Biallelic variants in HECT E3 paralogs, HECTD4 and UBE3C, encoding ubiquitin ligases cause neurodevelopmental disorders that overlap with Angelman syndrome.

PURPOSE: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified. METHODS: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes. RESULTS: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect. CONCLUSION: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome.

Prognostic value of TP53 expression and MGMT methylation in glioblastoma patients treated with temozolomide combined with other chemotherapies.

OBJECTIVE: To assess the recurrence interval and predictive significance of TP53 expression and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in glioblastomas treated with radiotherapy and combined chemotherapies, including temozolomide, lomustine, procarbazine and bevacizumab. METHOD: We reviewed the clinical outcomes of 52 totally resected glioblastoma patients, who received conventional radiotherapy and temozolomide with other chemotherapeutic agents. Correlation of TP53 expression and MGMT promotor methylation with recurrence interval was analyzed using Kaplan Meier estimates. RESULTS: No significant association was found between MGMT promotor methylation and TP53 expression in glioblastomas (P-value = 0.158). Patients with non-methylated MGMT who received temozolomide chemotherapy with other chemotherapeutic agents showed significantly later recurrence (P-value = 0.007) compared with patients with non-methylated MGMT who received temozolomide alone. No significant difference was found in recurrence interval among glioblastoma patients with methylated MGMT who received temozolomide alone or with other chemotherapies (P-value = 0.667). Moreover, patients with non-TP53-expressing tumors who received temozolomide with other chemotherapies had significantly later recurrence (P-value = 0.04) compared with patients who received temozolomide alone. CONCLUSION: Totally resected glioblastoma patients, with non-methylated MGMT or non-TP53-expressing tumors treated with radiotherapy and combined chemotherapies had a reduced chance of tumor recurrence and a more favorable outcome. Furthermore, both MGMT and TP53 are independent prognostic factors for glioblastoma.

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises.

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan-Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.

MC4R variants rs12970134 and rs17782313 are associated with obese polycystic ovary syndrome patients in the Western region of Saudi Arabia.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder causing infertility in reproductive-age women. The cause of PCOS is not fully understood but it is thought to be influenced by environmental and genetic factors. Obesity is greatly related to PCOS and its reduction is one of the major aims in treating PCOS. Melanocortin 4 receptor (MC4R) gene polymorphisms were detected to be associated with different levels of obesity. Therefore, we aimed to determine the genotype and allele frequency of MC4R variants rs12970134 (A/G) and rs17782313 (C/T) in PCOS and investigate their association with PCOS and its clinical variables. METHODS: A case-control study was conducted on 189 women, consisting of 95 PCOS cases and 94 controls. Genotyping was performed by real-time polymerase chain reaction (PCR) using TaqMan™ Genotyping assays. Quantitative data were presented as (median ± interquartile range (IQR) whereas qualitative data were presented as frequencies. The chi-squared test was used to observe the difference between SNPs within the study groups (PCOS and control subjects). Multinomial logistic regression was used to test the risk of obesity and development of PCOS considering p < 0.05 is statistically significant. RESULTS: Rs12970134 and rs17782313 are significantly associated with body mass index (BMI, kg/m2, p < 0.0001) in PCOS women but not associated with PCOS itself. Risk alleles in our population are A in rs12970134 and C in rs17782313 that are associated with high BMI (> 30 kg/m2) in obese women with PCOS (OR = 1.348, p = 0.002 and OR = 1.364, p = 0.002 respectively) in the homozygous state. In addition, we found that the other genotypes for non-obese PCOS group, AG/GG for rs12970134 and CT/TT for rs17782313, are associated with hirsutism, loss of hair, hyperandrogenism and anti-Müllerian hormone in PCOS. CONCLUSIONS: These findings demonstrate that MC4R single nucleotide polymorphisms, rs12970134 and rs17782313, are correlated with elevated BMI in PCOS but are not causative factors for PCOS among women in the western region of Saudi Arabia. Moreover, the reverse genotypes are associated with major clinical variants in non-obese (< 30 kg/m2) PCOS patients may demonstrate a poor prognosis for this group.

Membranous or Cytoplasmic HER2 Expression in Colorectal Carcinoma: Evaluation of Prognostic Value Using Both IHC & BDISH.

BACKGROUND: Human epidermal growth factor recptor-2 (HER2) was identified as a driver gene in several types of cancers with both prognostic and predictive value. However, the molecular association of HER2 gene mutation with HER2 gene amplification and/or protein expression in cancer tissues has not been clearly defined. Moreover, there is little information available on HER2 status role in tumor progression and metastasis in colorectal carcinoma (CRC) compared to other solid tumors. The aim of this study was to evaluate both HER2 amplification and protein expression profiles using immunohistochemistry (IHC) and bright-field dual in situ hybridization (BDISH) techniques, respectively. PATIENTS AND METHODS: Tissue microarray (TMA) was constructed to accommodate a total of 243 CRC formalin-fixed paraffin embedded (FFPE) samples of consent patients and stained by IHC and BDISH methods. The expression patterns of HER2 protein status were evaluated and correlated to HER2 gene amplification status and then assessed for its prognostic value. RESULTS: The expression profile of 58% samples showed cytoplasmic expression patterns of different categories. Interestingly, only 1% showed strong (+3) membranous expression pattern of HER2 with perfect match with their corresponding gene amplification status (>2). However, the cytoplasmic HER2 protein status did not show significant correlation with most clinicopathological features and survival outcomes except with age (p = 0.04) and tumor size (p = 0.03). CONCLUSION: We demonstrated that the membranous HER2 gene/protein status is infrequent, while the main fraction of HER2 overexpression was cytoplasmic and lacking prognostic value. This cytoplasmic HER2 overexpression was induced through a gene-amplification independent pathway, making the HER2 gene status evaluation approach in those cases not worthy. Further investigations about the molecular pathways of the cytoplasmic HER2 protein in CRC and its associations with survival outcomes are required to allow either a breakthrough in CRC management; or to confirm the hypothesis of a marginal role in CRC onset and progression.

Comprehensive molecular biomarker identification in breast cancer brain metastases.

BACKGROUND: Breast cancer brain metastases (BCBM) develop in about 20-30% of breast cancer (BC) patients. BCBM are associated with dismal prognosis not at least due to lack of valuable molecular therapeutic targets. The aim of the study was to identify new molecular biomarkers and targets in BCBM by using complementary state-of-the-art techniques. METHODS: We compared array expression profiles of three BCBM with 16 non-brain metastatic BC and 16 primary brain tumors (prBT) using a false discovery rate (FDR) p < 0.05 and fold change (FC) > 2. Biofunctional analysis was conducted on the differentially expressed probe sets. High-density arrays were employed to detect copy number variations (CNVs) and whole exome sequencing (WES) with paired-end reads of 150 bp was utilized to detect gene mutations in the three BCBM. RESULTS: The top 370 probe sets that were differentially expressed between BCBM and both BC and prBT were in the majority comparably overexpressed in BCBM and included, e.g. the coding genes BCL3, BNIP3, BNIP3P1, BRIP1, CASP14, CDC25A, DMBT1, IDH2, E2F1, MYCN, RAD51, RAD54L, and VDR. A number of small nucleolar RNAs (snoRNAs) were comparably overexpressed in BCBM and included SNORA1, SNORA2A, SNORA9, SNORA10, SNORA22, SNORA24, SNORA30, SNORA37, SNORA38, SNORA52, SNORA71A, SNORA71B, SNORA71C, SNORD13P2, SNORD15A, SNORD34, SNORD35A, SNORD41, SNORD53, and SCARNA22. The top canonical pathway was entitled, role of BRCA1 in DNA damage response. Network analysis revealed key nodes as Akt, ERK1/2, NFkB, and Ras in a predicted activation stage. Downregulated genes in a data set that was shared between BCBM and prBT comprised, e.g. BC cell line invasion markers JUN, MMP3, TFF1, and HAS2. Important cancer genes affected by CNVs included TP53, BRCA1, BRCA2, ERBB2, IDH1, and IDH2. WES detected numerous mutations, some of which affecting BC associated genes as CDH1, HEPACAM, and LOXHD1. CONCLUSIONS: Using complementary molecular genetic techniques, this study identified shared and unshared molecular events in three highly aberrant BCBM emphasizing the challenge to detect new molecular biomarkers and targets with translational implications. Among new findings with the capacity to gain clinical relevance is the detection of overexpressed snoRNAs known to regulate some critical cellular functions as ribosome biogenesis.

The Third International Genomic Medicine Conference (3rd IGMC, 2015): overall activities and outcome highlights.

The Third International Genomic Medicine Conference (3rd IGMC) was organised by the Centre of Excellence in Genomic Medicine Research (CEGMR) at the King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia (KSA). This conference is a continuation of a series of meetings, which began with the first International Genomic Medicine Conference (1st IGMC, 2011) followed by the second International Genomic Medicine Conference (2nd IGMC, 2013). The 3rd IGMC meeting presented as a timely opportunity to bring scientists from across the world to gather, discuss, and exchange recent advances in the field of genomics and genetics in general as well as practical information on using these new technologies in different basic and clinical applications. The meeting undoubtedly inspired young male and female Saudi researchers, who attended the conference in large numbers, as evidenced by the oversubscribed oral and poster presentations. The conference also witnessed the launch of the first content for npj Genomic Medicine, a high quality new journal was established in partnership by CEGMR with Springer Nature and published as part of the Nature Partner Journal series. Here, we present a brief summary report of the 2-day meeting including highlights from the oral presentations, poster presentations, workshops, poster prize-winners and comments from the distinguished scientists.

Clinical significance of frequent somatic mutations detected by high-throughput targeted sequencing in archived colorectal cancer samples.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with different molecular characteristics associated with many variables such as the sites from which the tumors originate or the presence or absence of chromosomal instability. Identification of such variables, particularly mutational hotspots, often carries a significant diagnostic and/or prognostic value that could ultimately affect the therapeutic outcome. METHODS: High-throughput mutational analysis of 99 CRC formalin-fixed and paraffin-embedded (FFPE) cases was performed using the Cancer Hotspots Panel (CHP) v2 on the Ion Torrent™ platform. Correlation with survival and other Clinicopathological parameters was performed using Fisher's exact test and Kaplan-Meier curve analysis. RESULTS: Targeted sequencing lead to the identification of frequent mutations in TP53 (65 %), APC (36 %), KRAS (35 %), PIK3CA (19 %), PTEN (13 %), EGFR (11 %), SMAD4 (11 %), and FBXW7 (7 %). Other genes harbored mutations at lower frequency. EGFR mutations were relatively frequent and significantly associated with young age of onset (p = 0.028). Additionally, EGFR or PIK3CA mutations were a marker for poor disease-specific survival in our cohort (p = 0.009 and p = 0.032, respectively). Interestingly, KRAS or PIK3CA mutations were significantly associated with poor disease-specific survival in cases with wild-type TP53 (p = 0.001 and p = 0.02, respectively). CONCLUSIONS: Frequent EGFR mutations in this cohort as well as the differential prognostic potential of KRAS and PIK3CA in the presence or absence of detectable TP53 mutations may serve as novel prognostic tools for CRC in patients from the Kingdom of Saudi Arabia. Such findings could help in the clinical decision-making regarding therapeutic intervention for individual patients and provide better diagnosis or prognosis in this locality.

Utilization of amplicon-based targeted sequencing panel for the massively parallel sequencing of sporadic hearing impairment patients from Saudi Arabia.

BACKGROUND: Hearing Impairment (HI) can have genetic or environmental causes and in some cases, an interplay of both. Genetic causes are difficult to determine as mutations in more than 90 genes have been shown recently to be responsible for HI. Providing a genetic diagnostic test for HI is therefore a challenge especially for ethnic groups where GJB2 mutations are shown to be rare. RESULTS: Here we show the design and implementation of an amplicon-based targeted sequencing panel that allows the simultaneous sequencing of 87 HI genes. Mutations identified included known pathogenic mutations and novel variants with unknown significance. The diagnostic rate of this panel is 28 % when only pathogenic variants were reported. However, an additional 28 % harbored recurrent combinations of novel or rare single nucleotide variants in the OTOF or PCDH15 genes. Such combinations were not identified in healthy individuals. CONCLUSIONS: Targeted sequencing approach is a very useful strategy for the identification of mutations affecting the HI genes because of its relatively fast turn-around time and cost effectiveness compared to whole-exome sequencing. Further novel or rare variants could be identified by implementing a large-scale screening of HI using our panel which will eventual lead to a higher diagnostic rate.

Identification of novel genetic variations affecting osteoarthritis patients.

BACKGROUND: Osteoarthritis (OA) is a progressive joint disease characterized by gradual degradation of extracellular matrix (ECM) components in the cartilage and bone. The ECM of cartilage is a highly specified structure that is mainly composed of type II collagen and provides tensile strength to the tissue via aggrecan and proteoglycans. However, changes in the ECM composition and structure can lead to loss of collagen type II and network integrity. Several risk factors have been correlated with OA including age, genetic predisposition, hereditary factors, obesity, mechanical injuries, and joint trauma. Certain genetic association studies have identified several genes associated with OA using genome-wide association studies (GWASs). RESULTS: We identified several novel genetic variants affecting genes that function in several candidate causative pathways including immune responses, inflammatory and cartilage degradation such as SELP, SPN, and COL6A6. CONCLUSIONS: The approach of whole-exome sequencing can be a promising method to identify genetic mutations that can influence the OA disease.

Prognostic value of HER2 status in bladder transitional cell carcinoma revealed by both IHC and BDISH techniques.

BACKGROUND: Her2/neu is an oncogene that plays an important role in the pathogenesis of many cancer types. In bladder carcinoma (BC), the clinical significance of Her2/neu status remains under-investigated and poorly linked to the patients' clinic-pathological features and survival status. Thus, the current study was conducted to assess Her2/neu status in a cohort of patients' in Saudi Arabia, and to explore its prognostic value in BC. METHODS: A total of 160 consent patients of transitional cell carcinoma (TCC) of bladder were arranged on a tissue microarray (TMA) and stained by immunohistochemistry (IHC) and bright-field dual in situ hybridization (BDISH) methods. The intensity of Her2/neu protein receptor immunostaining was evaluated, correlated to Her2/neu gene amplification status in TCC and assessed for potential clinical value by correlation measures. RESULTS: IHC data demonstrated that Her2/neu protein is expressed in 60 % (2+ and 3+) of our TCC patient's cohort from Saudi Arabia. Her2/neu gene amplification is detected in 25 % by BDISH. There was a strong association between Her2/neu protein levels and lymph node invasion (p = 0.04), tumor stage (p = 0.002), vascular invasion and borderline significance with distant metastasis (p = 0.07). Amplification of Her2/neu gene was associated with tumor grade (p = 0.03) and poor disease-specific survival (p = 0.02), in that, patients with non-amplified Her2/neu gene live longer. Interestingly, there was a reasonable concordance rate (71 %) between IHC and BDISH data in the analyzed cohort. CONCLUSION: The study showed that 25 % of our patients' cohort has Her2/neu over-expression. This Her2/neu (over-expression/amplification) status was concordant using either IHC or BDISH and significantly associated with disease aggressiveness and poor outcome. These findings suggested a potential impact of anti-Her2 targeted therapy in the treatment of bladder cancer with amplified/overexpressed HER2 that needs further investigation.

Exome Sequencing of Normal and Isogenic Transformed Human Colonic Epithelial Cells (HCECs) Reveals Novel Genes Potentially Involved in the Early Stages of Colorectal Tumorigenesis.

BACKGROUND: We have generated a series of isogenically derived immortalized human colonic epithelial cell (HCEC 1CT and HCEC 2CT) lines, including parental un-immortalized normal cell strains. The CDK4 and hTERT immortalized colonic epithelial cell line (HCEC 1CT) is initially karyotypically normal diploid and expresses a series of epithelial cell markers including stem cell markers. Under stressful tissue culture conditions, a spontaneous aneuploidy event occurred in the HCEC 1CT line, resulting in a single chromosomal change leading to a stable trisomy 7 cell line (1CT7). Trisomy 7 occurs in about 40% of all benign human adenomas (polyps) and thus this specific chromosomal change in diploid HCEC 1CT cells appears to be non random. In addition, we have partially transformed the HCEC 1CT line by introducing stable knockdown of wild type APC and TP53, and ectopically introducing a mutant Krasv12 and a mutant version of APC (A1309), all commonly found mutations in colorectal cancer (CRC). METHODS: Whole exome sequencing and bioinformatic analyses were performed to comprehensively examine the genetic background of these isogenic cell lines. RESULTS: Exome sequencing of these experimentally progressed cell lines recapitulates a list of genes previously reported to be involved in CRC tumorigenesis. In addition, sequencing revealed a collection of novel genes specifically detected in 1CT7 and A1309 cells but not normal diploid 1CT cells. CONCLUSION: This study demonstrates the utility of using isogenic experimentally derived HCEC lines as a model to recapitulate CRC initiation and progression. Exome sequencing reveals a collection of novel genes that may play important roles in CRC tumorigenesis.

Transcriptomics profiling study of breast cancer from Kingdom of Saudi Arabia revealed altered expression of Adiponectin and Fatty Acid Binding Protein4: Is lipid metabolism associated with breast cancer?

BACKGROUND: Breast cancer incidence rates are increasing at an alarming rate among Saudi Arabian females. Most molecular genetic discoveries on breast cancer and other cancers have arisen from studies examining European and American patients. However, possibility of specific changes in molecular signature among cancer patients of diverse ethnic groups remains largely unexplored. We performed transcriptomic profiling of surgically-resected breast tumors from 45 patients based in the Western region of Saudi Arabia using Affymetrix Gene 1.0 ST chip. Pathway and biological function-based clustering was apparent across the tissue samples. RESULTS: Pathway analysis revealed canonical pathways that had not been previously implicated in breast cancer. Biological network analysis of differentially regulated genes revealed that Fatty acid binding protein 4, adipocyte (FABP4), adiponectin (ADIPOQ), and retinol binding protein 4 (RBP4) were most down regulated genes, sharing strong connection with the other molecules of lipid metabolism pathway. The marked biological difference in the signatures uncovered between the USA and Saudi samples underpins the importance of this study. Connectivity Map identified compounds that could reverse an observed gene expression signature CONCLUSIONS: This study describes, to our knowledge, the first genome-wide profiling of breast cancer from Saudi ethnic females. We demonstrate the involvement of the lipid metabolism pathway in the pathogenesis of breast cancer from this region. This finding also highlights the need for strategies to curb the increasing rates of incidence of this disease by educating the public about life-style risk factors such as unhealthy diet and obesity.

Characterization of familial breast cancer in Saudi Arabia.

BACKGROUND: The contribution of genetic factors to the development of breast cancer in the admixed and consanguineous population of the western region of Saudi Arabia is thought to be significant as the disease is early onset. The current protocols of continuous clinical follow-up of relatives of such patients are costly and cause a burden on the usually over-stretched medical resources. Discovering the significant contribution of BRCA1/2 mutations to breast cancer susceptibility allowed for the design of genetic tests that allows the medical practitioner to focus the care for those who need it most. However, BRCA1/2 mutations do not account for all breast cancer susceptibility genes and there are other genetic factors, known and unknown that may play a role in the development of such disease. The advent of whole-exome sequencing is offering a unique opportunity to identify the breast cancer susceptibility genes in each family of sufferers. The polymorphisms/mutations identified will then allow for personalizing the genetic screening tests accordingly. To this end, we have performed whole-exome sequencing of seven breast cancer patients with positive family history of the disease using the Agilent SureSelect™ Whole-Exome Enrichment kit and sequencing on the SOLiD™ platform. RESULTS: We have identified several coding single nucleotide variations that were either novel or rare affecting genes controlling DNA repair in the BRCA1/2 pathway. CONCLUSION: The disruption of DNA repair pathways is very likely to contribute to breast cancer susceptibility in the Saudi population.

Frequent methylation of the KLOTHO gene and overexpression of the FGFR4 receptor in invasive ductal carcinoma of the breast.

Invasive ductal carcinoma of the breast is the most common cancer affecting women worldwide. The marked heterogeneity of breast cancer is matched only with the heterogeneity in its associated or causative factors. Breast cancer in Saudi Arabia is apparently an early onset with many of the affected females diagnosed before they reach the age of 50 years. One possible rationale underlying this observation is that consanguinity, which is widely spread in the Saudi community, is causing the accumulation of yet undetermined cancer susceptibility mutations. Another factor could be the accumulation of epigenetic aberrations caused by the shift toward a Western-like lifestyle in the past two decades. In order to shed some light into the molecular mechanisms underlying breast cancer in the Saudi community, we identified KLOTHO (KL) as a tumor-specific methylated gene using genome-wide methylation analysis of primary breast tumors utilizing the MBD-seq approach. KL methylation was frequent as it was detected in 55.3 % of breast cancer cases from Saudi Arabia (n = 179) using MethyLight assay. Furthermore, KL is downregulated in breast tumors with its expression induced following treatment with 5-azacytidine. The involvement of KL in breast cancer led us to investigate its relationship in the context of breast cancer, with one of the protagonists of its function, fibroblast growth factor receptor 4 (FGFR4). Overexpression of FGFR4 in breast cancer is frequent in our cohort and this overexpression is associated with poor overall survival. Interestingly, FGFR4 expression is higher in the absence of KL methylation and lower when KL is methylated and presumably silenced, which is suggestive of an intricate relationship between the two factors. In conclusion, our findings further implicate "metabolic" genes or pathways in breast cancer that are disrupted by epigenetic mechanisms and could provide new avenues for understanding this disease in a new context.

High fibroblast growth factor 19 (FGF19) expression predicts worse prognosis in invasive ductal carcinoma of breast.

Metabolic diseases like diabetes and obesity are major risk factors for breast cancer. Aberrant expression of metabolic effectors such as fibroblast growth factor 19 (FGF19) could be therefore associated with the disease. The expression of FGF19 was examined in 193 archival breast tumor samples by immunohistochemistry and evaluated semi-quantitatively by determining the staining index and correlating it with clinicopathological parameters using Fisher's exact test. The correlation between FGF19 expression and 5-year disease-specific survival rate was determined using the univariate Kaplan-Meier analysis. The prognostic value of FGF19 expression was evaluated using the multivariate Cox regression analysis. Of the 193 tumors analyzed, 40% were classified with low FGF19 expression, whereas 60% were categorized as tumors with high FGF19 expression. There was a highly significant correlation between high FGF19 expression and patients' age (p = 0.008) as well as 5-year disease-specific survival (p = 0.001). However, FGF19 expression did not show any significant correlations with other clinicopathological parameters, including hormonal status, tumor grade, tumor size, or lymph node status. Univariate Kaplan-Meier log rank analysis showed that patients with high FGF19 expression exhibited a significantly shorter disease-specific 5-year survival (p = 0.007). This effect was exacerbated by lymph node metastasis (p = 0.001), negative estrogen receptor (ER) status (p = 0.002), or old age (p = 0.013). Multivariate analysis showed that high FGF19 expression could be an independent prognostic marker of disease-specific survival in breast cancer patients (p = 0.030). Quantification of FGF19 expression appears to provide valuable prognostic information in breast cancer, particularly in older patients with lymph node metastasis and negative ER status.

Identification of a novel SBF2 missense mutation associated with a rare case of thrombocytopenia using whole-exome sequencing.

We describe in this report a case of a 6-years-old female who presented at the age of 1 month with a mucocutaneous bleeding and suspected thrombocytopenia. The patient's condition was refractory to the known idiopathic thrombocytopenic purpura treatments and congenital form of Thrombocytopenia was suspected following the delivery of a male sibling with the same phenotype. The patient also manifested fair colored hair and skin relative to her family however she did not have any detectable neurologic or other immunologic abnormalities. In order to further understand this condition, we have used whole-exome sequencing of the patient's DNA as well as her father's with the assumption that her condition is transmitted in an autosomal recessive manner. We have identified a missense change c.659C>G (p.Thr220Arg) in the SBF2 (also known as MTMR13) gene that causes a mutation in the DENN domain of the protein. This mutation was validated by traditional Sanger sequencing and analyzed in the remaining family members were it was found to segregate in the homozygous state in the affected siblings and in the heterozygous state in both parents. This novel mutation in the DENN domain of SBF2 maybe interfering with its putative association with the Rab family of small GTPases which are important mediators of vesicle transport and membrane trafficking. In conclusion, we have identified a novel mutation that is associated with severe thrombocytopenia. The fact that this mutation is found in SBF2 gene may indicate that the underlying cause of thrombocytopenia in our patient is either a new variant form of Griscelli syndrome (through the Rab GTPases action) or a variant Charcot-Marie-Tooth type 4 disease as SBF2 truncating mutations were previously identified in sufferers of this disease. This finding will help to accurately diagnose and classify similar cases of congenital thrombocytopenia and provide further proof to the power of whole-exome sequencing in personalizing patients management from the point of diagnosis to treatment and followup.

Five novel glucose-6-phosphate dehydrogenase deficiency haplotypes correlating with disease severity.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. An association between enzyme levels and gene haplotypes remains to be established. METHODS: In this study, we determined G6PD enzyme levels and sequenced the coding region, including the intron-exon boundaries, in a group of individuals (163 males and 86 females) who were referred to the clinic with suspected G6PD deficiency. The sequence data were analysed by physical linkage analysis and PHASE haplotype reconstruction. RESULTS: All previously reported G6PD missense changes, including the AURES, MEDITERRANEAN, A-, SIBARI, VIANGCHAN and ANANT, were identified in our cohort. The AURES mutation (p.Ile48Thr) was the most common variant in the cohort (30% in males patients) followed by the Mediterranean variant (p.Ser188Phe) detectable in 17.79% in male patients. Variant forms of the A- mutation (p.Val68Met, p.Asn126Asp or a combination of both) were detectable in 15.33% of the male patients. However, unique to this study, several of such mutations co-existed in the same patient as shown by physical linkage in males or PHASE haplotype reconstruction in females. Based on 6 non-synonymous variants of G6PD, 13 different haplotypes (13 in males, 8 in females) were identified. Five of these were previously unreported (Jeddah A, B, C, D and E) and were defined by previously unreported combinations of extant mutations where patients harbouring these haplotypes exhibited severe G6PD deficiency. CONCLUSIONS: Our findings will help design a focused population screening approach and provide better management for G6PD deficiency patients.

A paediatric dysembryoplastic neuroepithelial tumour (DNET) with deregulated stem cell markers: a case report.

Background: Dysembryoplastic neuroepithelial tumours (DNETs) are rare, with only a few reported lethal cases. Currently, there are focused efforts by neuro-oncology professionals to reveal the molecular characterisations of individual central nervous system tumours (CNSTs). Here, we report the status of cancer stem cell (CSC) genes associated with resilience and drug resistance in a paediatric DNET, since the deregulations and variations of CSC genes may prove critical to these tumours' molecular characterisations. Case Description: Immunofluorescence, clonogenic assay and whole exome sequencing (WES) were applied to the patient's tissue and its corresponding cell line. The case is for of a 6-year-old boy with intractable epilepsy and unremarkable physical and neurological examinations. Following magnetic resonance imaging (MRI) and histopathological tests, the patient was diagnosed with DNET. The child underwent a right posterior temporoparietooccipital neuronavigation-assisted craniotomy. Several CSC markers were upregulated in situ, including the metastasis-related protein, anterior gradient 2 (AGR2; 67%), and the Wnt-signalling-related protein, frizzled class receptor 9 (FZD9; 79%). The cell line possessed a similar DNA profile as the original tissue, stained positive for the tumorigenic marker [BMI1 proto-oncogene (BMI)] and CSC markers, and displayed drug resistance. Variants identified in the tissue DNA, which are listed in the catalogue of somatic mutations in cancer (COSMIC) database for genes previously known to be necessary for the development of the embryonic brain, included variants in the cell division cycle 27 (CDC27) gene. Conclusions: we report the in situ and in vitro presence of CSCs in a paediatric DNET.

The Diagnostic Value of Pan-Trk Expression to Detect Neurotrophic Tyrosine Receptor Kinase (NTRK) Gene Fusion in CNS Tumours: A Study Using Next-Generation Sequencing Platform.

Background: Neurotrophic tyrosine receptor kinase (NTRK) fusion has been detected in rare types of CNS tumours, which can promote tumorigenesis. The efficacy of Trk inhibitor became a significant therapeutic interest. Our aim was to investigate whether Pan-Trk immunohistochemistry (IHC) is a reliable and efficient marker for detecting NTRK-fusion in different brain tumours. Methods: This study included 23 patients diagnosed with different types of CNS tumours. Testing for Pan-Trk IHC with monoclonal Ab (EPR17341) has been performed on all FFPE tissues. Parallelly, NTRK-rearrangements were tested using both DNA and RNA-based next-generation sequencing (NGS) assay using TruSight Onco500 platform. Results: The cohort included eight pilocytic astrocytomas, one oligodendroglioma, six IDHwildtype glioblastomas, four IDHmutant grade four astrocytomas, and one sample of each (astroblastoma, central neurocytoma, medulloblastoma, and liponeurocytoma). The mean age was 35 years; seven cases were in the paediatric age group, and 16 were adult. Pan-Trk expression was detected in 11 (47.8%) tumours, and 12 (52.1%) tumours showed no Pan-Trk expression. Nine Cases (82%) with different Pan-Trk expressions did not reveal NTRK-rearrangement. The other two positively expressed cases (liponeurocytoma and glioblastoma) were found to have NTRK2-fusions (SLC O 5A1-NTRK2, AGBL4-NTRK2, BEND5-NTRK2). All the 12 cases (100%) with no Pan-Trk expression have shown no NTRK-fusions. There was no statistically significant association between Pan-Trk expression and NTRK-fusion (p = 0.217). The detection of NTRK- fusions using NGS had high specificity over NTRK-fusion detection by using Pan-Trk IHC. Conclusion: Pan-Trk IHC is not a suitable tissue-efficient biomarker to screen for NTRK-fusions in CNS tumours, however RNA-based NGS sequencing should be used as an alternative method.