Genetic determinants of response to aspirin and warfarin and development of silicon nanowire based genotyping

PhD ThesisChronic diseases such as cardiovascular diseases and colorectal cancer are the leading cause of mortality worldwide. Commonly used drugs such as aspirin and warfarin are shown to effective at reducing the risk of chronic diseases but have a narrow therapeutic window and are associated with adverse drug reactions, particularly, hemorrhage. Identification of pharmacogenetic markers such as single nucleotide polymorphisms (SNPs) that could help deliver personalized dose could help improve the risk-benefit ratio. Furthermore, development of a rapid point of care genotyping device consisting of a pharmacogenetic SNP panel for aspirin and warfarin could help implement personalized medicine in the clinical setting. Analysis of candidate SNPs in aspirin’s pharmacokinetic and pharmacodynamic pathways was carried out to explain variation in aspirin’s colorectal chemopreventive efficacy using two large population based case-control datasets. Associations and interactions were tested using logistic regression models and meta-analysis of the 2 datasets. A novel sitespecific association for rs1799853 (OR=0.73, 95% CI=0.60-0.90, P=0.003) and rs1105879 (OR=1.16, 95% CI=1.02-1.32, P=0.03) with colon cancer risk was observed. Furthermore, stratification by aspirin use showed increased risk of colorectal cancer in aspirin users but not in non-users carrying variant allele of the SNPs rs4936367 and rs7112513 in PAFAH1B2 gene and rs2070959 and rs1105879 in UGT1A6 gene (Pinteraction<0.05 for all). These results provide insight into aspirin’s differential chemopreventive efficacy and the neoplastic transformation of cells in colon and rectum. Utility of clinically validated pharmacogenetic dosing algorithms consisting of three warfarin dose associated SNPs from the European population needs to tested in the Gujarati Indians, an Indian sub-population. Dose prediction accuracy of the algorithms was compared between Gujarati Indian and European population. Mean squared difference of both pharmacogenetic algorithms was higher in Gujarati Indian compared to European population (Klein et al 2009, 216.3 v/s 160.7, P=0.05; Gage et al 2008, 170.6 v/s 143.2, P=0.07). Poor prediction accuracy could be explained by the presence of study subjects requiring dose for target INR range 2.5-3.5 and low frequency of the VKORC1 rs9923231 variant, which is the most important genetic determinant of warfarin dosing in Europeans. Therefore, the SNP panel and dosing algorithms developed from European populations cannot be assumed to have utility in the Gujarati Indian population. Finally, to help develop a rapid, point-of care, silicon nanowire (SiNW) based SNP genotyping device, a panel of isothermal melting probes were designed to genotype three warfarin dose associated SNPs. Testing of hybridization and washing conditions to have optimal hybridization kinetics between the probe and target DNA and high target sequence specificity was carried out using custom designed microarray platform. Accurate genotype calls for all 3 SNPs in 2 anonymised samples using empirically optimized hybridization and washing conditions was carried out successfully. Current work highlighted associations between probe characteristics and hybridization parameters, which would be useful in designing and testing probes on the SiNW platform. Identification, validation and testing of clinical utility of population specific pharmacogenetic markers along with development and deployment of ultra-rapid point of care genotyping technologies could help deliver personalized risk-benefit ratio for aspirin and warfarin

Identification of Novel Mutations in FAH Gene and Prenatal Diagnosis of Tyrosinemia in Indian Family

Carrier of tyrosinemia type I was diagnosed by sequencing FAH (fumarylacetoacetate hydrolase) gene. It leads to the identification of heterozygous status for both c.648C>G (p.Ile216Met) and c.1159G>A (p.Gly387Arg) mutations in exons 8 and 13, respectively, in the parents. The experimental program PolyPhen, SIFT, and MT predicts former missense point mutation as “benign” that creates a potential donor splice site and later one as “probably damaging” which disrupts secondary structure of protein

Prader-Willi syndrome - type 1 deletion, a consequence of an unbalanced translocation of chromosomes 13 and 15, easily to be mixed up with a Robertsonian translocation

BACKGROUND: Prader-Willi syndrome, due to microdeletion of proximal 15q, is a well-known cause of syndromic obesity. CASE CHARACTERISTICS: A couple with history of repeated first trimester abortions had a son with balanced Robertsonian translocation of chromosomes 13 and 15 according to cytogenetic banding technique. RESULTS: Chromosomal analysis for the couple was performed. A balanced translocation involving BP1-BP3 region of proximal 15q was observed in the father. DISCUSSION: Investigations of the parents is mandatory when a structural rearrangement is detected in a dysmorphic child

Pure interstitial dup(6)(q22.31q22.31) – a case report

‘Pure’ interstitial duplication of chr6q is rare. The varying size of duplication encompassing 6q22.31 is associated with the expressivity of dysmorphism and autism. Here, we report a unique case with facial dysmorphism, developmental delay, complex neurological impairment and spasticity unrelated to autism. Genetic analysis by aCGH exhibited a 627–971 kb dup(6)(q22.31q22.31) encompassing TRDN and NKAIN2 genes. The presence of the duplication was confirmed by quantitative PCR in the proband and phenotypically normal parents. With the current techniques, we cannot exclude presence of a deleterious homozygous point mutation in the proband where each copy would have been inherited from both parents

A small supernumerary marker chromosome present in a Turner syndrome patient not derived from X- or Y-chromosome: a case report

<p>Abstract</p> <p>Background</p> <p>Small supernumerary marker chromosomes (sSMC) can be present in numerically abnormal karyotypes like in a 'Turner-syndrome karyotype' mos 45,X/46,X,+mar.</p> <p>Results</p> <p>Here we report the first case of an sSMC found in Turner syndrome karyotypes (sSMC^T) derived from chromosome 14 in a Turner syndrome patient. According to cytogenetic and molecular cytogenetic characterization the karyotype was 46,X,+del(14)(q11.1). The present case is the third Turner syndrome case with an sSMC^Tnot derived from the X- or the Y-chromosome.</p> <p>Conclusion</p> <p>More comprehensive characterization of such sSMC^Tmight identify them to be more frequent than only ~0.6% in Turner syndrome cases according to available data.</p

Maculopathy and adult-onset ataxia in patients with biallelic MFSD8 variants

Background: Biallelic variants in the major facilitator superfamily domain containing 8 gene (MFSD8) are associated with distinct clinical presentations that range from typical late-infantile neuronal ceroid lipofuscinosis type 7 (CLN7 disease) to isolated adult-onset retinal dystrophy. Classic late-infantile CLN7 disease is a severe, rare neurological disorder with an age of onset typically between 2 and 6 years, presenting with seizures and/or cognitive regression. Its clinical course is progressive, leading to premature death, and often includes visual loss due to severe retinal dystrophy. In rare cases, pathogenic variants in MFSD8 can be associated with isolated non-syndromic macular dystrophy with variable age at onset, in which the disease process predominantly or exclusively affects the cones of the macula and where there are no neurological or neuropsychiatric manifestations. Methods: Here we present longitudinal studies on four adult-onset patients who were biallelic for four MFSD8 variants. Results: Two unrelated patients who presented with adult-onset ataxia and had macular dystrophy on examination were homozygous for a novel variant in MFSD8 NM_152778.4: c.935T>C p.(Ile312Thr). Two other patients presented in adulthood with visual symptoms, and one of these developed mild to moderate cerebellar ataxia years after the onset of visual symptoms. Conclusions: Our observations expand the knowledge on biallelic pathogenic MFSD8 variants and confirm that these are associated with a spectrum of more heterogeneous clinical phenotypes. In MFSD8-related disease, adult-onset recessive ataxia can be the presenting manifestation or may occur in combination with retinal dystrophy

Prenatal screening of cytogenetic anomalies – a Western Indian experience

BACKGROUND: Children born with congenital anomalies present a very high rate of perinatal death and neonatal mortality. Cytogenetic analysis is a convincing investigation along with clinical suspicion and biochemical screening tests. The current study was designed to characterize the prevalence and types of chromosomal abnormalities in high risk prenatal samples using different cytogenetic techniques. METHODS: This study was conducted on a total of 1,728 prenatal samples (1,324 amniotic fluids, 366 chorionic villi and 38 cord blood samples) from 1994 to 2014 at Institute of Human Genetics, Ahmedabad, India. Conventional karyotyping was conducted with GTG-banding. Molecular approaches were used (fluorescence in situ hybridization = FISH and/ or array-comparative genomic hybridization = aCGH) when indicated to detect karyotypic abnormalities. RESULTS: Abnormal karyotypes were detected in 125/1,728 (7.2%) cases. Trisomy 21 was the most common abnormality detected in 46 (2.7%) followed by trisomy 18 in 11 (0.6%) and trisomy 13 in 2 (0.1%) samples. Besides, structural abnormalities such as reciprocal and Robertsonian translocation were detected in 20 [1.2%] cases. Turner syndrome was diagnosed in seven (0.4%) cases; in six (0.34%) cases there was an inversion in the Y-chromosome. Heteromorphic variants were diagnosed in 22 (1.3%) cases. Finally, small supernumerary marker chromosomes (sSMC) were found in six (0.34%) cases. CONCLUSION: Conventional GTG-banding along with molecular cytogenetic techniques is useful in detecting genomic alterations and rearrangements. Comprehensive characterization of chromosomal rearrangements like sSMC has the potential to save potentially healthy fetuses from being terminated

Natural history of non-lethal raine syndrome during childhood

Background: Raine syndrome (RS) is a rare autosomal recessive disorder caused by biallelic loss-of-function mutations of FAM20C. The most common clinical features are microcephaly, exophthalmos, hypoplastic nose and severe midface hypoplasia, leading to choanal atresia. The radiological findings include generalized osteosclerosis and brain calcifications. RS is usually lethal during the neonatal period due to severe respiratory distress. However, there exists a non-lethal RS form, the phenotype of which is extremely heterogeneous. There is paucity of data about clinical course and life expectancy of these patients.Results: This is the first description of follow-up features of non-lethal RS patients. Moreover, we present three unpublished cases. There are five Asian and two Arab patients. All were born to consanguineous parents. The most common neonatal comorbidity was respiratory distress secondary to choanal atresia. A variable degree of neurodevelopmental delay was seen in the majority of our cases and seizures and hearing or vision involvement were also frequent. Neurological and orthopedic issues were the most frequent complications seen at follow-up in our group. Persistent hypophosphatemic rickets was the most striking endocrinological manifestation, which was scarcely responsive to therapy with phosphate salts and alfacalcidol. Life expectancy of our patients goes beyond childhood, with the oldest of those described being 18 years old at present.Conclusions: Manifestations of RS in those surviving the neonatal period are being increasingly recognized. Our study supports previous findings and provides clinical and biochemical observations and data from longer follow up. Finally, we propose multidisciplinary follow up for patients with non-lethal RS