ABSTRACT
PURPOSE: Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies. METHODS: AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene. RESULTS: We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable. CONCLUSION: Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
ABSTRACT
Background: Epidermal necrolysis (SJS/TEN) is a rare but acute severe drug reaction associated with high morbidity and mortality rates. Aims: To describe the clinical, molecular, biochemical, and therapeutic profile of these patients. Methods: A total of 24 acute SJS/TEN patients were recruited during their hospital stay and detailed clinical history and treatment course recorded. Blood samples collected were subjected to DNA and serum separation for molecular and biochemical analysis. Results: Of 24 patients, 18 (75%) were females and six (25%) were males with six SJS, six SJS-TEN overlap, and 12 TEN cases. The inciting drugs were non-steroidal anti-inflammatory (87.50%; n = 21) followed by antibiotics (66.67%; n = 16), antiepileptics (37.50%; n = 9), and others (37.50%; n = 9). Seventeen patients (77.2%) showed skin eruptions within 7 days after drug intake. Different co-morbidities were observed in 22 (91.6%) and 20 (83.3%) patients showed ocular manifestations. Length of hospital stay ranged from 8 to 55 days, 20 (83.3%) patients were treated with corticosteroids, and four (16.6%) received antimicrobial therapy. Interleukin polymorphisms revealed significantly low frequency of IL-4 in the patients, HLA-A locus typing revealed higher frequency of HLA-A*3301 (20.8%), HLA-A*02 (25%), HLA-A*2402 (14.6%), and sera showed raised levels of granulysin and sFas L in the patients compared to controls. Conclusions: The preliminary study illustrates the clinical, molecular, and biochemical features of acute SJS/TEN and provides a better understanding that helps to improve patient care at an earlier stage. It also highlights the use of corticosteroids and antimicrobial therapy for effective treatment of patients.
ABSTRACT
Purpose: This study sought to investigate the association of molecular markers with chronic ocular sequelae in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Methods: One hundred SJS/TEN patients (200 eyes) with confirmed diagnosis were enrolled between July 2011 and July 2015 from a tertiary eye-care hospital, and their clinical histories were noted. Each eye was scored for severity of manifestation on a scale of 0-5. Peripheral blood samples were collected for DNA followed by screening for interleukin (IL-4, IL-13, IL-4R) polymorphisms, HLA-A locus allele typing, and sera to detect levels of the apoptotic markers granulysin and sFas L. Results: Of the 100 enrolled patients (53 males/47 females; age range: 6-58 years), the incriminating drugs were non-steroidal anti-inflammatory (52%), antibiotics (10%), sulphonamides (8%), anti-epileptics (6%), and unknown (24%). Significant differences in the frequencies of IL-4R polymorphism, HLA-A*3301, HLA-A*02, and HLA-A*2402 alleles, and elevated levels of granulysin and sFas L were observed in patients compared to controls. The ocular complications of conjunctival keratinization (p=0.004) showed an association with IL-13 promoter region (IL-13a) genotypes. Conclusions: The study highlights the possible association of interleukin-13 with severity-graded chronic sequelae and the role of HLA-A alleles- HLA-A*3301, HLA-A*02, and HLA-A*2402 in SJS/TEN causation and manifestation. Screening of these alleles may help caregivers to identify markers associated with severe and lifelong ocular complications, and help in appropriate treatment and management of the condition.
