The Shapiro—Senapathy algorithm (S&S) is a computational method for identifying splice sites in eukaryotic genes. The algorithm employs a Position Weight Matrix (PWM) scoring formula to predict donor and acceptor splice sites in any given gene. This methodology has been used to discover splice sites and disease-causing splice site mutations in the human genome, and has become a standard tool in clinical genomics. The S&S algorithm has been cited in thousands of clinical studies, according to Google Scholar. It has also formed the basis of widely used software, including Human Splicing Finder, SROOGLE, and Alamut, which identify splice sites and splice site mutations that cause disease. The algorithm has uncovered splicing mutations in diseases ranging from cancers to inherited disorders, and predicted the deleterious effects of these mutations including exon skipping, intron retention, and cryptic splice site activation.
The algorithm A splice site defines the boundary between a coding exon and a non-coding intron in eukaryotic genes. The S&S algorithm employs a sliding window, corresponding to the length of the splice site motif, to scan a gene sequence and detect potential splice sites. For each sliding window, the algorithm calculates a score by comparing the nucleotide sequence to a Position Weight Matrix (PWM) derived from known splice sites. This formula generates a percentile score, indicating the likelihood that a given sequence functions as a donor or acceptor splice site. The majority of disease-causing mutations in the human genome are located in splice sites. Clinical genomics studies analyze the splice site scores generated by the S&S algorithm to predict the consequences of splice site mutations including exon skipping and intron retention. The algorithm's sensitivity to single-nucleotide changes allows it to determine mutations that may impact RNA splicing and contribute to disease. In addition to identifying real splice sites, the S&S algorithm has been used to discover cryptic splice sites — alternative splice sites activated by mutations — which may disrupt normal splicing. The algorithm detects mutations that lead to the activation of cryptic splice sites, which may be located proximal to real splice sites or deep within non-coding introns. It has thus been used to determine the causes of numerous diseases that are due to cryptic splicing.
Cancer gene discovery using S&S The S&S algorithm has been used to identify splice-site mutations in genes associated with several cancers. For example, genes causing commonly occurring cancers including breast cancer, ovarian cancer, colorectal cancer, leukemia, head and neck cancers, prostate cancer, retinoblastoma, squamous cell carcinoma, gastrointestinal cancer, melanoma, liver cancer, Lynch syndrome, skin cancer, and neurofibromatosis have been found. In addition, splicing mutations in genes causing less commonly known cancers including gastric cancer, gangliogliomas, Li-Fraumeni syndrome, Loeys–Dietz syndrome, Osteochondromas (bone tumor), Nevoid basal cell carcinoma syndrome, and Pheochromocytomas have been identified. Specific mutations in different splice sites in various genes causing breast cancer (e.g., BRCA1, PALB2), ovarian cancer (e.g., SLC9A3R1, COL7A1, HSD17B7), colon cancer (e.g., APC, MLH1, DPYD), colorectal cancer (e.g., COL3A1, APC, HLA-A), skin cancer (e.g., COL17A1, XPA, POLH), and Fanconi anemia (e.g., FANC, FANA) have been uncovered. The mutations in the donor and acceptor splice sites in different genes causing a variety of cancers that have been identified by S&S are shown in Table 1.
Discovery of genes causing inherited disorders using S&S Specific mutations in different splice sites in various genes that cause inherited disorders, including, for example, Type 1 diabetes (e.g., PTPN22, TCF1 (HCF-1A)), hypertension (e.g., LDL, LDLR, LPL), Marfan syndrome (e.g., FBN1, TGFBR2, FBN2), cardiac diseases (e.g., COL1A2, MYBPC3, ACTC1), eye disorders (e.g., EVC, VSX1) have been uncovered. A few example mutations in the donor and acceptor splice sites in different genes causing a variety of inherited disorders identified using S&S are shown in Table 2.
Genes causing immune system disorders More than 100 immune system disorders affect humans, including inflammatory bowel diseases, multiple sclerosis, systemic lupus erythematosus, bloom syndrome, familial cold autoinflammatory syndrome, and dyskeratosis congenita. The Shapiro–Senapathy algorithm has been used to discover genes and mutations involved in many immune disorder diseases, including Ataxia telangiectasia, B-cell defects, epidermolysis bullosa, and X-linked agammaglobulinemia. Xeroderma pigmentosum, an autosomal recessive disorder is caused by faulty proteins formed due to new preferred splice donor site identified using S&S algorithm and resulted in defective nucleotide excision repair. Type I Bartter syndrome (BS) is caused by mutations in the gene SLC12A1. S&S algorithm helped in disclosing the presence of two novel heterozygous mutations c.724 + 4A > G in intron 5 and c.2095delG in intron 16 leading to complete exon 5 skipping. Mutations in the MYH gene, which is responsible for removing the oxidatively damaged DNA lesion are cancer-susceptible in the individuals. The IVS1+5C plays a causative role in the activation of a cryptic splice donor site and the alternative splicing in intron 1, S&S algorithm shows, guanine (G) at the position of IVS+5 is well conserved (at the frequency of 84%) among primates. This also supported the fact that the G/C SNP in the conserved splice junction of the MYH gene causes the alternative splicing of intron 1 of the β type transcript. Splice site scores were calculated according to S&S to find EBV infection in X-linked lymphoproliferative disease. Identification of Familial tumoral calcinosis (FTC) is an autosomal recessive disorder characterized by ectopic calcifications and elevated serum phosphate levels and it is because of aberrant splicing.
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