Institute of Genetics and Hospital for Genetic Diseases

Recent genome-wide association studies (GWAS) have revealed genetic risk factors in autoimmune and inflammatory disorders. Several of the associated genes and underlying pathways are shared by various autoimmune diseases. Rheumatoid arthritis (RA) and coeliac disease (CD) are two autoimmune disorders which have commonalities in their pathogenesis. We aimed to replicate known RA loci in a Dutch RA population, and to investigate whether the effect of known RA and CD risk factors generalize across the two diseases. We selected all loci associated to either RA or CD in a GWAS and confirmed in an independent cohort, with a combined P-value cut-off P < 5 x 10(-6). We genotyped 11 RA and 11 CD loci in 1368 RA patients, 795 CD patients and 1683 Dutch controls. We combined our results in a meta-analysis with UK GWAS on RA (1860 cases; 2938 controls) and CD (767 cases; 1422 controls). In the Dutch RA cohort, the PTPN22 and IL2/IL21 variants showed convincing association (P = 3.4 x 10(-12) and P = 2.8 x 10(-4), respectively). Association of RA with the known CD risk variant in the SH2B3 was also observed, predominantly in the subgroup of rheumatoid factor-positive RA patients (P = 0.0055). In a meta-analysis of Dutch and UK data sets, shared association with six loci (TNFAIP3, IL2/IL21, SH2B3, LPP, MMEL1/TNFRSF14 and PFKFB3/PRKCQ) was observed in both RA and CD cohorts. We confirmed two known loci and identified four novel ones for shared CD-RA genetic risk. Most of the shared loci further emphasize a role for adaptive and innate immunity in these diseases.

BACKGROUND: Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s) from Nigella sativa with anti cancer activity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell. RESULTS: Methanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 microg/ml, 2.20 microg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL) assay. CONCLUSION: Western Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

Background: ) who are receiving medical follow-up, including colonoscopy surveillance, which aims to the achieve early diagnosis and treatment of cancers. Here we use the most recent PLSD cohort that is larger and has wider geographical representation than previous versions, allowing us to present mortality as an outcome, and median ages at cancer diagnoses for the first time. Methods: variants from 25 countries were included, providing 71,713 years of follow up. Cumulative cancer incidences at 65 years of age were combined with 10-year crude survival following cancer, to derive estimates of mortality up to 75 years of age by organ, gene, and gender. Findings: carriers, more deaths followed non-colorectal Lynch syndrome cancers than colorectal cancers. Interpretation: carriers undergoing colonoscopy surveillance, non-colorectal Lynch syndrome cancers were associated with more deaths than were colorectal cancers. Reducing deaths from non-colorectal cancers presents a key challenge in contemporary medical care in Lynch syndrome. Funding: We acknowledge funding from the Norwegian Cancer Society, contract 194751-2017.

Abstract De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF &lt; 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes ( p -value = 1.00 × 10 −5 ) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes ( p -value = 5.01 × 10 −4 ) in contrast to predicted benign de novo mutations. One gene we identify, RBM5 , is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men ( p -value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

A 21-year-old female with trisomy 21 Down's syndrome delivered a normal male child with 46, XY chromosome compliment. The case is described as a first report from India.

INTRODUCTION: Coronary artery disease is caused by the additive and interactive effects of inherited and environmental factors. Substantial evidence shows that reactive oxygen species (ROS) play a vital role in the aetiopathogenesis of atherosclerosis. Our study has been designed to evaluate the oxidative stress due to ROS and assess the antioxidant protection against ROS, in addition to the major risk factors, like lipid profiles, habit of smoking and conditions such as diabetes mellitus and hypertension, in myocardial infarction (MI) patients. METHODS: World Health Organisation criteria were followed in the selection of the subjects. 150 patients with MI were included in the study along with equal number of age- and gender-matched controls. Malondialdehyde (MDA) and nitrite/nitrate levels were measured as markers of oxidative stress of free radical induced injury, and total antioxidant status was determined to assess the antioxidant protection against ROS, along with the lipid profiles. RESULTS: The levels of total cholesterol, low density lipoprotein cholesterol, triglycerides, MDA and nitrite/nitrate were found to be significantly high, while high density lipoprotein cholesterol and total antioxidant capacity were significantly low in MI patients compared to controls. CONCLUSION: Our study revealed the importance of determining the total antioxidant status in MI, in addition to the markers of oxidative stress and lipid profiles to enable the formulation of specific antioxidant therapies for an early intervention and better management of the disease. The study also suggests initiating lifestyle modifications as a preventive measure to reduce the burden of the disease.

STUDY QUESTION: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders? SUMMARY ANSWER: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 10 out of 21 patients (48%) and variants in novel candidate genes in seven additional patients (33%). WHAT IS KNOWN ALREADY: Severe sperm motility disorders are a form of male infertility characterised by immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum that do not affect viability. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 22 genes. STUDY DESIGN, SIZE, DURATION: We performed exome sequencing in 21 patients with severe sperm motility disorders from two different clinics. PARTICIPANTS/MATERIALS, SETTING, METHOD: Two groups of infertile men, one from Argentina (n = 9) and one from Australia (n = 12), with clinically defined severe sperm motility disorders (motility <5%) and normal morphology values of 0-4%, were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on light and transmission electron microscopy. Sperm ultrastructural information was not available for the Australian cohort. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted. MAIN RESULTS AND ROLE OF CHANCE: In 10 of 21 patients (48%), we identified pathogenic variants in known sperm assembly genes: CFAP43 (3 patients); CFAP44 (2 patients), CFAP58 (1 patient), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (55% and 42%, respectively). Furthermore, we identified patients with variants in the novel human candidate sperm motility genes: DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we described patients with either a homozygous or two heterozygous candidate pathogenic variants in genes linked to sperm motility disorders. Due to unavailability of parental DNA, we have not assessed the frequency of de novo or maternally inherited dominant variants and could not determine the parental origin of the mutations to establish in all cases that the mutations are present on both alleles. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirm the likely causal role of variants in six known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility disorders (10/21 diagnosed; 48%). Furthermore, our analysis revealed six novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes. STUDY FUNDING/COMPETING INTEREST(S): This project was supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., J.A.V. and R.I.M.L., The Netherlands Organisation for Scientific Research (918-15-667) to J.A.V., the Royal Society and Wolfson Foundation (WM160091) to J.A.V., as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. and Grants from the National Research Council of Argentina (PIP 0900 and 4584) and ANPCyT (PICT 9591) to H.E.C. and a UUKi Rutherford Fund Fellowship awarded to B.J.H.

Pathogenic mitochondrial DNA mutations are most often implicated in inherited and acquired hearing impairment. The current review mainly focuses on the 12S rRNA mitochondrial gene mutations associated with non-syndromic deafness without or after aminoglycosides exposure. Aminoglycoside-induced and nonsyndromic deafness has been shown to have a genetic susceptibility and the pathogenic mitochondrial 12S rRNA A1555G mutation was identified as the primary factor underlying the hearing loss in many familial as well as in genetically unrelated cases, particularly in Asian populations where aminoglycoside antibiotics are commonly used even for minor infections. Many families were shown to transmit the aminoglycoside ototoxicity through matrilineal inheritance and the A1555G mutation in the 12S rRNA gene was frequently identified. The aminoglycoside antibiotics are believed to target the mitochondrial ribosome in the cochlea resulting in abnormal RNA processing or decreased efficiency of translation thereby leading to irreversible auditory dysfunction. Such cases may have a genetic predisposition to aminoglycoside ototoxicity following autosomal dominant, autosomal recessive, X-linked, or mitochondrial pattern of inheritance.

AIM: Acute coronary syndrome (ACS) is an inflammatory disease. Cytokines are the central regulators of inflammation and may be a cause or marker of atherosclerosis. Accumulating evidence suggests that polymorphisms at promoter regions of various cytokine genes are known to be associated with their expression levels. In the present study we investigated whether variants at -1082G→A (rs1800896) and -592C→A (rs1800872) of interleukin-10 (IL-10), -1188A→C (rs3212227) of IL-12 p40, -308G→A of tumor necrosis factor-α (TNF-α) (rs1800629), -174G→C of IL-6 (rs1800795) and +874A→T of interferon-γ (IFN-γ) genes (rs2430561) are associated with ACS. MATERIALS AND METHODS: DNA samples were collected from 1083 subjects and IL-10-1082G→A, -592A→C, TNF-α-308G→A, IL-12 p40-1188 A→C, and IFN-γ+874A→T polymorphisms were identified by amplified refractory mutation system polymerase chain reaction and IL-6-174 G/C, restriction fragment length polymorphism based on standard methods. RESULTS: Six hundred and fifty one ACS patients along with 432 age and sex matched controls were analyzed for various gene polymorphisms. The "low-producer" IL-10-1082 AA (χ(2)=9.45; p=0.0021; odds ratio [OR]=1.472; 95% confidence interval [CI]=1.15-1.884), "high producer" IL-10-592 CC (χ(2)=39.42; p=0.001, OR=2.26; 95% CI=1.748-2.292), "low producer"IFN-γ+874AA (χ(2)=28; p<0.00154; OR=2.36 & 95% CI=1.713-3.251), and "high producer" TNF-α -308AA (χ(2)=3.213, p=0.073; OR=1.515) genotypes may be responsible for the regulation of immune response leading to inflammation in ACS patients. However, -1188 of the IL-12 gene was not associated with the disease. CONCLUSION: The polymorphisms at -308G→A of TNF-α, -174G→C of IL-6, +874A→T of IFN-γ and -1082G→A, and -592C→A of IL-10 genes evaluated in the present study are important risk factors for the development of ACS in the South Indian population from Andhra Pradesh. The better understanding of these variants conferring susceptibility to ACS may aid in early diagnosis and development of new methods to create personalized medicine.

1. Twenty-five male workers occupationally exposed to DDT, BHC malathion, parathion, dimethoate, fenitrothion, urea and gromor were selected as subjects for the analysis of chromosomal aberrations and sister chromatid exchanges (SCE) in peripheral lymphocytes. 2. Blood samples were collected from 30 normal healthy males from the same age group and socioeconomic class for the control. 3. The frequency of chromosomal aberrations and SCEs increased significantly irrespective of the duration of exposure to pesticides, when compared to controls.

AIM: To determine the frequency of hypothyroidism in women with recurrent pregnancy loss in first trimester in the Indian population. SETTINGS AND DESIGN: The study included 163 non-pregnant women with recurrent pregnancy loss in a gestational age up to <or=12 weeks verified by a pregnancy test or ultrasonography, and a total of 170 age matched women with at least one successful pregnancy and no history of miscarriages were selected as controls. METHODS: Levels of thyroid hormones T3, T4 and TSH were estimated in non-pregnant women with RPL and controls. RESULTS: Hypothyroidism was found in seven (4.12%) women with RPL and one in control group. The differences in the levels of serum T3, T4 and TSH between euthyroid and hypothyroid women were found significant in women with RPL in first trimester. STATISTICAL ANALYSIS: The statistical analyses were performed with the use of student's two-tailed t-test. CONCLUSION: The study demonstrates that hypothyroidism has a statistically significant relationship with recurrent pregnancy loss in the first trimester and suggests that diagnosis of hypothyroidism could help couples with recurrent pregnancy loss to have a successful outcome in subsequent pregnancies.

Hyperhomocysteinaemia, a risk factor for recurrent pregnancy loss, is related either to a hereditary defect within the methionine-homocysteine pathway or it might be acquired as a result of deficiencies of vitamin B(12) and folate (B(9)). Because hyperhomocysteinaemia seems to be determined by both genetic and environmental factors, the current study was undertaken to find out the interactions between folate status and MTHFR mutation on the homocysteine concentration in 24 women experiencing unexplained three or more consecutive recurrent pregnancy losses. The median fasting total plasma homocysteine concentration in the study group was 10.23 micro mol/l compared to 8.95 micro mol/l; P = 0.096 in the controls. Elevated homocysteine levels > 18 micro mol/l, which was considered to be a risk factor for recurrent early pregnancy loss, was found in four women in the study group and none among the controls. Lower red cell folate levels (normal range >/= 160 ng/ml) were observed in nine (37.5%) women among the study group, compared to five (20.84%) women among controls. The mean +/- SD red cell folate levels in the study group was found to be 154.37 +/- 37.07, while in the controls it was 159.0 +/- 28.97. In the present study six women in the study group and two among controls were found to be carriers for the C677T MTHFR mutation. None were homozygous for the mutant (TT) allele. The highest values of homocysteine concentration were found in women experiencing recurrent pregnancy loss with both the CT genotype and folate deficiency. Identification of hyperhomocysteinaemia in women with recurrent pregnancy loss may help in therapeutic normalisation and might permit a normal birth.

In the present study 61 male pesticide applicators who worked in cotton fields and regularly sprayed pesticides such as DDT, BHC, endosulfan, malathion, methyl parathion, phosphamidon, dimethoate, monocrotophos, quinalphos fenvelrate, and cypermethrin were analyzed for sister chromatid exchanges, mitotic index, and cell cycle kinetics in peripheral lymphocytes. Subjects who handled pesticides were non-smokers and teetotalers and the data were compared with the matched control group. Statistical analysis revealed that the frequency of sister chromatid exchanges was significantly higher among the pesticide applicators at all the durations of exposure when compared to controls. Subjects exposed to pesticides also showed cell cycle delay and decrease in mitotic index when compared to the control group.

Pentylenetetrazol (PTZ)-induced oxidative stress results in disturbance of the antioxidant enzyme status accompanied by neuronal injury and the development of epilepsy in rats. The present study evaluated the antioxidant effects of curcumin against PTZ-induced convulsions. Over a period of 30 days, i.p. injections of subconvulsive doses of PTZ on alternate days resulted in the development of a well-known kindling model of epilepsy. Spectrophotometric analysis revealed a markedly elevated activity of the antioxidant enzymes malondialdehyde (MDA), catalase and glutathione S-transferase (GST) in the cerebrum and cerebellum of epileptic rats due to PTZ-induced oxidative stress. Oral supplementation of curcumin at a dose of 2 g/kg for 30 days resulted in a transient decrease in MDA, catalase and GST levels in the rat cerebrum and cerebellum. Piperine (20 mg/kg orally) was administered along with curcumin to enhance the bioavailability of the latter up to 20-fold more. Combined treatment with curcumin and carbamazepine (3.6 mg/kg orally) also gave similar results, indicating that the potent antioxidant curcumin can be used as an adjuvant in antiepileptic therapy.

OBJECTIVE: To compare colorectal cancer (CRC) incidences in carriers of pathogenic variants of the MMR genes in the PLSD and IMRC cohorts, of which only the former included mandatory colonoscopy surveillance for all participants. METHODS: CRC incidences were calculated in an intervention group comprising a cohort of confirmed carriers of pathogenic or likely pathogenic variants in mismatch repair genes (path_MMR) followed prospectively by the Prospective Lynch Syndrome Database (PLSD). All had colonoscopy surveillance, with polypectomy when polyps were identified. Comparison was made with a retrospective cohort reported by the International Mismatch Repair Consortium (IMRC). This comprised confirmed and inferred path_MMR carriers who were first- or second-degree relatives of Lynch syndrome probands. RESULTS: In the PLSD, 8,153 subjects had follow-up colonoscopy surveillance for a total of 67,604 years and 578 carriers had CRC diagnosed. Average cumulative incidences of CRC in path_MLH1 carriers at 70 years of age were 52% in males and 41% in females; for path_MSH2 50% and 39%; for path_MSH6 13% and 17% and for path_PMS2 11% and 8%. In contrast, in the IMRC cohort, corresponding cumulative incidences were 40% and 27%; 34% and 23%; 16% and 8% and 7% and 6%. Comparing just the European carriers in the two series gave similar findings. Numbers in the PLSD series did not allow comparisons of carriers from other continents separately. Cumulative incidences at 25 years were < 1% in all retrospective groups. CONCLUSIONS: Prospectively observed CRC incidences (PLSD) in path_MLH1 and path_MSH2 carriers undergoing colonoscopy surveillance and polypectomy were higher than in the retrospective (IMRC) series, and were not reduced in path_MSH6 carriers. These findings were the opposite to those expected. CRC point incidence before 50 years of age was reduced in path_PMS2 carriers subjected to colonoscopy, but not significantly so.

OBJECTIVE: The present investigation was taken up to evaluate the 8-oxo-7,8-dihydro-2'-deoxyguanosine and malondialdehyde as markers of oxidative stress, the levels of antioxidants and the correlations between these oxidative stress markers and antioxidants in lung cancer patients. METHODS: The study included 222 patients (158 men and 64 women, age ranging from 32 to 85 years) and 207 control subjects (153 men and 54 women, aged 30-80 years) for the analysis of urinary excretion of 8-oxodG using an ELISA assay, plasma malondialdehyde using spectrophotometer and red cell Cu-Zn SOD and GPx activities by kit methods. RESULTS: The levels of 8-oxodG and malondialdehyde were significantly higher (p < 0.001) and red cell superoxide dismutase and glutathione peroxidase activities (p < 0.001) were significantly lower in lung cancer patients than in controls. There was a significantly positive correlation between 8-oxodG and malondialdehyde (r=0.912, p < 0.001) and a negative correlation between 8-oxodG and antioxidants. CONCLUSIONS: Our results demonstrate that an increased rate of oxidative stress might play a role in the pathogenesis of lung cancer as evidenced by a failure in the oxidant/antioxidant balance in favour of lipid peroxidation and DNA damage.

A class of small, non-coding transcripts called microRNAs (miRNAs) that play a major role in post-transcriptional gene regulation has recently emerged and become the focus of intense research. MicroRNAs are abundant in the nervous system, where they have key roles in development and are likely to be important mediators of plasticity. A highly conserved pathway of miRNA biogenesis is closely linked to the transport and translatability of mRNAs in neurons. MicroRNAs have been shown to modulate programmed cell death during development. Although there are nearly 750 known human miRNA sequences, each of only approximately 20-25 nucleotides in length that bind to multiple mRNA targets, the accurate prediction of miRNA targets seems to lie just beyond our grasp. Nevertheless, the identification of such targets promises to provide new insights into many facets of neuronal function. In this review, we briefly describe miRNA biogenesis and the principle approaches for studying the function of miRNAs and potential application of miRNAs as biomarkers, diagnostic targets, and potential therapeutic tools of human diseases in general and neurological disorders in particular.

The pathogenesis of a number of diseases like cardiovascular diseases, cancer and neurological disorders, has been associated with changes in the balance of certain trace elements. In this study we aimed at investigating the levels of trace elements like calcium, copper, iron and zinc, in ischemic stroke patients in comparison with healthy controls. Serum samples were collected from 256 ischemic stroke patients and 180 healthy, age and sex matched controls. Trace element levels were detected using commercially available kits and an Auto-Analyzer (ChemWell 2910, Awareness Technology, US). The concentrations of calcium, copper and iron were not significantly different in patients when compared to healthy controls. The concentration of zinc was significantly lower in stroke patients (P = 0.001) as compared to normal subjects. To conclude, patients with acute ischemic stroke have reduced levels of serum zinc. Zinc may represent an independent risk factor for stroke and therefore a possible target for prevention. Additional studies are needed to further examine the role of zinc in the pathogenesis of stroke.

The aim of this study was to examine whether an association exists between glutathione S-transferase Mu-1 (GSTM1) gene polymorphism and idiopathic male infertility. Forty-two men with infertility and 43 fertile men were recruited for this study. GSTM1 gene was analysed using PCR technique. The frequency of GSTM1 null (-) genotype was observed to be 45.2% in infertile men as against 20.09% in fertile men. Subjects with the GSTM1 null genotype had lower sperm concentrations and motility when compared with the subjects with GSTM1-positive genotype in both the groups. This study shows that the frequency of GSTM1 null (-) genotype is significantly high in infertile males when compared with the frequency in fertile males (OR = 0.32, P = 0.017, 95% CI = 0.124-0.831).

Stroke is a complex disease comprising of a heterogenous group of disorders with multiple risk factors. Genetic predisposition to stroke does occur and has been documented in both animal models and human beings. However, a precise definition of genetic factors responsible for stroke is still lacking because research into the genetic basis of stroke presents some unique challenges. More commonly it seems to be a multifactorial polygenic disorder. Mutations in some candidate genes are likely to predispose or give protection against stroke. Several mutations in various genes have been found to be associated with stroke. However, we have a long way to go before we can accurately pinpoint the genes responsible for multifactorial stroke. Recently, the deCODE group has suggested an association between the phosphodiesterase 4D (PDE4D) gene and the risk of stroke in Icelanders. PDE4D is the first putative gene associated with common polygenic stroke. Specific variants of this gene have been shown to present risk for ischemic stroke in Icelanders. Replication studies in non-Icelanders have yielded variable results. There may be obvious racial differences in the prevalence of these mutations but still many questions remain unsolved regarding the role of PDE4D in stroke development.