Pattern of nucleotide variants of TP53 and their correlation with the expression of p53 and its downstream proteins in a Sri Lankan cohort of breast and colorectal cancer patients.

BACKGROUND: Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. METHODS: 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. RESULTS: Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. CONCLUSION: The prevalence of pathogenic somatic TP53 variants was 31 and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5-8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

Exploring novel protein-based biomarkers for advancing breast cancer diagnosis: A review.

This review provides a contemporary examination of the evolving landscape of breast cancer (BC) diagnosis, focusing on the pivotal role of novel protein-based biomarkers. The overview begins by elucidating the multifaceted nature of BC, exploring its prevalence, subtypes, and clinical complexities. A critical emphasis is placed on the transformative impact of proteomics, dissecting the proteome to unravel the molecular intricacies of BC. Navigating through various sources of samples crucial for biomarker investigations, the review underscores the significance of robust sample processing methods and their validation in ensuring reliable outcomes. The central theme of the review revolves around the identification and evaluation of novel protein-based biomarkers. Cutting-edge discoveries are summarised, shedding light on emerging biomarkers poised for clinical application. Nevertheless, the review candidly addresses the challenges inherent in biomarker discovery, including issues of standardisation, reproducibility, and the complex heterogeneity of BC. The future direction section envisions innovative strategies and technologies to overcome existing challenges. In conclusion, the review summarises the current state of BC biomarker research, offering insights into the intricacies of proteomic investigations. As precision medicine gains momentum, the integration of novel protein-based biomarkers emerges as a promising avenue for enhancing the accuracy and efficacy of BC diagnosis. This review serves as a compass for researchers and clinicians navigating the evolving landscape of BC biomarker discovery, guiding them toward transformative advancements in diagnostic precision and personalised patient care.

Association of selected genetic variants in CBS and MTHFR genes in a cohort of children with homocystinuria in Sri Lanka.

BACKGROUND: Homocystinuria is an inherited, inborn error of homocysteine metabolism, which leads to the abnormal accumulation of homocysteine and its metabolites in blood and urine, resulting in various complications. Variants in the cystathionine ß-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) genes interrupt the formation of the corresponding enzymes and prevent homocysteine from being metabolised; hence, the homocysteine levels in plasma increase than the optimum levels. MATERIALS AND METHODS: In the current study, eight clinically confirmed children with homocystinuria were detected to study the chosen variants in the CBS gene (c.833 T>C and c.19del) and in the MTHFR gene (c.665 C>T, c.1286 A>C) using SNaPshot mini-sequencing and direct sequencing. RESULTS: After screening eight patients, none had the c.833T>C, but four patients were in the homozygous state for the c.19del variant in the CBS gene. Furthermore, seven were heterozygous for c.1286A>C, while one patient was heterozygous for c.665C>T in the MTHFR gene. CONCLUSION: According to the results, c.19del is common in the studied cohort of Sri Lankan children, while c.833T>C is absent, whereas c.1286A>C was more frequent than c.665C>T. To our knowledge, the current study was the first report to discuss the genetic impact of homocystinuria in Sri Lanka; further comprehensive studies are necessary with a larger sample size to establish the association of these variants with the disease in Sri Lanka, which can be beneficial in enhanced patient care and for prospective studies.

Interaction of Gut Microbiome and Host microRNAs with the Occurrence of Colorectal and Breast Cancer and Their Impact on Patient Immunity.

Breast and colorectal cancers are two primary malignancies on which most of the research done worldwide investigates the potential genetic and environmental risk factors and thereby tries to develop therapeutic methods to improve prognosis. Breast cancer is the most diagnosed cancer type in women, while colorectal cancer is diagnosed in males as the third most and females as the second most cancer type. Though these two cancer types are predominantly seen in adult patients worldwide, in the current context, these malignancies are diagnosed at a younger age with a significant rate of incidents than previous. Such early-onset cancers are generally present at an advanced stage of the most aggressive type with a poor prognosis. In the past, the focus of the research was mainly on studying possible candidate genes to understand the onset. However, it is now recognized that genetics, epigenetics, and other environmental factors play a pivotal role in cancer susceptibility. Thus, most studies were diversified to study the behavior of host microRNAs, and the involvement of gut microbiota and good communication between them surfaced in the occurrence and state of the disease. It is understood that the impact of these factors affects the outcome of the disease. Out of the adverse outcomes identified relating to the disease, immunosuppression is one of the most concerning outcomes in the current world, where such individuals remain vulnerable to infections. Recent studies revealed that microbiome and microRNA could create a considerable impact on immunosuppression. This review focused on the behavior of host microRNAs and gut microbiome for the onset of the disease and progression, thereby influencing an individual's immunosuppression. Understanding the interactions among microRNA, microbiome, presentation of the disease, and impact on the immune system will be immensely useful for developing future therapeutic strategies based on targeting host microRNA and the patient's gut microbiome. Therapies such as inhibitory-miRNA therapies, miRNA mimic-based therapeutics, immune checkpoint blockade therapies, and bacteria-assisted tumor-targeted therapies help modulate cancer. At the same time, it paid equal attention to potential noninvasive biomarkers in diagnosis, prognosis, and therapeutics in both cancers.

Overview of the genetic basis toward early detection of breast cancer.

Cancer is a socioeconomical burden in any nation. Out of that, breast cancer is identified as the most common malignancy worldwide among women irrespective of age. As women are an important segment in a community, the weakening of their strength toward the development of a nation is a critical problem in each nation. In this review, it was aimed to discuss the characteristics of cancer genome, cancer genetics, and cancer epigenetics in general and then focus on discussing both genetic and nongenetic factors responsible for the predisposition of breast cancer in humans. More emphasis was placed on genes responsible for the early onset of the disease and which can be used as genetic tools in the identification of the disease at an early stage. Then the context of genetic involvement toward the breast cancer occurrence before age of 40 years was highlighted accordingly. In addition to genetic testing, the review paid adequate attention to mention novel liquid biopsy techniques and other clinical, laboratory, and radiologic assessments. These techniques can be used in early detection and recurrence as well as the surveillance of the patients after primary therapies.

Nucleotide variants and protein expression of TP53 in a Sri Lankan cohort of patients with head and neck cancer.

Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3' untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno­positive, whereas, samples with nonsense and frameshift TP53 variants were immuno­negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Novel gross deletion at the GHRHR gene locus possibly mediated by Alu specific microhomology identified in a Sri Lankan patient with isolated growth hormone deficiency.

OBJECTIVE: Characterization of a deletion in the exon 1 and 5' regulatory region of the GHRHR gene in a proband with isolated growth hormone deficiency. METHODS: Multiple ligation dependent probe amplification (MLPA) assay was carried out to confirm the homozygous deletion which was suspected during screening of the GHRHR gene by single strand conformation polymorphism. A series of short range PCR amplifications were carried out to map the approximate location of the break points of the deletion. Sanger sequencing was carried out to locate the break points and to identify the length of the deletion. Long range PCR amplification was carried out to confirm the length of the deletion and to screen the parents of the proband for the deletion. RESULTS: A homozygous deletion was confirmed via MLPA assay. Zones of sequence similarity between upstream intergenic region and intron 1 of the GHRHR gene were identified. Break points of the deletion were identified within perfectly matching 32 bp repeat sequences ie: microhomologies in the specified zones. The novel deletion may have arisen via Alu specific microhomology mediated non-recurrent rearrangement in the maternal lineage of the proband. The deletion being reported in this study include, last 3118 bp from the upstream intergenic region and complete exon 1 and first 2620 bp from intron 1 and one of the 32 bp microhomologies. The total length of the deleted segment was 5875 bp. As the deleted region contained significant elements essential for gene expression, the identified deletion is being reported as likely pathogenic. The same deletion was identified in the mother in heterozygous state. CONCLUSION: We have characterized a novel deletion that seems to have arisen via Alu specific microhomology mediated non-recurrent rearrangement at GHRHR gene locus. HGVS nomenclature of the deletion is c.-3166_58-2057del. This novel structural variant was identified to be the cause of IGHD of the affected proband.

Pathogenic and likely pathogenic genetic alterations and polymorphisms in growth hormone gene (GH1) and growth hormone releasing hormone receptor gene (GHRHR) in a cohort of isolated growth hormone deficient (IGHD) children in Sri Lanka.

OBJECTIVE: Genetic alterations in GH1 and GHRHR genes are known to cause isolated growth hormone deficiency (IGHD). Of these, GHRHR codon 72 mutation has been reported to be highly prevalent in the Indian subcontinent, but among Sri Lankans its prevalence was low compared to reports from neighboring countries. The present study was therefore carried out to identify genetic alterations in the GH1 gene and rest of the GHRHR gene in a cohort of Sri Lankan IGHD patients who tested negative for GHRHR codon 72 mutation. METHODS: Fifty five IGHD children negative for codon 72 (GHRHR) mutation were screened for gross GH1 gene deletion by polymerase chain reaction (PCR) and restriction fragment length polymorphism technique. The coding, intronic and promoter regions of the GH1 gene were sequenced in children who were negative for GH1 deletion (N=53). In a subset (N=40), coding, flanking intronic and promoter regions of the GHRHR gene were screened by single strand conformation polymorphism/sequencing. Identified coding region and intronic variants were subjected to in silico analysis to ascertain pathogenicity. Family members available were screened for the significant variants observed in the index child. RESULTS: Gross GH1 gene deletions, 6.7kb and 7.0kb were observed in one child each. One novel and 24 reported single nucleotide variants (SNVs) were observed in the GH1 gene and its promoter. These included one reported pathogenic splice site mutation (c.172-2A>T) and one reported likely pathogenic missense mutation (c.406G>T). One large novel deletion of 5875 base pairs that included exon 1, one likely pathogenic novel SNV (c.211G>T) and 18 reported SNVs were observed in the GHRHR gene. Fourteen variants observed were of uncertain significance (8 in GH1 and 6 in GHRHR), twenty three variants were likely benign (11 in GH1 and 12 in GHRHR) and four variants were benign (4 in GH1 and none in GHRHR). CONCLUSION: In a cohort of IGHD children, six pathogenic or likely pathogenic genetic alterations of either GH1 gene or GHRHR gene were found. These affected a total of six children. Pathogenic status of four of these had been reported in the literature. Novel SNV in the GHRHR gene was predicted to be pathogenic through in silico analysis. The large novel deletion is likely to be pathogenic as it included exon 1 of GHRHR gene. Analysis of other genes will be needed to ascertain the genetic cause of IGHD in the remaining children.

Novel and reported pathogenic variants in exon 11 of BRCA2 gene in a cohort of Sri Lankan young breast cancer patients.

Women with breast carcinoma diagnosed before 40 years of age with a strong familial risk have a greater prevalence of germline BRCA1 or BRCA2 variants than late onset breast cancer. Previously germline variants in BRCA1 and BRCA2 genes were characterized in a cohort of Sri Lankan breast cancer patients unselected for age of onset. This study focused on young breast cancer patients who were screened for previously identified hotspot regions in BRCA2 gene. A total of 48 young breast cancer patients with family history of cancer and 25 healthy controls were studied. Direct sequencing was used to detect pathogenic and other sequence variants in the hotspot regions of BRCA2 gene. Thirty-six sequence variants including seven pathogenic (c.2411_2412delAA/p.Glu804Valfs*2, c.2500_2501insG/p.Leu834Cysfs*4, c.3881T>G/p.Leu1294*, c.4768A>T/p.Lys1590*, c.5645C>G/p.Ser1882*, c.5747delC/p.His1916Phefs*3, c.6728C>T/p.Ser2243Phe) and two likely pathogenic (c.1922C>T and c.3378A>T) variants, two intronic variants of unknown significance (c.1910-74T>C, c.1910-51G>T), two variants of uncertain significance (c.2324C>T c.5104C>T) and 23 benign variants were detected. Among them, seven were novel (pathogenic 5 and likely pathogenic 2). Prevalence of pathogenic and likely pathogenic variants in the hotspots regions of BRCA2 was 23 and 6.3 % respectively in this cohort. This justifies BRCA2 variant testing in young breast cancer patients with family history of cancer in Sri Lanka.

Analysis of BRCA1and BRCA2 large genomic rearrangements in Sri Lankan familial breast cancer patients and at risk individuals.

BACKGROUND: Majority of mutations found to date in the BRCA1/BRCA2 genes in breast and/or ovarian cancer families are point mutations or small insertions and deletions scattered over the coding sequence and splice junctions. Such mutations and sequence variants of BRCA1 and BRCA2 genes were previously identified in a group of Sri Lankan breast cancer patients. Large genomic rearrangements have been characterized in BRCA1 and BRCA2 genes in several populations but these have not been characterized in Sri Lankan breast cancer patients. FINDINGS: A cohort of familial breast cancer patients (N = 57), at risk individuals (N = 25) and healthy controls (N = 23) were analyzed using multiplex ligation-dependent probe amplification method to detect BRCA1 and BRCA2 large genomic rearrangements. One familial breast cancer patient showed an ambiguous deletion in exon 6 of BRCA1 gene. Full sequencing of the ambiguous region was used to confirm MLPA results. Ambiguous deletion detected by MLPA was found to be a false positive result confirming that BRCA1 large genomic rearrangements were absent in the subjects studied. No BRCA2 rearrangement was also identified in the cohort. CONCLUSION: Thus this study demonstrates that BRCA1 and BRCA2 large genomic rearrangements are unlikely to make a significant contribution to aetiology of breast cancer in Sri Lanka.

Novel sequence variants and common recurrent polymorphisms of BRCA2 in Sri Lankan breast cancer patients and a family with BRCA1 mutations.

We previously reported BRCA1 mutations and sequence variants in Sri Lankan breast cancer patients. Mutations and sequence variants of the BRCA2 gene were studied in 149 study participants from the same cohort. There were 55 familial and 54 sporadic breast cancer patients, 20 at-risk individuals and 20 healthy controls. Direct sequencing (exon 11) and sequencing of abnormal bands after screening with single-strand conformation polymorphism (remaining exons) were used to detect mutations and sequence variants. Twenty-three sequence variants were found in the BRCA2 gene. Two novel pathogenic frame-shift additions resulting in a premature stop codon (c.2403 insA/exon 11, c.2667 insT/exon 11) were identified. Possibly pathogenic two novel missense mutations (c.1191 A>C/exon 10, c.5695 A>C/exon 11) one novel intronic variant (IVS15-21 insTT), four novel silent mutations (c.969 C>T/exon 9, c.1353 C>T/exon 10, c.2766 A>C/exon 11 and c.7452 A>G/exon 14) and one novel missense mutation (c.971 C>G/exon 9) were observed. One previously reported possibly pathogenic intronic variant (IVS81 G>C) and several previously reported silent mutations, missense mutations, and one 5' UTR polymorphism were detected. Pathogenic and possibly pathogenic mutations were more frequent in the BRCA2 gene among Sri Lankan familial breast cancer patients when compared to our previous findings for the BRCA1 gene.