27 patients and was found to be false negative in 7 (14.8%) patients. Also, 6 out of 47 cases had Y chromosome translocation detected on CMA that failed detection by karyotype. On enrichment analysis, thirty KEGG pathways were found to be enriched by the overlapping genes between TS-interactome and the interactome constructed by genes located within 14q11.2, and 14q32.33 67% of genes (212) in this network overlap with TS-interactome. Conclusions: CMA is a superior diagnostic modality for TS than karyotyping. Functional interactomes between Chromosome X and Chromosome 14 on enrichment analysis reveal novel pathways underlying phenotypic manifestations.