LncRNAs are noncoding RNA species with more than 200 nucleotides in length, involved in regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels. Dysregulation of lncRNA expression can cause malignancies such as breast, liver, colon, and lung cancer and leukaemia. 1 HOX transcript antisense intergenic RNA (HOTAIR) is one of the lncRNAs with an oncogenic impact on developing different cancers, especially breast, gastric, pancreatic, colorectal, hepatocellular, lung, and cervical cancer. 2 Some studies on SNP analysis in the HOTAIR gene have found a relationship between SNPs and BC susceptibility. The functional single nucleotide polymorphisms (SNPs) located in the intronic region of the gene (rs920778, rs4759314, and rs1899663) are involved in the overexpression of HOTAIR relating to the development and progression of some cancers, including estrogen-dependent cancers such as breast cancer and gastrointestinal cancer in the Asian population. 3 The current study focused on detecting HOTAIR functional genetic variants in sporadic breast cancer patients who acquired mutations in genes during their life, representing the majority of breast cancer diagnosed when compared to hereditary breast cancers. tissues of sporadic breast cancer patients (previously collected for a Doctor of Philosophy study done by Manoharan, 2020 under the ethical approval of EC/14/160, using an AllPrep® DNA/RNA/Protein Mini Kit (Cat. No./ ID: 8000, Qiagen). 4 Nine DNA samples from sporadic breast cancer patients and 5 DNA samples from healthy individuals were used.

Two primer sets were designed to amplify the regions containing the two selected SNPs using the online NCBI/ Primer-BLAST software. Then PCR products were outsourced for sequencing to Genelabs Medical, Colombo, Sri Lanka. The sequencing results were analyzed using BioEdit® and Mutation Surveyor® v4.0.10 by aligning with the Human HOTAIR NCBI Reference Sequence: NC_000012.12 at the National Centre for Biotechnology Information (NCBI).

Direct sequencing was carried out to detect the rs920778 C>T and rs1899663 G>T in nine sporadic breast cancer and 5 healthy samples. Tables 1 and 2 summarize the status of the genotype of each SNP in the patient with tumur features and healthy individual cohort.

It was understood that these two variants were reported to be involved in the overexpression of HOTAIR concerning the development and progression of estrogen-dependent cancers. 3 A study done on Indian pre-menopausal women by Rajagopal et al. (2022) reported that TC and CC genotypes of rs920778 increased BC risk 5 while the study on the southeast Iranian population found a significant association between rs920778 and BC. 6 H1 TT GG H2 TT GG H3 TC GG H4 CC GG H5 TC GG

In addition, this pattern was observed in the study done on the Turkish population, which showed the CC genotype of HOTAIR rs920778 was associated with an elevated risk of BC and clinicopathological characteristics of the tumour. 6 Conversely, a population-based study in China reported that the T allele of rs920778 significantly increased BC risk. 7 In addition, a meta-analysis done by 8 supported the fact that individuals in Asian populations with either the T allele or TT genotype of rs920778 C>T had a significantly increased cancer risk in digestive and gynecological cancers. 8 In the current study, TT and TC genotypes were prominent in the cohort of sporadic breast cancer samples with an allele frequency of T = 0.89 and a C = 0.11, showing the fact that TT and TC genotypes may have a possible association with breast cancer risk in the studied cohort. These results are aligned with the results of a study on the Chinese population which showed an association between T allele and breast cancer risk. When considering the hormone receptor status of the patients in the studied cohort, based on the available information, four patients (sample no: 5, 6, 7, and 8) have ER+ PR+ HER2 -status, where three of them showed the TT genotype and the remaining cases showed the TC genotype. Out of the three patients with TT genotype, sample 7 had invasive ductal carcinoma-NG -II, T3N0Mx, and sample 8 had invasive ductal carcinoma NG -I, T4bN0Mx, whereas sample 6 tumour feature was not available. Sample 5 with the TC genotype for rs920778 had invasive ductal carcinoma-NG-2, T4N2Mx. The patients with available data for receptor status showed invasive ductal carcinoma with estrogen-positive. Thus, it was found that elevated levels of HOTAIR expression can be seen in aggressive ductal carcinoma in situ (DCIS) and estradiol (E2) induces HOTAIR expression in an estrogen receptor-dependent manner. 9 In addition, elevated HOTAIR expression showed poor prognosis in estrogen-positive tumours. 10 Furthermore, the studied healthy control showed allele frequency of wild allele (C) of 0.4 while altered allele frequency (T) of 0.6, aligned with the results of the 1000 Genome project concerning the South Asian population, where the C allele frequency was 0.445 and the T allele frequency was 0.555. The results are more supported by the Global allele frequency of 0.4519 for C and 0.5481 for T in the 1000 Genome project. Therefore, the involvement of genotype (TT/TC) of rs920778 in the BC risk of the studied patients was significant. Further studies are needed to confirm the association with a larger cohort on a casecontrol basis. With respect to rs1899663G>T SNP in a cohort of sporadic breast cancer patients and healthy controls, none of the cases or controls showed the mutant allele (T).