Heterozygous Form Research Articles

Profiling Fanconi Anemia Gene Mutations among Iranian Patients.

Fanconi anemia (FA) is a rare genetic syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. FA is usually inherited as an autosomal recessive condition. This disease is genetically heterogeneous and mutations in 16 different genes have been identified in FA patients to date. An accurate diagnosis needs detection of pathogenic variations in the FA genes along with positive results from chromosome breakage test. In this study, 48 families with at least 2 affected FA patients and positive chromosome breakage test were enrolled from the Iranian population. Molecular analysis of FA genes was performed using Next Generation Sequencing (NGS) method and Multiple Ligation Dependent Probe Amplification (MLPA). Causal mutations for 30 (63%) patients were identified in homozygous or compound heterozygous forms. FANCA had the highest mutation frequency rate (83%) followed by FANCG (10%), FANCD2 (3%) and FANCL (3%). A significant proportion (44%) of FANCA mutations were large rearrangements. Genetic testing for FA patients improves the accuracy of diagnosis and also will be essential for genetic counselling and prenatal diagnosis for future pregnancies in the family. Availability of NGS technology has made the screening of all known FA genes at once more practical and affordable.

An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model.

A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.

Impact of the A2V Mutation on the Heterozygous and Homozygous Aβ1-40 Dimer Structures from Atomistic Simulations.

The A2V mutation was reported to protect from Alzheimer's disease in its heterozygous form and cause an early Alzheimer's disease type dementia in its homozygous form. Experiments showed that the aggregation rate follows the order A2V > WT (wild-type) > A2V-WT. To understand the impact of this mutation, we carried out replica exchange molecular dynamics simulations of Aβ1-40 WT-A2V and A2V-A2V dimers and compared to the WT dimer. Our atomistic simulations reveal that the mean secondary structure remains constant, but there are substantial differences in the intramolecular and intermolecular conformations upon single and double A2V mutation. Upon single mutation, the intrinsic disorder is reduced, the intermolecular potential energies are reduced, the population of intramolecular three-stranded β-sheets is increased, and the number of all α dimer topologies is decreased. Taken together, these results offer an explanation for the reduced aggregation rate of the Aβ1-40 A2V-WT peptides and the protective effect of A2V in heterozygotes.

The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran.

Background:β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran.Methods:Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed.Results:The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels.Conclusions:This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations.

Computational simulations of the effects of the G229D KCNQ1 mutation on human atrial fibrillation.

Atrial fibrillation (AF) is related to mutations at the genetic level. This includes mutations in genes that encode KCNQ1, a subunit of the I Ks channel. Here, we investigate the mechanism of gain-of-function in I Ks towards the occurrence of AF. We used the Courtemanche-Ramirez-Nattel (CRN) human atrial cell model (Am J Physiol Heart Circ Physiol 275:H301-H321, 1998) and applied the modification proposed by Hasegawa et al. (Heart Rhythm 11:67-75, 2014) to fit the behavior of I Ks due to the G229D mutation in KCNQ1 under a heterozygous mutant form. This was incorporated into two-(2D) and three-dimensional (3D) tissue models, where the mutation sustained a reentrant wave. However, under the wild-type condition, the reentrant wave terminated before the end of our simulations (in 2D, the spiral wave terminated before 10s, while in 3D, the spiral wave terminated before 13s). Sustained reentry under the mutation conditions also resulted in a spiral wave breakup in the 3D model, which was sustained until the end of the simulation (20s), indicating AF.

Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in field populations.

Paraoxnase1 Gene Polymorphism in Childhood Idiopathic Nephrotic Syndrome

Background: Paraoxonase1 (PON1) is a serum enzyme bound to high-density lipoproteins with antioxidant properties. Molecular studies of PON1 revealed 2 polymorphic sites at amino acids 55 and 192 resulting in 2 different allozymes, the L and M-genotype at residue 55 and A and B at site 192, respectively. We have studied the association between PON1 gene polymorphisms and the minimal change nephrotic syndrome/focal segmental glomerulosclerosis (MCNS/FSGS) types of idiopathic nephrotic syndrome (INS) in Kuwaiti Arab children. Methods: The PON1 gene, 55 and 192 polymorphisms were analyzed in 50 children with INS (32 MSCN, 18 FSGS) and compared to 50 controls. Serum creatinine, albumin and lipids were measured in all subjects. Results: The LL genotype was detected in 50% of the INS patients compared to 48% of controls (p = 0.84). The heterozygous LM genotype was detected in 42% of INS patients compared 36% of controls (p = 0.68). The MM-genotype was detected in 8% of INS patients and 16% of controls (p = 0.35). The L-allele frequency in its homozygous and heterozygous forms was found in 71% of INS patients compared to 66% controls (p = 0.54). The L-allele frequency (LM and LL) was significantly higher in FSGS compared to MCNS patients (p = 0.0001) and when compared to controls (p = 0.0007). All patients and controls had the AA form of the 192 PON1 gene polymorphism. Conclusion: Our data demonstrate a strong association between the L-allele of PON1 gene 55 polymorphism with FSGS in Kuwaiti Arab children with INS. PON1 genotyping can help in the early prediction of FSGS, which might guide clinicians to a better therapeutic approach.

α-Actinin-4 Gene Mutations, Steroid Responsiveness and FSGS in Adult Onset-Nephrotic Syndrome

Focal segmental glomerulosclerosis (FSGS) is a common pattern of injury seen in primary as well as secondary renal disorders and is a major cause of steroid- resistant nephrotic syndrome (SRNS) and end-stage kidney disease (ESKD). SRNS is defined by resistance to standard steroid therapy and it remains one of the most intractable causes of kidney failure. Mutations in α-actinin-4 gene (ACTN4) represent a frequent cause of SRNS associated with an adult onset form of FSGS. We screened a cohort of 374 subjects from the South Indian population for the presence of two missense mutations, K255E and S262P in exon 8 of ACTN4 gene encoding α-actinin-4, an actinfilament crosslinking and bundling protein. Our results revealed that these two mutations were seen only among the patients (4%) in heterozygous form and absent in the controls suggesting that, these mutations were found to be disease causing in nature and the regulation of the actin cytoskeleton of glomerular podocytes may be altered in the patients. Further categorization revealed that K255E and S262P mutations were together responsible for 5% of SRNS and 15% of FSGS cases in our study group. Conversely, these mutations were not found in the controls as well as in the patients with steroid–sensitive nephrotic syndrome (SSNS). This is the first report from India, more studies are warranted to establish the frequency of ACTN4 mutations in our population and such analysis may help in developing mutation(s) specific therapeutic interventions in future.

The role of proprotein convertase subtilisin/kexin type 9 inhibitors in managing cardiovascular risk

Hypercholesterolaemia and dyslipidaemia, marked by decreased levels of high-density lipoprotein and elevated levels of lowdensity lipoprotein (LDL), increase the risk of cardiovascular disease. Familial hypercholesterolaemia (FH), diagnosed based on the clinical features seen in patients with a positive family history, constitutes a heritable disorder involving a single gene. FH can exist in either the heterozygous or homozygous form, and may be differentiated based on clinical features and genetic studies. A novel drug target, proprotein convertase subtilisin/kexin type 9 (PCSK9), has resulted in the development and subsequent approval of new, targeted monoclonal antibodies in the treatment of FH. Targeting PCSK9 with monoclonal antibodies, i.e. evolocumab and alirocumab, inhibits the degradation of LDL receptors, and against a background of optimised statin therapy, increases the life expectancy of patients with hypercholesterolaemia by reducing the incidence and severity of coronary artery disease.

Moyamoya Disease: A Rare Sickle Cell Trait Neurological Complication

Introduction: Sickle cell disease is an inherited blood disorder with multisystem complications, including cerebrovascular accidents primarily due to micro infarcts. However, people with sickle cell trait, the heterozygous form of sickle cell disease, usually lead a normal, healthy life. Very rarely, severe tissue hypoxia, acidosis, dehydration, and hypothermia in sickle cell trait increase red blood cell polymerization and sickling, which could lead to micro infarcts in blood vessels. Moyamoya disease is a rare neurovascular complication caused by repeated strokes after arterial cerebral infarction. It commonly manifests as ischemic stroke in young patients, but mainly as intracranial hemorrhage in adults. Case presentation: A 14-year-old girl with sickle cell trait, who had been living a normal and healthy life, started to complain of severe persisting headache, migraine, drowsiness, and a tendency to sleep. Brain magnetic resonance imaging and angiography revealed moyamoya disease. Hematological treatment with hydroxyurea, aspirin, and an NSAID was commenced for this patient.

A novel mutation on the transferrin gene abolishes one N-glycosylation site and alters the pattern of transferrin isoforms, mimicking that observed after excessive alcohol consumption

ObjectivesIn the process of obtaining a driver's license, a healthy 28year old man presented increased levels of disialo-transferrin (TF) (approx. 20%, ref. value<2) by HPLC analysis of TF isoforms (%CDT), while other markers of excessive alcohol consumption (PEth, MCV and γ-GT) were in the normal range. The objective of this study was to determine the cause of the increased %CDT levels. Design and methodsSerum TF isoforms were re-analyzed by LC–MS. All coding exons of the TF gene were Sanger sequenced. ResultsAnalysis of TF isoforms by LC–MS confirmed the presence of increased disialo-TF and revealed a discrepancy in the mass difference between disialo-TF and tetrasialo-TF which suggested the presence of a genetic TF isoform with one abolished N-glycosylation site. Sanger sequencing of the TF gene revealed the presence of two missense mutations in heterozygous form: c.1295A>G (p.N432S) and c.1765C>T (p.P589S). p.N432S is a novel mutation that abolishes one N-glycosylation site of TF, while p.P589S is the polymorphism that defines the C2 isoform of TF. The sum of mass shifts caused by both amino acid substitutions agrees with the mass shift observed by LC–MS, which indicates that both variants are located in cis. ConclusionsAn individual initially suspected of alcohol abuse based on elevated %CDT was shown to be carrier of a novel mutation in the TF gene that abolishes the N-glycosylation site at position p.N432. The presence of this genetic variant has to be kept in mind when interpreting TF isoform patterns.

The Effect of Proteasome Subunit Beta Type 1 P11A Polymorphism on the Survival of Multiple Myeloma Patients Treated with First Line Bortezomib Based Chemotherapy

Background: Proteasome inhibitors have a fundamental role in the treatment of multiple myeloma however there are still patients who fail to achieve a good response and have a dismal prognosis. Markers predicting the outcome of individual patients are needed to identify those who would benefit more from a different approach. Proteasome subunit beta type 1 (PSMB1) rs12717 single nucleotide polymorphism (C/G substitution resulting in a P11A change) was reported recently to be associated with greater clinical benefit in relapsed follicular lymphoma patients treated with bortezomib containing combination (Coiffier et al Clin Cancer Res 2013). To the best of our knowledge there is only one report with limited number of myeloma patients showing no association between PSMB1 P11A genotype and survival (Lichter et al Blood 2012).Aims and Methods: We analyzed the association of PSMB1 P11A polymorphism and treatment outcome of 220 consecutive myeloma patients having had first line chemotherapy in our unit. PSMB1 P11A polymorphism was tested using LightCycler melting analysis. Statistical analyses were performed using the SPSS (version 20.0) software package.Results: The distribution of presentation prognostic markers such as age, ISS and FISH were even among the three genotype groups. 149 patients had bortezomib-based chemotherapy, mainly VTD (n=76) and MPV (n=47); 63% of these patients had ASCT consolidation. 71 patients had non-bortezomib-containing induction with either thalidomide, lenalidomide or other protocols including MP and VAD; only 28% of these patients had ASCT. In a recessive model the median PFS was 727 (635-818) days in carriers of the wild type C allele either in homozygous of heterozygous form (C/C&C/G, n=188), and only 491 (366-615) days in the homozygous variant G allele carriers (G/G, n=32, p=0.01). In multivariate analysis, C allele carriers had favorable PFS compared to G/G genotype patients with a hazard ratio of 1.402 (1.093-1.799), p=0.008 besides age, ISS, FISH, bortezomib treatment and ASCT. When we analyzed separately the bortezomib-treated and non-bortezomib-treated patients the benefit in PFS was only present in the bortezomib group [Fig 1, PFS 747 (656-837) days in C/C&C/G and 448 (279-616) days in G/G patients, p=0.002], and not in patients treated without bortezomib (p=0.559). We performed statistical interaction testing which showed significant interaction between bortezomib exposure and PSMB1 P11A (p=0.035).Conclusion: PSMB1 P11A is a novel predictive marker in multiple myeloma which could help to identify patients having a suboptimal response to bortezomib who might benefit from alternative management. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Superoxide Dismutase and Glutathione Peroxidase Enzyme Polymorphisms in Familial Mediterranean Fever Patients

Familial Mediterranean Fever (FMF) is a fairly common inflammatory disease in communities with mediterranean origin. It is characterized with autosomal recessive, recurrent short-term episodes of fever, peritoneal, pleural, synovial membrane involvement and skin lesions. The aim of the present study was to investigate possible associations between FMF and Ala-9Val polymorphism of MnSOD and Pro198Leu polymorphism of GPx1. The study included 129 FMF patients who has mutations (E148Q, P369S, F479L, M680I(G/C), M680I(G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H) in the heterozygous or homozygous form and 95 healthy subjects. To identify MnSOD Ala-9Val and GPx1 Pro198Leu SNPs, genotyping was performed using PCR amplification, and polymorphisms were detected with hybridization probes labeled with fluorescent dyes. Genotype and allele frequencies of Ala-9Val polymorphism of MnSOD and Pro198Leu polymorphism of GPx1 were detected. The MnSOD Val allele frequency is 132 (51.16%) in the FMF and 115 (60.52%) in the control group (p<0.05). The GPx1 Leu allele is 83 (32.17%) in the FMF and 61 (32.11%) in the control group (p=0.988). No significant differences were found between genotype frequencies of GPx1 and MnSOD polymorphisms.According to our findings MnSOD Val allele may be a genetic factor involved in the pathogenesis of FMF. The fact that there are only few studies in literature, we need more patients, other enzyme levels and works about polymorphism to support our study.

Carrier Testing in Known Autosomal Recessive Intellectual Disability Genes in an Iranian Healthy Individual Using Exome Sequencing.

Intellectual Disability (ID) is one of the most common disabling impairments worldwide. Autosomal recessive ID (ARID), a genetically heterogeneous disorder, is more common in countries such as Iran where the rate of consanguineous marriages is high. Considering the social-economic burden of ARID in our country, it is crucial to find out whether couples who are cousins are carriers for disease causing mutations, in order to prevent the birth of an affected child. Using exome sequencing, we screened known ARID genes in a normal individual to identify possible mutations in heterozygous form. We identified four protein coding alleles which possibly affect protein function, in different ID genes: PMM2, RBM28, SLC19A3, and VPS13B. These findings can be used to prevent the birth of children with ARID by checking the other partner for possible disease causing variants.

19. Parathyroidectomy in Pregnancy: A single centre 10-year experience

19. Parathyroidectomy in Pregnancy: A single centre 10-year experience

20.Anatomic study of the external branch of the superior laryngeal nerve. Anatomic findings and its implication in thyroid surgery

20.Anatomic study of the external branch of the superior laryngeal nerve. Anatomic findings and its implication in thyroid surgery

A genotyping assay for missense mutation in WISP3 gene associated with childhood onset pseudorheumatoid arthropathy

Introduction: Progressive pseudorheumatoid dysplasia (PPD) is an autosomal recessive genetic disorder reported to be caused by gene alterations of the Wnt1-inducible signaling pathway protein 3 corresponding gene (WISP3) located on chromosome position 6q22. Up to date, there is only a handful of WISP3 mutations identified in Europe, whereas most mutations are identified in Asia and Middle East. According to our knowledge, this is the first report of genetic dissection of WISP3 associated with spondyloepiphyseal dysplasia tarda from Bosnia and Herzegovina. Based on clinical examination findings (general manifestations, physical examination, characteristics of their bones on X-ray and laboratory results), an index patient was directed to WISP3 genotyping for confirmation of suspected diagnosis of PPD.Methods: DNA was extracted from peripheral blood leukocytes. All 5 exons and their exon-intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) and sequenced by Sanger method. Segregation analysis was done to confirm the familial carrier status.Results: A missense mutation (C223G) - homozygous T to G transition at c.667 in exon 4 was identified in index patient. This mutation changed codon CAG to TAG and resulted in a subsequent change of the cysteine to glycine codon. Same mutation was observed in both parents in heterozygous form confirming the familial segregation.Conclusion: Due to its nature, the identified mutation C223G in exon 4 in WISP3 gene is the most probably causative for PPD in described patient. Here we describe the PCR based method for genotyping of specific mutation in WISP3 gene. The identification of this mutation might be a valuable addition to a regional databases on rare genetic variant although a functional analysis should be performed to explain its pathological effect.

Complete hematologic response of early T-cell progenitor acute lymphoblastic leukemia to the γ-secretase inhibitor BMS-906024: genetic and epigenetic findings in an outlier case.

Notch pathway antagonists such as γ-secretase inhibitors (GSIs) are being tested in diverse cancers, but exceptional responses have yet to be reported. We describe the case of a patient with relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) who achieved a complete hematologic response following treatment with the GSI BMS-906024. Whole-exome sequencing of leukemic blasts revealed heterozygous gain-of-function driver mutations in NOTCH1, CSF3R, and PTPN11, and a homozygous/hemizygous loss-of-function mutation in DNMT3A. The three gain-of-function mutations were absent from remission marrow cells, but the DNMT3A mutation persisted in heterozygous form in remission marrow, consistent with an origin for the patient's ETP-ALL from clonal hematopoiesis. Ex vivo culture of ETP-ALL blasts confirmed high levels of activated NOTCH1 that were repressed by GSI treatment, and RNA-seq documented that GSIs downregulated multiple known Notch target genes. Surprisingly, one potential target gene that was unaffected by GSIs was MYC, a key Notch target in GSI-sensitive T-ALL of cortical T-cell type. H3K27ac super-enhancer landscapes near MYC showed a pattern previously reported in acute myeloid leukemia (AML) that is sensitive to BRD4 inhibitors, and in line with this ETP-ALL blasts downregulated MYC in response to the BRD4 inhibitor JQ1. To our knowledge, this is the first example of complete response of a Notch-mutated ETP-ALL to a Notch antagonist and is also the first description of chromatin landscapes associated with ETP-ALL. Our experience suggests that additional attempts to target Notch in Notch-mutated ETP-ALL are merited.

Genotyping Reduces Leukopenia in TPMT-Deficient Patients

Patients with thiopurine methyl transferase (TPMT) deficiency, which in its heterozygous form affects about 10% of the population, are at increased

Simultaneous Estimation of Additive and Mutational Genetic Variance in an Outbred Population of Drosophila serrata.

How new mutations contribute to genetic variation is a key question in biology. Although the evolutionary fate of an allele is largely determined by its heterozygous effect, most estimates of mutational variance and mutational effects derive from highly inbred lines, where new mutations are present in homozygous form. In an attempt to overcome this limitation, middle-class neighborhood (MCN) experiments have been used to assess the fitness effect of new mutations in heterozygous form. However, because MCN populations harbor substantial standing genetic variance, estimates of mutational variance have not typically been available from such experiments. Here we employ a modification of the animal model to analyze data from 22 generations of Drosophila serrata bred in an MCN design. Mutational heritability, measured for eight cuticular hydrocarbons, 10 wing-shape traits, and wing size in this outbred genetic background, ranged from 0.0006 to 0.006 (with one exception), a similar range to that reported from studies employing inbred lines. Simultaneously partitioning the additive and mutational variance in the same outbred population allowed us to quantitatively test the ability of mutation-selection balance models to explain the observed levels of additive and mutational genetic variance. The Gaussian allelic approximation and house-of-cards models, which assume real stabilizing selection on single traits, both overestimated the genetic variance maintained at equilibrium, but the house-of-cards model was a closer fit to the data. This analytical approach has the potential to be broadly applied, expanding our understanding of the dynamics of genetic variance in natural populations.