Clinical Exome Gene Panel Analysis of a Cohort of Urothelial Bladder Cancer Patients from Sri Lanka.

BACKGROUND: Bladder cancer has a high rate of recurrence and high mortality rates in those who progress to muscle invasive disease. Biomarkers and molecular sub classification of tumours beyond standard histopathology has been proposed to address therapeutic dilemmas. The Cancer Genome Atlas project and other studies have contributed to the enhanced knowledge base of the mutational landscape of urothelial bladder cancer. Once again, these are mostly from Caucasian and Chinese patients, with data from the rest of Asia and Sri Lanka being sparse. The objective of this study was to assess the genomic variations of a cohort of urothelial bladder cancer patients in Sri Lanka. METHODS: The molecular genetic study was conducted on formalin fixed paraffin embedded tumour samples of 24 patients, prospectively enrolled from 2013 to 2017. The samples were sequenced and variant distribution performed based on a 70-gene panel. RESULTS: Total number of filtered mutations in the 24 patients was 10453. Median mutations per patient were 450 (range 22-987). The predominant mutational change was C>T and G>A. The top 5 mutated genes in our cohort were SYNE1, SYNE2, KMT2C, LRP2, and ANK2. The genes were clustered into 3 groups dependent on the number of mutations per patient per gene. The genes of cluster 1 and 2 mapped to Chromatin modifying enzymes and Generic Transcription Pathway. The chromatin remodelling pathway accounted for the largest proportion (22%) of mutations. CONCLUSIONS: Clinical exome sequencing utilising a gene panel yielded a high mutation rate in our patients. The predominant mutational change was C>T and G>A. Three clusters of genes were identified. SYNE1 was the gene with the most mutations. The mutations comprised predominantly of genes of the chromatin remodelling pathway.

Genetic associations of inflammatory bowel disease in a South Asian population.

AIM: To estimate prevalence and phenotypic associations of selected inflammatory bowel disease (IBD)-associated genetic variants among Sri Lankan patients. METHODS: A case study of histologically confirmed ulcerative colitis (UC) or Crohn's disease (CD) patients with ≥ 1 year disease duration, who were compared to unrelated, gender-matched, healthy individuals as controls, was conducted at four major centers in Sri Lanka. Phenotypic data of the cases were obtained and all participants were genotyped for 16 selected genetic variants: IL12B:rs1045431, IL23R:rs11805303, ARPC2:rs12612347, IRGM:rs13361189, IL26/IL22:rs1558744, CDH1:rs1728785, IL10:rs3024505, FCGR2A:rs3737240, PTGER4:rs4613763, IL17REL/PIM3:rs5771069, HNF4a:rs6017342, STAT3:rs744166, SMURF1:rs7809799, LAMB1:rs886774, HLA-DRB5, DQA1, DRB1, DRA:rs9268853, MST1, UBA7, and APEH:rs9822268. The genotypes of all variants were in Hardy-Weinberg Equilibrium (P > 10-3). To account for multiple hypothesis testing, P-values < 0.003 were considered significant. RESULTS: A total of 415 patients and 465 controls were recruited. Out of the single nucleotide polymorphisms (SNPs) tested, the majority were not associated with IBD in Sri Lankans. Significant positive associations were noted between rs886774 (LAMB1-gene) and UC (odds ratio (OR) = 1.42, P = 0.001). UC patients with rs886774 had mild disease (OR = 1.66, P < 0.001) and remained in remission (OR = 1.48, P < 0.001). A positive association was noted between rs10045431 (IL 12B gene) and upper gastrointestinal involvement in CD (OR = 4.76, P = 0.002). CONCLUSION: This confirms the heterogeneity of allelic mutations in South Asians compared to Caucasians. Most SNPs and disease associations reported here have not been described in South Asians.

The INSR rs2059806 single nucleotide polymorphism, a genetic risk factor for vascular and metabolic disease, associates with pre-eclampsia.

Pre-eclampsia is a risk factor for later life vascular and metabolic diseases. This study postulates that this reflects a common genetic cause, and investigates whether the INSR rs2059806 single nucleotide polymorphism (SNP) (a risk factor for essential hypertension, type 2 diabetes and metabolic syndrome) is also associated with pre-eclampsia. The association of INSR rs2059806 with pre-eclampsia was tested in two cohorts - a Caucasian case control group (123 pre-eclamptic mother-father-baby trios and 1185 mother-father-baby trios from uncomplicated pregnancies) and an independent cohort of Sinhalese women (175 women with pre-eclampsia and 171 women with uncomplicated pregnancies). In the Caucasian cohort, the prevalence of the INSR rs2059806 AA genotype was greater among pre-eclamptic women compared with the uncomplicated pregnancies (12.7% versus 4.7%, OR[95%CI] = 3.1[1.6-5.8], P = 0.0003). In the Sinhalese cohort, maternal INSR rs2059806 AA genotype was greater among pre-eclamptic women who delivered small for gestational age infants compared with the uncomplicated pregnancies (10.8% versus 4.2%, OR[95%CI] = 2.8[1.0-7.4], P = 0.03). Thus, it was found that the INSR rs2059806 SNP is also associated with pre-eclampsia phenotypes in two independent cohorts suggesting that genetic susceptibility may be implicated in the link between pre-eclampsia and subsequent vascular and metabolic diseases.

Polymorphisms in the inflammatory pathway genes and the risk of preeclampsia in Sinhalese women.

OBJECTIVE: Elevated pro-inflammatory cytokines play an important role in the pathogenesis of preeclampsia. We investigated the prevalence of functional polymorphisms in genes regulating inflammation in preeclamptic women. METHODS: One hundred seventy-five nulliparous Sinhalese women with preeclampsia (cases) and 171 normotensive women matched for age, ethnicity, parity and body mass index (BMI) (controls) were recruited. Preeclampsia was diagnosed using international guidelines. Genotyping was performed on DNA extracted from peripheral blood using the Sequenom MassARRAY system. RESULTS: The prevalence of the CT genotype of IL1A rs17561 polymorphism was increased in preeclamptic women compared with controls {p = 0.04, odds ratio (OR) [95% class interval (CI)] = 1.6 (1.0-2.5)}. The prevalence of the CT genotype [p = 0.01, OR (95% CI) = 1.8 (1.1-2.8)] and the dominant model (CT + TT) [p = 0.03, OR (95% CI) = 1.6 (1.1-2.5)] of the IL1A rs1800587 polymorphism were increased in preeclamptic women compared with controls. The prevalence of the GA genotype [p = 0.04, OR (95% CI) = 0.6 (0.4-0.9)] and the dominant model (GA + AA) [p = 0.03, OR (95% CI) = 0.6 (0.4-0.9)] of the MBL1 rs1800450 polymorphism were reduced in preeclamptic women compared to controls. CONCLUSION: Genotypes conferring a pro-inflammatory phenotype are increased in preeclamptic women.

The obesity-related FTO gene variant associates with the risk of recurrent miscarriage.

OBJECTIVE: To investigate the association of the fat mass and obesity associated gene (FTO) rs9939609 single nucleotide polymorphism with recurrent miscarriage. DESIGN: Candidate gene association study. SETTING: Human Genetics Unit, Colombo, Sri Lanka. POPULATION: A total of 202 Sinhalese women with two or more first-trimester miscarriages and no living children (cases) and 202 age- and ethnicity-matched women with no history of miscarriage and having two or more living children (controls). METHODS: Peripheral blood was collected from the participants and DNA was extracted. Genotyping was performed at the Australian genome Research Facility using the Sequenom MassARRAY system. Genotype and allele frequencies of cases were compared with controls using chi-squared testing. MAIN OUTCOME MEASURES: The prevalence of the single nucleotide polymorphism in cases and controls. RESULTS: The mean age of the women in the recurrent miscarriage group was 31.9 ± 0.4 years and that of the control group was 32.3 ± 0.3 years. Of the women in the recurrent miscarriage group, 140 (69.3%) had experienced three or more first-trimester miscarriages. The prevalence of the AA genotype [p = 0.0002, odds ratio (95% CI) = 3.8 (1.8-8.0)] and A allele [p = 0.002, odds ratio (95% CI) = 1.6 (1.2-2.2)] of the FTO rs9939609 single nucleotide polymorphism were increased in women in the recurrent miscarriage group compared with the control group. CONCLUSION: The obesity-related FTO rs9939609 single nucleotide polymorphism associates with recurrent miscarriage. This finding warrants further investigation with controlling for important factors such as body mass index, diabetes and cardiovascular disease status. The single nucleotide polymorphism may be useful in predicting the risk of recurrent miscarriage.

Cytogenetic analysis of chromosomal abnormalities in Sri Lankan children.

BACKGROUND: Cytogenetic analysis is a valuable investigation in the diagnostic work up of children with suspected chromosomal disorders. The objective of this study was to describe the prevalence of various types of chromosomal abnormalities in Sri Lankan children undergoing cytogenetic analysis. METHODS: Cytogenetic reports of 1554 consecutive children with suspected chromosomal disorders who underwent karyotyping in two genetic centers in Sri Lanka from January 2006 to December 2011 were reviewed retrospectively. RESULTS: A total of 1548 children were successfully karyotyped. Abnormal karyotypes were found in 783 (50.6%) children. Numerical and structural abnormalities accounted for 90.8% and 9.2%, respectively. Down syndrome was the commonest aneuploidy identified. Other various autosomal and sex chromosomal aneuploidies as well as micro-deletion syndromes were also detected. CONCLUSIONS: The prevalence of chromosomal abnormalities in Sri Lankan children undergoing cytogenetic analysis for suspected chromosomal disorders was relatively higher than that in Caucasian and other Asian populations.

Microbiome of the placenta in pre-eclampsia supports the role of bacteria in the multifactorial cause of pre-eclampsia.

AIM: This study was aimed at detecting, identifying, quantifying and comparing the bacteria present in the placental tissues of women with pre-eclampsia with that of normotensive pregnant women. MATERIAL AND METHODS: Placental tissue samples were collected from 55 primiparous women with pre-eclampsia (cases) and 55 matched primiparous normotensive pregnant women (controls) at the time of delivery by cesarean section. Genotyping was carried out in two stages. First the samples were screened for the presence of bacteria by polymerase chain reaction (PCR) for the 16S rRNA gene. Next, the samples that were PCR-positive for the 16S rRNA gene were screened by next-generation sequencing on an Illumina MiSeq platform. RESULTS: Seven (12.7%) placental tissue samples from women with pre-eclampsia were PCR-positive. All the placental samples from control women were negative (P = 0.006). The complete microbiome of the seven samples was revealed through next-generation sequencing. The organisms that were present included Bacillus cereus, Listeria, Salmonella, Escherichia (all of which are usually associated with gastrointestinal infection); Klebsiella pneumonia and Anoxybacillus (both of which are usually associated with respiratory tract infections); and Variovorax, Prevotella, Porphyromonas, and Dialister (all of which are usually associated with periodontitis). CONCLUSIONS: This study confirms the presence of bacteria in the placental tissues of a subset of women with pre-eclampsia and supports the role of bacteria in the multifactorial cause of pre-eclampsia.

Polymorphisms in the fibrinolytic pathway genes and the risk of recurrent spontaneous abortion.

Impaired fibrinolytic activity is implicated in the pathogenesis of recurrent spontaneous abortion (RSA). This case-control study assessed the prevalence of polymorphisms in fibrinolytic system genes in RSA. Cases comprised 202 Sinhalese women who had experienced at least two first-trimester spontaneous abortions and had no living children; controls were 202 women with no history of spontaneous abortion and two or more living children. The groups were matched for age and ethnicity. DNA was genotyped using the Sequenom MassARRAY system. The PLAUR rs4251923 A (OR 95% CI 2.3 [1.3 to 4.0]), SERBP2 rs6098 A (OR 95% CI 1.4 [1.1 to 1.9]) and SERBP2 rs6103 C alleles (OR 95% CI 1.4 [1.1 to 1.9]) were increased in the RSA group compared with controls. The prevalence of PLAUR rs4251923/ SERBP2 rs6098/ SERBP2 rs6103 GG/AA/CC (OR 95% CI 2.4 [1.2 to 4.9], GA/GA/GC(OR 95% CI 3.9 [1.3 to 11.2]), GA/AA/CC (OR 95% CI 2.9 [1.0 to 8.6] and GA/GG/GG (OR 95% CI 21.3 [1.1 to 410.3]) genotypes were also increased in cases. Polymorphisms in the fibrinolytic system genes are associated with RSA in Sinhalese women. These likely impair implantation.

A child with a novel de novo mutation in the aristaless domain of the aristaless-related homeobox (ARX) gene presenting with ambiguous genitalia and psychomotor delay.

The objective of this study was to identify disease-causing mutations in a Sri Lankan male child presenting with ambiguous genitalia and psychomotor delay using the exome sequencing approach. A novel mutation in the aristaless-related homeobox (ARX) gene causing a hemizygous nucleotide substitution in exon 5 was identified (NM_139058.2 (ARX): c.1614G>T; p.K538N). This change causes a nonsynonymous substitution in the aristaless domain within the ARX protein which is predicted to be deleterious. This is the first reported case of ambiguous genitalia and psychomotor delay associated with this novel missense mutation within the ARX protein, and it highlights the value of exome sequencing even in sporadic cases.

Polymorphism in the epidermal growth factor gene is associated with pre-eclampsia and low birthweight.

AIM: We have previously reported that polymorphism in the epidermal growth factor (EGF) gene is associated with pre-eclampsia and birthweight based on case-control association studies involving two single nucleotide polymorphisms (SNP). We extended that work to investigate other SNP in the EGF gene for their association with pre-eclampsia and the weight of babies at birth. MATERIAL AND METHODS: A population-based DNA collection was genotyped to determine whether the selected SNP were polymorphic in the study population. In total, 175 women with pre-eclampsia and 171 matched normotensive controls were genotyped for the polymorphic SNP using polymerase chain reaction/restriction fragment length polymorphism and MassARRAY Sequenom iPLEX methodology. RESULTS: The rs3756261A, rs4444903G, rs2237051G haplotype was associated with the highest increased risk of pre-eclampsia (odds ratio: 3.70, 95% confidence interval: 1.38-9.94; P = 0.016). The rs3756261A allele was the one that contributed to this high degree of significance. The same allele was present in the haplotype rs3756261A, rs11568943G, rs2237051G, rs11569017A, rs4698803T (likelihood ratio statistic = 20.4671, d.f. = 3, P-value = 0.0001), which was associated with the lower birthweight. CONCLUSIONS: In this study we found further evidence for the association of polymorphism in the EGF gene with pre-eclampsia and the weight of babies at birth and identified rs3756261A>G as the SNP that makes the most significant contribution to this association. Bioinformatic analysis showed that this effect may be mediated by caudal type homeohox-2, a transcriptional repressor expressed in the trophoblast, for which a binding site is created at this polymorphic site when the rs3756261A allele is present.

Anatomical landmarks for safer carpal tunnel decompression: an experimental cadaveric study.

BACKGROUND: Carpal tunnel syndrome is a common presentation to surgical outpatient clinics. Treatment of carpal tunnel syndrome involves surgical division of the flexor retinaculum. Palmar and recurrent branches of the median nerve as well as the superficial palmar arch are at risk of damage. METHODOLOGY: Thirteen cadavers of Sri Lankan nationality were selected. Cadavers with deformed or damaged hands were excluded. All selected cadavers were preserved with the conventional arterial method using formalin as the main preservative. Both hands of the cadavers were placed in the anatomical position and dissected carefully. We took pre- determined measurements using a vernier caliper. We hypothesized that the structures at risk during carpal tunnel decompression such as recurrent branch of the median nerve and superficial palmar arch can be protected if simple anatomical landmarks are identified. We also hypothesized that an avascular area exists in the flexor retinaculum, identification of which facilitates safe dissection with minimal intra operative bleeding. Therefore we attempted to characterize the anatomical extent of such an avascular area as well as anatomical landmarks for a safer carpal tunnel decompression.Ethical clearance was obtained for the study. RESULTS: In a majority of specimens the recurrent branch was a single trunk (n =20, 76.9%). Similarly 84.6% (n = 22) were extra ligamentous in location. Mean distance from the distal border of the TCL to the recurrent branch was 7.75 mm. Mean distance from the distal border of TCL to the superficial palmar arch was 11.48 mm. Mean length of the flexor retinaculum, as measured along the incision, was 27.00 mm. Mean proximal and distal width of the avascular area on TCL was 11.10 mm and 7.09 mm respectively. CONCLUSION: We recommend incision along the radial border of the extended ring finger for carpal tunnel decompression. Extending the incision more than 8.16 mm proximally and 7.75 mm distally from the corresponding borders of the TCL should be avoided. Incision should be kept to a mean length of 27.0 mm, which corresponds to the length of TCL along the above axis. We also propose an avascular area along the TCL, identification of which minimizes blood loss.

Clinical and genetic features of Huntington disease in Sri Lanka.

BACKGROUND: Huntington disease was one of the first neurological hereditary diseases for which genetic testing was made possible as early as 1993. The study describes the clinical and genetic characteristics of patients with Huntington disease in Sri Lanka. METHODS: Data of 35 consecutive patients tested from 2007 to 2012 at the Human Genetics Unit, Faculty of Medicine, University of Colombo was analyzed retrospectively. Clinical data and genetic diagnostic results were reviewed. Statistical analysis was performed using descriptive statistics. RESULTS: Thirty patients had fully penetrant (FP) CAG repeat mutations and 5 had reduced penetrant (RP) CAG repeat mutations. In the FP group mean ages of onset and diagnosis were 37.5 and 40.4 years, while in the RP group it was 63.0 and 64.8 years respectively. The age of diagnosis ranged from 15 to 72 years, with 2 patients with Juvenile onset (<20 years) and 3 with late onset (>60 years) Huntington disease. The symptoms at diagnosis were predominantly motor (32/35 -91%). Three patients had psychiatric and behavioral disorders. The age difference between onset and genetic diagnosis showed significant delay in females compared to males (p < 0.05). Twenty two (62.8%) had a positive family history, with 13/22 (59.1%) showing a paternal inheritance of the disease. In both groups, those with a family history had a significantly lower age of presentation (p < 0.05). The mean CAG repeat length in patients with FP alleles was 44.6 ± 5 and RP alleles was 37.2 ± 1.1. Age of onset and CAG repeat length of the HTT gene showed significant inverse correlation (p < 0.0005, R2 = 0.727). CONCLUSIONS: The clinical and genetic features seen in patients with Huntington disease in the Sri Lankan study population were similar to that previously reported in literature.

Autosomal dominant hereditary ataxia in Sri Lanka.

BACKGROUND: Spinocerebellar ataxias (SCA) are a group of hereditary neurodegenerative disorders. Prevalence of SCA subtypes differ worldwide. Autosomal dominant ataxias are the commonest types of inherited ataxias seen in Sri Lanka. The aim of the study is to determine the genetic etiology of patients with autosomal dominant ataxia in Sri Lanka and to describe the clinical features of each genetic subtype. METHODS: Thirty four patients with autosomal dominant ataxia were recruited. For every patient the following was done: recording of clinical details and genotyping for SCA 1, 2, 3, 6, 7, 8, 12, and 17. RESULTS: Sixty one per cent of the subjects were identified as SCA1. One subject had SCA2, 12 remain unidentified. Mean age at onset was 34.8 ± 10years for SCA1 and 32.7 ± 9.8 for non SCA1. 76% of SCA1 patients and 50% of non SCA1 were using walking aids. Quantification of symptoms and signs were similar in the SCA1 and non SCA1 groups. Clinical depression was evidenced in 68.4% of SCA1 and 75% non SCA-1 patients. Mean CAG repeat length in SCA1 patients was 52.0 ± 3.8, with greater anticipation seen with paternal inheritance. CONCLUSION: SCA1 was the predominant subtype and showed similar phenotype to previous reports. However, disease severity was higher and depression more prevalent in this population than previously described.

Prevalence of chromosomal abnormalities in Sri Lankan women with primary amenorrhea.

AIM: Chromosomal abnormalities are implicated in the etiology of primary amenorrhea. The underlying chromosomal aberrations are varied and regional differences have been reported. The objective of this study is to describe the prevalence of various types of chromosomal abnormalities in Sri Lankan women with primary amenorrhea. MATERIAL AND METHODS: Medical records of all patients diagnosed with primary amenorrhea referred for cytogenetic analysis to two genetic centers in Sri Lanka from January 2005 to December 2011 were reviewed. Chromosome culture and karyotyping was performed on peripheral blood samples obtained from each patient. Data were analyzed using standard descriptive statistics. RESULTS: Altogether 338 patients with primary amenorrhea were karyotyped and mean age at testing was 20.5 years. Numerical and structural chromosomal abnormalities were noted in 115 (34.0%) patients which included 45,X Turner syndrome (10.7%), Turner syndrome variants (13.9%), XY females (6.5%), 45,X/46,XY (0.9%), 46,XX/46,XY (0.6%), 47,XXX (0.3%), 47,XX,+ mar (0.3%), 46,X,i(X)(p10) (0.3%), 46,XX with SRY gene translocation on X chromosome (0.3%) and 46,XX,inv(7)(p10;q11.2) (0.3%). Short stature, absent secondary sexual characteristics, neck webbing, cubitus valgus and broad chest with widely spaced nipples were commonly seen in patients with Turner syndrome and variant forms. Neck webbing and absent secondary sexual characteristics were significantly associated with classical Turner syndrome than variant forms. CONCLUSION: A considerable proportion of women with primary amenorrhea had chromosomal abnormalities. Mean age at testing was late suggesting delay in referral for karyotyping. Early referral for cytogenetic evaluation is recommended for the identification of underlying chromosomal aberrations in women with primary amenorrhea.

Vascular endothelial growth factor family gene polymorphisms in preeclampsia in Sinhalese women in Sri-Lanka.

OBJECTIVE: To investigate the association of polymorphisms in the vascular endothelial growth factor (VEGF) family genes (VEGFA rs699947, VEGFA rs3025039, PGF rs1042886, KDR rs2071559 and KDR rs2305948) with preeclampsia in Sinhalese women in Sri-Lanka. METHODS: We conducted a case-control study where 175 nulliparous Sinhalese women with preeclampsia and 171 normotensive women matched for age, ethnicity, parity and BMI were recruited in tertiary care maternity hospitals in Sri-Lanka. Preeclampsia was diagnosed using international guidelines. DNA extracted from peripheral venous blood and was genotyped using the Sequenom MassARRAY system. χ(2)-test was used to compare the distribution of allele and genotype frequencies between the cases and the control subjects. RESULTS: The frequency of PGF rs1042886 variant allele (odds ratio (OR) 1.5, 95% confidence interval (CI) 1.1-2.1) and dominant genotype model (aOR 1.6, 95% CI 1.0-2.4) were increased in preeclamptic women compared to controls. VEGFA rs699947, VEGFA rs3025039, KDR rs2071559, and KDR rs2305948 polymorphisms were not associated with preeclampsia. CONCLUSION: Maternal PGF rs1042886 polymorphism is associated with preeclampsia in Sinhalese women in Sri-Lanka.

An infant born to a mother with gestational diabetes presenting with 49,XXXXY syndrome and renal agenesis-a case report.

49,XXXXY is a rare sex chromosome polysomy with an incidence of 1 in 85 000 male births. It has a characteristic triad of mental retardation, skeletal malformation and hypogonadism. This is the first case report of a child with 49,XXXXY syndrome and renal agenesis. This child was referred for genetic testing at 14 years of age due to facial dysmorphism and hypergonadotropic hypogonadism. He had coarse facial features, cryptorchidism of the right testis, genu valgus deformities, and patent ductus arteriosus which are known associations of 49,XXXXY syndrome. He also had agenesis of the right kidney, hydronephrosis of the left kidney with hydroureter which is not a known association of 49,XXXXY syndrome. The patient was the offspring of a mother with gestational diabetes. There is a strong correlation between maternal diabetes and congenital anomalies, especially renal and cardiovascular anomalies. Additionally, it has been noted that gestational diabetes increases the incidence of chromosomal aneuploidies. The teratogenic effects of maternal diabetes during embryogenesis may be the causative factor for the final phenotype of 49,XXXXY syndrome and renal agenesis.

A child with mosaicism for deletion (14)(q11.2q13).

In this case report we describe a child with a de novo deletion in the (q11.2q13) region of chromosome 14. The child presented with dysmorphic features - anophthalmia, microcephaly, and growth retardation. Cytogenetic studies showed mosaicism. The karyotype was 46,XX,del(14)(q11.2;q13) [16] /46,XX [9]. We compared the features observed in this child with that of others with the same deletion reported in scientific literature and found that this is the first report of a child mosaic for this deletion. It is also the first time it has been reported in association with anophthalmia.