Enzymatic and Genetic Study in Blood Among Some Children with β – Thalassaemia in Mosul City, Iraq

The present study aimed to demonstrate the extent to which the activity of a number of enzymes and genetic variation of β-globin genes were affected in the blood of 65 children patient with β - thalassemia major of both sexes, their ages ranged between ( 2 – 15 ), who registered in the Thalassemia Center at Ibn Al-Atheer Teaching Hospital for Children in the city of Mosul / Iraq and who are continuing treatment, after they were diagnosed by specialist doctors, as well as 30 healthy children of both sexes with same ages of the patients and it was considered as a control group. The results showed a significant increase (P≤0.05) in the activity of Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), Glucose 6- phosphate dehydrogenase (G6PD) and Adenosine deaminase (ADA) in the serum of treated children patients with β - Thalassaemia major by 73% , 53%, 8%, 9% and 54% respectively, compared with the healthy children group (control group). Also the results showed a significant increase in the activity of G6PD and ADA in the hemolysis of RBC of the same children patients by 7% and 43% compared with control group. When determining the genetic variation of the β-globin gene the result which depends on PCR technique did not show any genetic variation in the size of PCR band ,while the result of the sequencing showed variation in the nucleotides and included converted the nucleotide (A) to (C) in position (250) , change nucleotide (T) to (C) in position (426), replacement (C) nucleotide to (A) in position (623), change (G) nucleotide to (A) in position (630) and replacement (T) to (A) in the position (724), also the result detection three Transversion mutation and two transition mutation in β-globin gene in babies with β-thalassemia

Treatment of a metabolic liver disease by in vivo prime editing in mice

Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of pathogenic mutations. However, correction of a disease phenotype in vivo in somatic tissues has not been demonstrated thus far. Here, we establish proof-of-concept for in vivo prime editing and repair the metabolic liver disease phenylketonuria (PKU) in mice. We first developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (PE2-deltaRnH), and a linker- and NLS-optimized intein-split PE construct (PE2 p.1153) for delivery by adeno-associated virus (AAV) vectors. Systemic dual AAV-mediated delivery of this variant into the liver of neonatal mice enabled installation of a transversion mutation at the Dnmt1 locus with an average efficiency of 15%, and delivery of unsplit PE2-deltaRnH using human adenoviral vector 5 (AdV5) further increased editing rates to 58%. PE2-deltaRnH-encoding AdV5 was also used to correct the disease-causing mutation of the phenylalanine hydroxylase (Pah)enu2 allele in phenylketonuria (PKU) mice with an average efficiency of 8% (up to 17.3%), leading to therapeutic reduction of blood phenylalanine (L-Phe) levels. Our study demonstrates in vivo prime editing in the liver with high precision and editing rates sufficient to treat a number of metabolic liver diseases, emphasizing the potential of prime editing for future therapeutic applications.

A novel mutation in the PRPH2 gene in a Chinese pedigree with retinitis pigmentosa and angle-closure glaucoma

Background Retinitis pigmentosa (RP) is a rare, progressive, and hereditary disorder that leads to the progressive loss of vision and visual field, and in some cases blindness. The specific relationship between RP and glaucoma has been debated for decades. Methods In this study, we examined a Han RP family with concomitant angle-closure glaucoma (ACG), performed an inductive analysis of their clinical features and assistant results, and applied whole-exome sequencing (WES) technology for a molecular diagnosis. Results A novel transversion mutation (c.626 T > A) was identified in the peripherin-2 (PRPH2) gene in the proband, resulting in the substitution of Valine to aspartic acid in codon 209. A full ophthalmic examination showed that the proband with the c.626 T > A mutation had a typical RP manifestation, with close angles; however, the proband’s elder brother, who lacked the novel mutation, had a normal fundus and open angles. Conclusion Our results extend the genetic mutation spectrum of PRPH2 in RP, and provide evidence to support a genetic correlation between RP and ACG.

A Novel Mutation in the PRPH2 Gene in a Chinese Pedigree with Retinitis Pigmentosa and Angle-closure Glaucoma

Background: Retinitis pigmentosa (RP) is a rare, progressive, and hereditary disorder that leads to the progressive loss of vision and visual field, and in some cases blindness. The specific relationship between RP and glaucoma has been debated for decades.Methods: In this study, we examined a Han RP family with concomitant angle-closure glaucoma (ACG), performed an inductive analysis of their clinical features and assistant results, and applied whole-exome sequencing (WES) technology for a molecular diagnosis. Results: A novel transversion mutation (c.626T>A) was identified in the peripherin-2 (PRPH2) gene in the proband, resulting in the substitution of tyrosine to aspartic acid in codon 209. A full ophthalmic examination showed that the proband with the c.626T>A mutation had a typical RP manifestation, with close angles; however, the proband’s elder brother, who lacked the novel mutation, had a normal fundus and open angles. Conclusion: Our results extend the genetic mutation spectrum of PRPH2 in RP, and provide evidence to support a genetic correlation between RP and ACG.

Exon 9 LEPR Gene SNP Polymorphism of Hybrid Chickens F2Kambro Crossbreeds of ♀ F1Kambro with ♂ F1Kambro

The implementation of the T-ARMS PCR method in the detection of single nucleotide polymorphisms (SNPs) in the LEPR gene in chicken DNA samples has never been conducted. This research aims to design a specific protocol for exon 9 LEPR gene SNPs detection and detect LEPR gene expression or LEPR SNPs in Pelung chicken samples, F1Pelung, Layer, Broiler Cobb 500, F1Kambro chicken and F2Kambro chicken using the T-ARMS PCR method. Determination of LEPR gene correlation degree on Body Weight (BT) and Egg Productivity (PT) in F1 Kambro population and F2Kambro. Qualitative phenotype parameters showed six groups of segregated phenotypes compared to F1Kambro chicken. Growth of F2Kambro chicken weight reached 753.36 ± 155.31 grams in 8 weeks was not significant for F1Kambro chicken due to inbreeding depression (Fx = 25%, IR = 4.925%) and transversion of A LEPR allele mutations. Specific protocol detection of exon 9 LEPR gene SNPs using the T-ARMS PCR method can detect C127A LEPR mutations with IP: OP ratio 10:1 pmol / µM, chicken DNA template concentration of 100 ng / µL with annealing temperature of 55.7° C / 30s. The transversion mutation of C127A of LEPR exon 9 SNP were detected in DNA samples of F1Kambro hens (80%), F2Kambro roosters (20%), Broiler Cobb 500 hens (75%). The mutations were not detected in Layer, Pelung Blirik Hitam chicken and F1Pelung populations.

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation

Activation-induced deaminase (AID) initiates hypermutation of Ig genes in activated B cells by converting C:G into U:G base pairs. G1-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G1 phase to inhibit viral replication. To derestrict G1-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. Samhd1 inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1’s restriction of dNTP supply enhances AID’s mutagenicity and that the evolution of Ig hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.

NUCLEOTIDE VARIANCE OF MITOCHONDRIAL DNA D-Loop 126 bp (nt: 34-159) REGION IN MADURESE

Endogamy continues to occur among the Madurese people in rural areas of the island of Madura, especially those areas of the smallest islands around the mainland of Madura. Endogamy as seen from a genetic standpoint will increase the frequency of homozygous genotypes. With regard to genetic variations, STRs of nuclear DNA and polymorphisms in mtDNA are frequently examined. Mitochondrial variations in the human undergo an evolutionary process through the accumulation of changes in DNA sequence, i.e. through the process of nucleotide substitutions that evolves in number with the directional development of lineage. So far, the genetic variations among the populations in Madura Island have not been known. The present study was an observational analytical research with the purpose of determining the genetic variations in the polymorphisms of 126-bp mtDNA D-Loop HV2 (nt: 34-159) in the populations of Madura Island. Results indicated that, based on the homology analysis with rCRS sequence, there were 9 variants consisting of two transition mutations, 6 transversion mutations, and one insertion mutation. This indicates that a transversion mutation had a higher probability than transition and insertion mutations. According to Mustama (2007), a gene pool is not only a collection of genes but a dynamic system organized and containing the past history of a population. Any genetic information has certain historical, anthropological and statistical aspects necessitating an interdisciplinary coordination and collaboration.

Base Excision Repair Gene OGG1 Affects the Relapse Risk of Acute Myeloid Leukemia

Background: Approximately 80% of acute myeloid leukemia (AML) patients can achieve complete remission, but around half of them relapse within five years. Recent studies have shown that AML relapse is associated with additional genetic mutation calls gclonal evolutionh in leukemic cell population (Ding, et al. Nature. 2012 & Parkin B, et al. Blood. 2013). These studies suggested that cytotoxic chemotherapy damaged cellular DNA and caused genetic mutation. In fact, anthracycline can produce 8-oxoguanine (8-OG) through induction of oxidative stress. 8-OG is most common DNA damage, which cause G:C to T:A transversion mutation. It is reported that transversion mutation more frequently observed in relapsed AML than primary AML. Base excision repair (BER) plays important role to correct base lesion including 8-OG and suppress genetic mutation. Therefore, we hypothesized BER gene polymorphisms may affect the risk of AML relapse, and focused five major functional polymorphisms: OGG1 S326C, MUTYH Q324H, APE1 D148E, XRCC1 R194W and XRCC1 R399Q. Material & Method: Ninety-four consecutive adults with AML who had achieved their first complete remission were recruited (male: 52, female: 42, age: 15-83 years, median age: 55.7 years). To remove the bias of the group, we also evaluated the risk in patients of non-M3 and under 65 years old (male: 22, female: 19, age: 15-64 years, median age: 49.0 years) (trimming group). These patients treated on Japan Adult Leukemia Study Group (JALSG) treatment protocols consisted of daunorubicin or idarubicin plus cytarabine (JALSG AML95, AML97, AML201, AML209). Genotyping was performed by PCR-RFLP method. The X2-test was used to compare the distribution of genotype and allele frequencies in patients. Leukemia-free survival (LFS) was calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. In multivariate analysis, a stepwise selection procedure was performed using the proportional hazards Cox model for LFS. The variables were chosen with reference to previous studies; age, sex, white blood cell count and lactate dehydrogenase at diagnosis, number of induction courses, stem cell transplantation, MRC classification and history of tumor. This study was approved by the Institutional Review Board of Gunma University Hospital. Results: The OGG1 S326C CC genotype was observed significantly more often in the relapsed group (28.9% vs. 8.9%, OR = 4.10, 95% CI = 1.35-12.70, p = 0.01). In trimming group, the CC genotype was also observed more frequently in the relapsed group (50.0% vs. 6.9%, OR = 13.5, 95% CI = 2.17-84.0, p = 0.002). In addition, the OGG1 S326C CC genotype experienced a shorter median LFS than those with a non-CC genotype (CC vs. non-CC = 27.0 months vs. not reached, p = 0.02) (Figure 1). This genotype was also associated with poor LFS in trimming group (CC vs. non-CC = 11.0 months vs. not reached, p < 0.001) (Figure 1). Furthermore, multivariate analysis of LFS revealed OGG1 S326C CC genotype as an independent prognostic factor (HR = 4.32, 95% CI = 1.70-11.0, p = 0.002), like age, number of induction courses, and MRC classification (Table 1). Other polymorphisms had no significant effect on the risk of relapse. Conclusion: We previously reported that mutations by 8-OG were more efficiently suppressed in OGG1-S326 transduced cells than in OGG1-C326 transduced cells. Therefore, we hypothesized that low OGG1 activity promotes relapse of AML. To the best of our knowledge, this is the first report to show an association between BER gene polymorphisms and the relapse of AML. Our data suggest that OGG1 S326C can be a prognostic factor for AML relapse. Disclosures No relevant conflicts of interest to declare.